structural and functional studies on e.coli diacylglycerol
TRANSCRIPT
Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
vorgelegt beim Fachbereich 14
der Johann Wolfgang Goethe – Universität in Frankfurt am Main
Structural and Functional Studies
on E.coli Diacylglycerol Kinase
by MAS NMR Spectroscopy
von
Kristin Möbius
aus Riesa
Frankfurt am Main, 2018
(D30)
vom Fachbereich 14
der Johann Wolfgang Goethe – Universität als Dissertation angenommen
Dekan: Prof. Dr. Clemens Glaubitz
Erster Gutachter: Prof. Dr. Clemens Glaubitz
Zweiter Gutachter: Prof. Dr. Volker Dötsch
Datum der Disputation:
Summary
Summary
The focus of this thesis is the integral membrane protein Escherichia coli diacylglycerol
kinase (DGK). It is located within the inner membrane, where it catalyzes the ATP-
dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It plays an
important role in recycling DAG during the biosynthesis of membrane-derived
oligosaccharides (MDOs) [1, 2] and lipopolysaccharides (LPSs) [3]. DGK is a unique
enzyme, which does not share any sequence homology with typical kinases. With
43 kDa, it is the smallest known kinase [4]. It features a notable complexity in structure
and function [4-7] as well as a remarkable stability [8, 9]. It is a homotrimer, in which
each monomer contains three transmembrane helices and one N-terminal amphiphilic
surface helix [5]. The trimer features three active sites arranged around the
membrane/cytoplasm interface [5]. Each active site is formed by two adjacent
protomers, leading to an unusual catalytic site architecture of the composite shared site
model [5, 7, 10]. Several mutational studies [6, 7, 10], MD simulations [6, 11], solution
NMR studies in detergent micelles [7] and particularly the 3D structure determination
by X-ray crystallography [5, 6] offer insights into a possible catalytic mechanism of
DGK. However, important long-standing questions remain unsolved.
The aim of this thesis is the investigation of DGK’s structure and function at an atomic
level directly within the native-like lipid bilayer using MAS NMR. This way, a deeper
understanding of DGK’s catalytic mechanism should be obtained.
First, the preparation of DGK was optimized, in order to achieve high amounts of pure,
active, stable and homogeneously reconstituted protein, which provides well-resolved
MAS NMR spectra (chapter 3). For this purpose, the preparation protocol that has been
used before in this lab was optimized successively, defining the DGK construct, type of
detergent, lipid composition, reconstitution method and protein-to-lipid ratio. The quality
of the sample was characterized by SDS- and BN-PAGE, SEC, LILBID-MS, sucrose
density gradient centrifugation, the coupled activity assay and MAS NMR. The
optimization resulted in a efficient protocol that provides a DGK sample, which is more
native than in previous studies and, in addition, features MAS NMR spectra of high
resolution and sensitivity.
The high quality MAS NMR spectra formed the foundation for the second step, the
resonance assignment of DGK’s backbone and side chains (chapter 4). The
assignment was performed at high magnetic field (1H frequency 850 MHz). The
Summary
sequential assignment of immobile domains was carried out using a combination of
dipolar coupling based 3D experiments, NCACX, NCOCX and CONCA. The
measurement time could be reduced by paramagnetic doping with Gd3+-DOTA [12] in
combination with a custom-built E-free probehead (Bruker). The sequential assignment
was mainly performed using a uniformly labelled sample (U-13C,15N-DGK). Residual
ambiguities could be resolved by reverse labelling of isoleucine, leucine and valine (U-
13C,15N-DGK-I,L,V). The carbon and nitrogen resonances could be assigned for
approximately 82% of the residues, from which 74% were completely assigned. For
validation, ssFLYA was applied, which is a generally applicable algorithm for the
automatic assignment of protein solid state NMR spectra [13]. Its principal applicability
for demanding systems as membrane proteins could be proven in this study for the first
time. Overall, 91.5% of the backbone and 89.1% of all (backbone + side chains)
manually obtained assignments could be confirmed by ssFLYA. For the completion of
DGK’s assignment, scalar coupling based 2D experiments, 1H-13C/15N HETCOR and
13C-13C TOBSY, were carried out to detect highly mobile residues. This way, residues
of the two termini and the cytosolic loop, which were not detectable by dipolar coupling
based experiments, could be assigned tentatively. Whereupon, peaks for arginine and
lysine were assigned unambiguously to Arg9 and Lys12. Overall, 84% of the residues
located in transmembrane and extramembranous regions could be assigned with the
used NMR strategy.
During the assignment procedure, no systematic peak doublets or triplets were
detected, indicating that the DGK trimer adopts a symmetric conformation in its apo
state. This is in contrast to the X-ray structure, which shows asymmetries between the
three subunits. The differences are most likely attributed to different experimental
conditions. Especially, crystal packing may be a potential source for structural
asymmetries.
Based on the resonance assignment, a secondary structure analysis was carried out. It
showed substantial similarities between wild-type DGK, its thermostable mutant [14]
determined both by MAS NMR and the crystal structure of wtDGK [6]. However, there
are few differences. In contrast to both MAS NMR secondary structures, the crystal
structure shows small deviations around the flexible regions. The largest discrepancy
occurs at the cytosolic loop (CL) concerning its length and position. It is shifted from the
residues 83-87 in the MAS NMR structure of wtDGK to the residues 83–90 (subunit A),
86-91 (subunit B) and 82-87 (subunit C) of the X-ray structure.
In addition to 13C/15N detected experiments at a moderate MAS rate, very first 1H
detected experiments in combination with ultra-fast MAS at 111 kHz were carried out
as well, demonstrating its general applicability on fully protonated DGK in lipid bilayers.
Summary
On the basis of the nearly complete assignment of DGK, the apo state was compared
with the substrate bound states (chapter 5). Perturbations in peak position and intensity
of the substrate bound states were analysed for each of the 101 assigned residues in
3D and 2D heteronuclear correlation spectra. The nucleotide-bound state was
emulated by adenylylmethylenediphosphonate (AMP-PCP), a non-hydrolysable ATP
analogue, whereas the DAG-bound state was mimicked by 1,2-dioctanoyl-sn-glycerol
(DOG, chain length n = 8). For finding saturation conditions, a competitive Mg*ATP
inhibition assay was performed by monitoring the ATPase activity as a function of
Mg*AMP-PCP concentration. Additionally, the binding of AMP-PCP and DOG was
verified by 31P-cross polarization (CP) experiments.
Significant chemical shift perturbations and altered peak intensities could be observed
in both the AMP-PCP and DOG bound state. These data provide evidence that all
three active sites are occupied at the same time. Additionally, it could be demonstrated
that the nucleotide substrate induces a substantial conformational change. This most
likely supports the enzyme in binding of the lipid substrate, indicating positive
heteroallostery. For DGK bound with either AMP-PCP + DOG or only AMP-PCP, a
similar spectral fingerprint was observed. This implies that binding of the nucleotide
seems to set the enzyme into a catalytic active state, triggering the actual phosphoryl
transfer reaction.
The investigation of DGK’s remarkable stability and the cross-talk between its subunits
forms the last part of this thesis (chapter 6). This demands for the identification of key
intra- and interprotomer contacts, which are of structural or functional importance. For
this purpose, 13C-13C DARR and 2D NCOCX spectra with long mixing times were
recorded using high field MAS NMR. Additionally, DNP-enhanced 13C−15N TEDOR
experiments were conducted on mixed labelled DGK trimers to enable the visualization
of interprotomer contacts. In order to generate mixed labelled trimers, a procedure was
established using SDS. With the applied NMR strategy, functionally relevant intra-
(Arg32 - Trp25/ Glu28/ Ala29 and Trp112 - Ser61) and interprotomer (ArgNn,e -
AspCg/ GluCd/ AsnCg) long-range interactions could be identified. Based on the
crystal structure, the interprotomer contacts can be most likely attributed to Arg81-
Glu88 and Arg92-Asn27/Glu28. The identified interactions may stabilize the active sites
and/or transmit information about substrate binding or changes of the surrounding lipid
bilayer within and/or between protomers.
Zusammenfassung
Zusammenfassung
Im Fokus dieser Dissertation steht das integrale Membranprotein Escherichia coli
Diacylglycerolkinase (DGK). Es befindet sich in der inneren Membran, wo es die ATP-
abhängige Phosphorylierung von Diacylglycerol (DAG) zu Phosphatidylsäure (PA)
katalysiert. Es spielt eine wichtige Rolle im Recycling von DAG während der
Biosynthese von Membran-abgeleiteten Oligosacchariden (MDOs) [1, 2] und
Lipopolysacchariden (LPSs) [3]. MDOs werden beispielsweise unter Bedingungen
geringer Osmolarität in großen Mengen produziert [15, 16], während LPSs die
Hauptbestandteile der bakteriellen Außenmembran darstellen [17]. Beide
Komponenten dienen dem Schutz des gram-negativen Bakteriums. E.coli DGK ist ein
einzigartiges Enzym, welches weder Struktur- noch Sequenzähnlichkeiten zu anderen
Kinasen aufweist. Mit 43 kDa ist es die kleinste bekannte Kinase [4]. DGK verfügt über
eine bemerkenswerte Komplexität in seiner Struktur und Funktion [4-7] sowie in seiner
Stabilität [8, 9]. Li et al. bestimmten 2013 die 3D Kristallstruktur von DGK in lipidisch-
kubischen Phasen bestehend aus Monoacylglycerolen, die auch als Substrat fungieren
[5]. Die Kristallstruktur zeigt DGK als Homotrimer, in welchem jedes Monomer drei
Transmembranhelices und eine N-terminale amphiphile Oberflächenhelix besitzt. Das
Trimer hat drei aktive Stellen, welche um die Membran/Zytoplasma-Kontaktfläche
angeordnet sind. Jede aktive Stelle wird von den Transmembranhelices einer
Untereinheit und der Oberflächenhelix einer benachbarten Untereinheit gebildet.
Daraus ergibt sich eine ungewöhnliche Architektur der katalytisch-aktiven Stellen
basierend auf dem sogenannten „composite shared site model“ [5, 7, 10].
Verschiedene Mutationsstudien [6, 7, 10], MD Simulationen [6, 11], Lösungs-NMR-
Studien in Detergenz-Mizellen [7] und vor allem die 3D Strukturbestimmung durch
Röntgenkristallographie [5, 6] liefern Einblicke in einen möglichen katalytischen
Mechanismus von DGK. Allerdings bleiben wichtige, seit Langem bestehende Fragen
unbeantwortet. Es ist beispielsweise unklar, ob das DGK-Trimer eine symmetrische
oder asymmetrische Konformation einnimmt. Außerdem ist noch nicht bekannt, ob sich
alle drei aktiven Zentren von DGK während der Katalyse im selben oder in
unterschiedlichen Zuständen befinden. Desweiteren ist noch nicht erwiesen, ob DGK
eine entscheidende Konformationsänderung vor dem eigentlichen Phoshoryltransfer
durchläuft. Eine Vermutung diesbezüglich wurde bereits 1997 von Badola und Sanders
angestellt [18], konnte allerdings bisher noch nicht belegt werden. Betrachtet man die
hohe Stabilität des Enzyms und die Tatsache, dass jedes aktive Zentrum durch zwei
benachbarte Untereinheiten gebildet wird, ergibt sich zudem die Frage, ob spezifische
Zusammenfassung
Intra- und Interprotomerkontakte existieren. Die vorliegende Dissertation widmet sich
diesen Fragen unter Verwendung von Festkörper-Nuklearmagnetischer
Resonanzspektroskopie (FK-NMR). Im Detail wurde MAS NMR Spektroskopie
angewandt, welche ein Drehen der Probe im sogenannten magischen Winkel von
54.74° impliziert. Hierbei handelt es sich um eine etablierte Methode für Studien an
Membranproteinen, welche zunehmend an Bedeutung in der Strukturbiologie gewinnt
und wesentliche komplementäre Daten zur Röntgenkristallographie,
Kryoelektronenmikroskopie und Lösungs-NMR liefert. Basierend auf der hohen
Sensitivität der NMR-Signale bzgl. der lokalen Umgebung können selbst schwache
Ligandenbindungen durch Änderungen in der chemischen Verschiebung analysiert
werden, was wiederum Struktur-Aktivitäts-Korrelationen erlaubt. MAS NMR bietet
zudem den Vorteil, dass es nicht die strukturelle Plastizität des entsprechenden
Membranproteins einschränkt, welche in den meisten Fällen von funktionaler
Bedeutung ist. Weiterhin bietet MAS NMR die Möglichkeit Membranproteine direkt in
der Lipiddoppelschicht zu untersuchen. Damit wird das zu untersuchende System
physiologischen Bedingungen näher gebracht als in anderen Membran-imitierenden
Umgebungen wie beispielsweise in Detergenz-Mizellen. Die Lipiddoppelschicht stellt
einen wichtigen strukturellen Faktor dar und ist zudem in den meisten Fällen direkt mit
der katalytischen Aktivität des Membranproteins verbunden [11, 19-21].
Das Ziel dieser Dissertation ist die Untersuchung der Struktur und Funktion von DGK
auf atomarem Level, direkt in der Lipiddoppelschicht mittels MAS NMR. Auf diesem
Weg soll ein tieferes Verständnis von DGK‘s katalytischem Mechanismus erlangt
werden.
Hierfür wurde zunächst die Präparation von DGK optimiert, um hohe Ausbeuten an
reinem, aktivem, stabilem und homogen-rekonstituiertem Protein zu erzielen, welches
gut-aufgelöste FK-NMR Spektren liefert (Kapitel 3). Dazu wurde das
Präparationsprotokoll schrittweise optimiert, welches zuvor in dieser Arbeitsgruppe
verwendet wurde. Hierbei wurden das DGK-Konstrukt, die Art des Detergenzes, die
Lipidzusammensetzung, die Rekonstitutionsmethode und das Protein-Lipid-Verhältnis
neu definiert: Es wurde Wildtyp-DGK, dessen hohe Stabilität bereits nachgewiesen
worden ist, anstelle der thermostabilen Quadrupelmutante (Δ4-DGK: I53C, I70L, M96L,
V107D) verwendet. Außerdem wurde das schonende, nicht-ionische Detergenz n-
Dodecyl-β-d-Maltopyranosid (DDM) anstatt des harschen zwitter-ionischen
Detergenzes Dodecyl Phosphocholin (DPC) genutzt. Die Reinheit von Wildtyp-DGK in
Detergenz-Mizellen wurde durch SDS-PAGE und SEC charakterisiert, während der
Zusammenfassung
oligomere Zustand mittels BN-PAGE und LILBID-MS bewertet wurde. Die Ausbeute an
DGK konnte um 50% von 20-30 mg DGK pro Liter Zellkultur auf 30-45 mg/l erhöht
werden. Weiterhin wurde die Rekonstitutionsmethode geändert. Statt einer
langwierigen Dialyse wurde zur hydrophoben Absorption mittels BioBeads gewechselt,
was den Zeitaufwand von zwei Wochen auf zwei Tage reduzierte. Zudem wurde die
Lipidkomposition modifiziert. Anstelle von 67.3mol% DMPC/ 32.7mol% Cholesterol
wurde die dem System besser entsprechende Lipidzusammensetzung aus 90mol%
DMPC/ 10mol% DMPA verwendet, die zudem zu einer höheren spektralen Auflösung
führte. Außerdem wurde das molare Protein-Lipid-Verhältnis von 1:80 auf 1:50 erhöht.
Somit konnten 30% mehr Protein in den Rotor gepackt werden, was die spektrale
Sensitivität verbesserte. Die Sucrose-Dichtegradienten-Zentrifugation wurde genutzt,
um eine homogene Proteinrekonstitution in die Liposomen zu überprüfen. Die Qualität
der optimierten Proteoliposomprobe wurde außerdem mit Hilfe eines gekoppelten
Aktivitätsassays und MAS NMR charakterisiert. Die Optimierung führte zu einem
effizienten Protokoll, welches DGK-Proben liefert, die nativer sind als in bisherigen
Studien und zudem FK-NMR-Spektren von hoher Auflösung und hoher Sensitivität
aufweisen.
Die qualitativ-hochwertigen FK-NMR-Spektren bildeten die Grundlage für den zweiten
wichtigen Schritt, die Resonanz-Zuordnung von DGK’s Rückgrat und Seitenketten
(Kapitel 4). Die Zuordnung wurde mittels multidimensionaler FK-NMR bei hohem
Magnetfeld (1H Frequenz von 850 MHz) durchgeführt. Die sequentielle Zuordnung der
immobilen Domänen erfolgte durch eine Kombination aus 3D Experimenten (NCACX,
NCOCX, CONCA), die auf der dipolaren Kopplung basieren. Die Messzeit konnte
durch paramagnetisches Doping mit Gd3+-DOTA in Kombination mit einem speziell
angefertigten E-freien Probenkopf (Bruker) reduziert werden. Die Zuordnung erfolgte
hauptsächlich an einer uniform-markierten Probe (U-13C,15N-DGK). Verbleibende
Ambiguitäten konnten mittels reverse labelling von Isoleucin, Leucin und Valin (U-
13C,15N-DGK-I,L,V) beseitigt werden. Die 13C- und 15N-Resonanzen konnten für ca.
82% der Reste zugeordnet werden. Davon wurden 74% vollständig zugeordnet. Zur
Validierung wurde ssFLYA angewandt. Hierbei handelt es sich um einen allgemein
anwendbaren Algorithmus zur automatischen Zuordnung von Protein-FK-NMR-
Spektren. Seine prinzipielle Anwendbarkeit für anspruchsvolle Systeme wie
Membranproteine konnte mittels dieser Studie nachgewiesen werden. Insgesamt
wurden 91,5% der manuell erhaltenen Rückgratzuordnungen und 89,1% aller
(Rückgrat und Seitenketten) manuellen Zuordungen durch ssFLYA bestätigt. Zur
Vervollständigung der Zuordnung von DGK wurden zudem J-Kopplung basierte 2D
Zusammenfassung
Experimente (1H-13C/15N HETCOR und 13C-13C TOBSY) zur Detektion von mobilen
Resten durchgeführt. Hierbei konnten Reste der beiden Termini und des zytosolischen
Loops, welche durch Experimente, die auf der dipolaren Kopplung basieren, nicht
detektierbar sind, tentativ zugeordnet werden. Wobei Signale für Arginin und Lysin
eindeutig Arg9 und Lys12 zugeordnet wurden. Insgesamt konnten 84% der Reste,
welche sich in den Transmembran- und Extramembran-Regionen befinden, durch die
angewandte NMR-Strategie zugeordnet werden.
Während des Zuordnungsprozesses wurden keine systematischen Signal-Dubletts
bzw. Tripletts detektiert, was darauf schließen lässt, dass das DGK-Trimer eine
symmetrische Konformation im Apo-Zustand einnimmt. Das steht im Gegensatz zur
Kristallstruktur, welche Asymmetrien zwischen den Untereinheiten aufweist [5]. Die
Abweichungen können auf unterschiedliche experimentelle Bedingungen zurückgeführt
werden. Hierbei scheint vor allem die dichte Packung der Kristalle eine mögliche
Quelle für strukturelle Asymmetrien zu sein.
Die Sekundärstrukturanalyse zeigte substantielle Ähnlichkeiten zwischen Wildtyp-DGK,
seiner thermostabilen Mutante, wobei beide Sekundärstrukturen durch FK-NMR
bestimmt wurden, und der Kristallstruktur von Wildtyp-DGK (PDB 3ZE4). Allerdings
konnten auch einige Unterschiede festgestellt werden. Im Gegensatz zu beiden FK-
NMR-Sekundärstrukturen, zeigt die Kristallstruktur geringe Abweichungen im Bereich
der flexiblen Regionen. Der größte Unterschied tritt im zytoplasmatischen Loop
bezüglich Position und Länge auf. Er ist von den Resten 83-87 in der MAS NMR
Struktur des Wildtyps zu den Resten 83–90 (Untereinheit A), 86-91 (Untereinheit B)
und 82-87 (Untereinheit C) in der Kristallstruktur verschoben.
Neben 13C/15N-detektierten Experimenten bei einer moderaten MAS-Rate, wurden
erste 1H-detektierte Experimente in Kombination mit ultra-schnellem MAS bei 111 kHz
(0.7 mm Rotor) durchgeführt. Die Ergebnisse indizieren eine erfolgsversprechende
Basis für zukünftige Experimente dieser Art an vollständig-protoniertem DGK in
Lipiddoppelschichten.
Auf der Basis der nahezu vollständigen Zuordnung von DGK, wurde der Apo-Zustand
mit den Substrat-gebundenen Zuständen verglichen (Kapitel 5). Es wurden
Änderungen in Peakposition und -intensität der Substrat-gebundenen Zustände für
jeden der 101 zugeordneten Reste in 3D und 2D heteronuklearen Korrelationsspektren
analysiert. Der Nukleotid-gebundene Zustand wurde durch Adenylyl
Methylendiphosphonat (AMP-PCP), einem nicht-hydrolysierbaren ATP-Analogon,
emuliert, während der DAG-gebundene Zustand durch 1,2-Dioctanoyl-sn-glycerol
(DOG, Kettenlänge n=8) imitiert wurde. Zur Ermittlung geeigneter
Zusammenfassung
Sättigungsbedingungen wurde ein kompetitiver Mg*ATP-Inhibierungsassay
durchgeführt, in dem die ATPase Aktivität als Funktion der Mg*AMP-PCP-
Konzentration beobachtet wurde. Außerdem wurde die Bindung von AMP-PCP und
DOG durch 31P-Kreuzpolarisationsexperimente überprüft.
Es konnten sowohl im AMP-PCP- als auch im DOG-gebundenen Zustand eindeutige
Änderungen der chemischen Verschiebung sowie der Peakintensitäten beobachtet
werden. Diese Daten liefern Hinweise darauf, dass alle drei aktiven Stellen gleichzeitig
besetzt sind. Außerdem konnte gezeigt werden, dass das Nukleotidsubstrat eine
weitreichende Konformationsänderung hervorruft. Diese dient sehr wahrscheinlich der
Bindung des Lipidsubstrates an das Enzym und indiziert somit eine positive
Heteroallosterie. Zudem weisen der AMP-PCP+DOG-gebundene und der
ausschließlich AMP-PCP-gebundene Zustand den gleichen spektralen Fingerabdruck
auf. Das deutet daraufhin, dass das Nukleotid das Enzym in einen katalytisch-aktiven
Zustand zu versetzen scheint, welcher die eigentliche Phosphoryltransferreaktion
einleitet.
Die Untersuchung der hohen Stabilität von DGK sowie der Kommunikation zwischen
den Untereinheiten bildet den letzten Teil der Dissertation (Kapitel 6). Dies erforderte
die Identifizierung von entscheidenden Intra- und Interprotomerkontakten, welche eine
strukturelle und funktionelle Bedeutung haben. Hierfür wurden 13C-13C DARR- und 2D
NCOCX-Spektren mit langen Mischzeiten unter Verwendung von Hochfeld-NMR
aufgenommen. Außerdem wurden DNP-verstärkte 13C−15N TEDOR-Experimente
durchgeführt, um Interprotomerkontakte in gemischt-markierten DGK-Trimeren
nachzuweisen. Zur Erzeugung von gemischt-markierten DGK-Trimeren wurde ein
Verfahren etabliert, welches SDS nutzt. Mit Hilfe der angewandten NMR-Strategie
konnten so funktional-relevante Intra (Arg32 - Trp25/ Glu28/ Ala29 and Trp112 -
Ser61)- und Inter (ArgNn,e - AspCg/ GluCd/ AsnCg)-protomerinteraktionen identifiziert
werden. Basierend auf der Kristallstruktur können die Interprotomerkontakte sehr
wahrscheinlich Arg81-Glu88 und Arg92-Asn27/Glu28 zugeordnet werden [5]. Die
identifizierten Interaktionen stablisieren hierbei möglicherweise die aktiven Zentren
und/oder übermitteln Informationen über die Substratbindung bzw. über Änderungen in
der umgebenden Lipiddoppelschicht innerhalb und/oder zwischen den einzelnen
Protomeren.
Table of Contents
1
Table of Contents
1 Introduction ................................................................................ 5
1.1 Diacylglycerol kinases in Eukaryotes and Prokaryotes.......................... 6
1.1.1 Escherichia coli diacylglycerol kinase ............................................................... 9
1.1.1.1 Location and function ................................................................................................ 9
1.1.1.2 Enzymology ............................................................................................................. 10
1.1.1.3 Stability/ folding and misfolding ............................................................................... 12
1.1.1.4 Structure .................................................................................................................. 14
1.1.1.5 Mapping of the active site........................................................................................ 19
1.1.1.6 Proposed catalytic mechanism ............................................................................... 24
1.2 Aim of thesis ............................................................................................. 27
1.3 Solid state nuclear magnetic resonance (ssNMR) spectroscopy ........ 29
1.3.1 Theoretical background .................................................................................. 29
1.3.1.1 Interactions in ssNMR ............................................................................................. 29
1.3.1.1.1 Chemical shift and dipolar coupling ................................................................. 29
1.3.1.2 Magic angle spinning (MAS) ................................................................................... 31
1.3.1.3 Cross polarization .................................................................................................... 32
1.3.1.4 Multidimensional NMR experiments ........................................................................ 34
1.3.2 High-field MAS NMR ...................................................................................... 35
1.3.2.1 Dipolar coupling based experiments for the detection of immobile residues .......... 35
1.3.2.1.1 Homonuclear correlation experiments based on proton driven spin diffusion . 35
1.3.2.1.2 Heteronuclear correlation experiments for the sequential assignment ............ 37
1.3.2.1.2.1 3D NCACX and NCOCX ........................................................................... 37
1.3.2.1.2.2 3D CONCA ................................................................................................ 38
1.3.2.2 Scalar coupling based experiments for the detection and tentative assignment of
highly mobile residues ............................................................................................ 39
1.3.2.2.1 2D 1H-
13C/
15N HETCOR ................................................................................... 39
1.3.2.2.2 2D 13
C-13
C TOBSY ........................................................................................... 40
1.3.3 Dynamic nuclear polarization (DNP)-enhanced MAS NMR ............................. 40
1.3.3.1 13
C-15
N TEDOR experiments for the dectection of interprotomer contacts ............. 42
2 Materials and Methods ............................................................. 45
2.1 Constructs and cells ................................................................................ 45
2.2 Molecular Cloning .................................................................................... 46
2.2.1 Transformation ............................................................................................... 48
2.2.2 Glycerol stocks ............................................................................................... 48
Table of Contents
2
2.3 Protein expression and purification ....................................................... 48
2.3.1 Samples for high field MAS NMR ................................................................... 48
2.3.2 Mixed labelled samples for DNP-enhanced MAS NMR .................................. 51
2.4 Protein reconstitution .............................................................................. 53
2.4.1 Liposome preparation ..................................................................................... 53
2.4.2 Reconstitution via BioBeads ........................................................................... 54
2.5 Sample characterization .......................................................................... 54
2.5.1 SDS-PAGE ..................................................................................................... 54
2.5.2 SEC ................................................................................................................ 55
2.5.3 BN-PAGE ....................................................................................................... 55
2.5.4 LILBID-MS ...................................................................................................... 56
2.5.5 Sucrose density gradient centrifugation .......................................................... 57
2.5.6 Coupled activity assay .................................................................................... 57
2.6 Preparing substrate-bound states of DGK ............................................. 58
2.7 MAS NMR .................................................................................................. 59
2.7.1 MAS NMR at high field ................................................................................... 59
2.7.1.1 Manual resonance assignment ............................................................................... 59
2.7.1.2 Substrate bound states ........................................................................................... 60
2.7.1.3 Data analysis ........................................................................................................... 60
2.7.2 Automatic resonance assignment by ssFLYA ................................................. 61
2.7.3 DNP-enhanced MAS NMR ............................................................................. 63
3 Sample optimization ................................................................. 65
3.1 Introduction .............................................................................................. 65
3.2 Results and Discussion ........................................................................... 66
3.2.1 DGK construct: Quadruple mutant (Δ4-DGK) vs. wild-type DGK .................... 66
3.2.2 Type of detergent: DPC vs. DDM ................................................................... 67
3.2.3 Characterizing the yield, purity and oligomeric state of wtDGK in DDM .......... 68
3.2.4 Reconstitution method: Dialysis vs. BioBeads ................................................ 69
3.2.5 Lipid composition, protein-to-lipid ratio and functional characterization ........... 70
3.2.6 Evaluating the optimized DGK proteoliposomes for MAS NMR application .... 75
3.3 Summary ................................................................................................... 76
4 Resonance assignment ............................................................ 77
4.1 Introduction .............................................................................................. 77
Table of Contents
3
4.1.1 Applied isotope labelling strategy ................................................................... 79
4.1.2 Applied strategy for improvements of the sensitivity and resolution ................ 81
4.1.2.1 High magnetic fields ................................................................................................ 81
4.1.2.2 Paramagnetic doping in combination with an E-free probehead ............................ 81
4.1.3 Applied assignment procedure ....................................................................... 82
4.1.3.1 Sequential assignment of immobile domains .......................................................... 83
4.1.3.1.1 Automatic assignment of immobile domains by ssFLYA ................................. 84
4.2 Results and Discussion ........................................................................... 85
4.2.1 Spectral resolution and isotope labelling ......................................................... 85
4.2.2 Paramagnetic doping in combination with an E-free probehead ..................... 86
4.2.3 Sequential assignment of immobile domains .................................................. 87
4.2.3.1 Automatic assignment of immobile domains by ssFLYA ........................................ 90
4.2.4 Tentative assignment of highly mobile regions ............................................... 92
4.2.5 Summary of the assignment ........................................................................... 94
4.2.6 Secondary structure analysis .......................................................................... 95
4.2.7 DGK forms a symmetric trimer in its apo state ................................................ 96
4.3 Outlook ...................................................................................................... 97
4.3.1 Further labelling strategies ............................................................................. 97
4.3.2 Perspective: 1H detection in combination with ultra-fast MAS ......................... 98
5 Functional studies based on chemical shift perturbations . 103
5.1 Introduction ............................................................................................ 103
5.2 Results .................................................................................................... 104
5.2.1 Establishing nucleotide- and DAG-bound states of DGK for NMR analysis .. 104
5.2.2 DGK forms a symmetric trimer in its substrate bound states ......................... 106
5.2.3 Substrate-induced chemical shift and peak intensity perturbations ............... 107
5.2.3.1 AMP-PCP bound state .......................................................................................... 107
5.2.3.2 DOG bound state .................................................................................................. 109
5.2.3.3 AMP-PCP + DOG bound state .............................................................................. 112
5.3 Discussion .............................................................................................. 112
5.3.1 DGK forms a symmetric trimer in its substrate-bound states ........................ 112
5.3.2 AMP-PCP bound state ................................................................................. 113
5.3.2.1 Comparison of the AMP-PCP bound state with solution NMR data ..................... 114
5.3.3 DOG bound state ......................................................................................... 115
5.4 Summary and Outlook ........................................................................... 116
Table of Contents
4
6 Long-range contacts .............................................................. 117
6.1 Introduction ............................................................................................ 117
6.2 Intraprotomer contacts visualized by high field MAS NMR ................ 118
6.2.1 Results and Discussion ................................................................................ 118
6.3 Interprotomer contacts visualized by DNP-enhanced MAS NMR ...... 119
6.3.1 Introduction .................................................................................................. 119
6.3.2 Results ......................................................................................................... 121
6.3.2.1 Creating mixed labelled trimers of DGK ................................................................ 121
6.3.2.2 Validation of the application of AMUPol as biradical ............................................. 124
6.3.2.3 DNP-enhanced 15
N−13
C TEDOR experiments ...................................................... 125
6.3.2.3.1 Finding the best mixing time using 1D TEDOR spectra ................................. 125
6.3.2.3.2 Visualizing interprotomer contacts using 2D TEDOR spectra ....................... 127
6.3.2.3.3 Attemps to assign the cross-peak by RxA-mutants ....................................... 130
6.3.2.3.4 AMP-PCP bound state of mixed labelled DGK .............................................. 133
6.3.3 Discussion .................................................................................................... 133
6.3.3.1 Creating mixed labelled trimers of DGK ................................................................ 133
6.3.3.2 Statistical analysis of unique interfaces in mixed labelled DGK ........................... 134
6.3.3.3 DNP-enhanced 15
N-13
C TEDOR experiments ....................................................... 136
6.3.3.4 Attemps to assign the cross-peak by RxA-mutants .............................................. 136
6.3.3.4.1 Drawback of mutations ................................................................................... 137
6.3.3.5 Assessing the interprotomer contacts during nucleotide binding .......................... 138
6.4 Summary and Outlook ........................................................................... 139
Appendix .................................................................................... 141
Supplementary tables .................................................................................. 141
List of abbreviations .................................................................................... 157
List of figures ................................................................................................ 160
List of tables ................................................................................................. 172
References ................................................................................. 174
Declaration of contributions ..................................................... 192
Acknowledgements ...................... Fehler! Textmarke nicht definiert.
Curriculum vitae ........................... Fehler! Textmarke nicht definiert.
Chapter 1: Introduction
5
1 Introduction
Membrane proteins represent between 20 to 30% of the genes in most organisms [22,
23]. They are essential for both cellular life and human health. In detail, they are critical
to cell physiology, playing roles in signalling, trafficking, transport, adhesion, and
recognition. Due to their importance, membrane proteins are major targets of
biomedical research. An analysis performed by Overington et al. concluded that
membrane proteins provide more than 60% of drug targets [24]. Thus, drug discovery
efforts aim to understand their biological functions and take advantage of their
therapeutic potential. In order to achieve this goal, molecular structure determination is
necessary. In recent years, structural biology of membrane proteins has progressed
remarkably. X-ray crystallography, electron microscopy (EM) and nuclear magnetic
resonance (NMR) have all contributed fundamental and complementary structural data
[25-28], each with individual advantages and particular challenges. X-ray
crystallography has made considerable contributions to membrane protein structural
biology, with remarkable achievements in the field of G protein coupled receptors
(GPCRs) [29-31]. EM has long been used to investigate the structures of membrane
proteins in proteolipid two-dimensional (2D) crystals [32]. The recent development of
single-particle cryo-EM [33-35] provides higher resolution structures of membrane
proteins without the need of the preparation of large, well-ordered crystalline samples
[26, 36]. NMR has a long history as a key technology in enhancing our understanding
of the structural, chemical, and dynamical characteristics of lipid bilayer membranes.
Early NMR studies offered elementary information about the structures and dynamics
of phospholipid formations, and the impact of membrane proteins and several other
membrane components on lipid bilayers [37-40]. NMR plays also a fundamental role in
membrane protein structural biology, providing methods for the investigation of
membrane proteins in a large variety of samples, including soluble detergent micelles,
detergent-free lipid bilayers, and native cell envelope preparations [7, 28, 41-45]. The
wide range of sample types mirrors the versatility of NMR as a tool for characterizing
the structures, dynamics, and functional interactions of biomolecules. NMR is also
versed at exploring intrinsically disordered regions of proteins [46]. Additionally, based
on the high sensitivity of NMR signals to the local environment, they are extremely
useful for analysing even weak ligand binding by chemical shift changes, providing
structure activity correlations for binding processes or conformational changes [47].
Another considerable advantage of NMR as a technique for structural analysis is that it
enables the investigation of membrane proteins without attenuating its structural
plasticity that is in most cases integral to the biological function. This is contrary to X-
Chapter 1: Introduction
6
ray and single particle cryo-EM studies, which stabilize a single molecular conformation
by the crystallization process itself and/or by the application of cryogenic temperatures.
In addition, they often demand elaborate sample engineering, such as antibody
stabilization and protein mutations, truncations, insertions and modifications [26, 36,
48-52]. Moreover, regarding EM, three-dimensional (3D) reconstruction persists
demanding for proteins smaller than ~100 kDa. Recent progress in NMR structural
studies of membrane proteins reflect large developments in the areas of recombinant
protein expression, sample preparation, pulse sequences for high resolution
spectroscopy, radio-frequency probes, high-field magnets and computational methods.
All enable monitoring single atomic sites in membrane proteins with ever-expanding
accuracy. Thus, far-reaching and detailed information addressing structural and
dynamical changes can be obtained. Solution NMR methods can be used for structure
determination of membrane proteins in detergent micelles or detergent/lipid mixed
micelles [7, 41, 42]. Whereas solid state NMR, in particular MAS (magic angle
spinning) NMR, offers the possibility to explore membrane proteins in detergent-free
lipid bilayers, which brings the investigated system closer to physiological conditions
compared to other membrane mimicking environments such as detergent micelles. The
membrane environment is of key importance as it is a strong structural factor. In most
cases, it is also directly linked to the catalytic activity of the membrane protein [11, 19-
21].
In this study, the structure and dynamics of the membrane protein diacylglycerol kinase
(DGK) embedded into the lipid bilayer is investigated by MAS NMR.
1.1 Diacylglycerol kinases in Eukaryotes and Prokaryotes
Diacylglycerol kinases (DGKs) are members of a conserved family of intracellular lipid
kinases that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to
phosphatic acid (PA) [53, 54]. DAG and PA are intermediates in lipid biosynthetic
pathways and two main signalling molecules. DAG is a common second messenger.
Its cellular levels are increased through hydrolysis of phosphoinositides by
phospholipase C (PLC) in response to a variety of extracellular stimuli including growth
factors and hormones. DAG is most known as an activator of protein kinase C (PKC)
[55], which plays a key role in biological processes like cell proliferation and
differentiation [56]. DAG also interacts with other effector proteins, such as α- and β-
chimaerins (having Rac-GAP activity) [57], guanyl nucleotide-exchange factors for Ras
and Rap, i.e., RasGRP [57-60] and CalDAG-GEFI [60], respectively, as well as
Chapter 1: Introduction
7
nonselective cation channels (TRPC6 and -3) [61]. Thus, DGK regulates the presence
of PKC at the membrane [62], and/or terminates receptor-induced PKC activation and
thereby the signalling pathways downstream of PKC. Moreover, DGK indirectly
regulates small molecular weight G proteins via their nucleotide exchange factors or
GTPase-activating proteins (e.g. chimaerins for Rac), which are activated by DAG. Not
only DGK’s substrate, but also its product, PA, serves as a second messenger. PA
interacts with various target proteins, including Raf-1 kinase [63], PKC-ζ [64],
phosphatidylinositol-4-phosphate-5-kinase [65, 66] and protein tyrosine-phosphatase
[67, 68]. Because of their importance, it is crucial that the intracellular levels of DAG
and PA are tightly regulated for maintenance of normal physiological conditions, which
is accomplished by the diacylglycerol kinases. Multiple forms of DGK are found in most
eukaryotic organisms [69, 70]. In mammalian species, 10 different water soluble
isozymes of DGK have been identified. They are denoted as α, β, γ, δ, ε, z, η, θ, ι as
well as κ, and differ in their biochemical properties, tissue distributions, and lengths
(ranging from 567 aa to >1150 aa) [56, 69]. Based on sequence similarities between
these isozymes and the presence or absence of specific functional domains, they have
been grouped into five different classes [70]. All of these isozymes feature a large
catalytic domain, which is sometimes divided into two parts - catalytic and accessory
domain. Additionally, they have at least two cysteine rich domains (CRDs), which
enable to recruit the protein to the membrane and to bind the lipid substrate, which is
present in the membrane [70]. The simplest and shortest (567 aa) of these isozymes is
DGK-ε, the sole member of class III. It contains only the commonly shared catalytic
domain and two CRDs. In addition, DGK-ε also features a transmembrane helix near
its N-terminal end consisting of 20 - 40 amino acids [71], which most likely plays a role
in membrane interaction [72]. DGKs of class I (α, β and γ isozymes), contain in addition
to the commonly shared domains two EF-hand motifs and a conserved domain of
unknown function near the N-terminal end [70], while DGKs of class II (δ, η and κ
isozymes) feature a plecstrin homology (PH) domain, a sterile α motif (SAM) domain
and a large insert within the catalytic domain separating it into two parts. DGKs of class
IV (z and ι isozymes) are distinguished from the others by a sequence homologous to
the MARCKS phosphorylation site domain and four ankyrin repeats near the C-terminal
end. Lastly, DGK-θ is the sole member of class V. It contains three C1 domains, a Gly-
Pro rich domain and a PH-domain-like region with an overlapping RAS-associating
domain. All these unique domains among the DGK isozymes represent a large
structural diversity, indicating a distinct mechanism of regulation for each isoform.
Recent studies have demonstrated that DGK isozymes play crucial roles in a wide
variety of mammalian signal transduction pathways conducting growth factor/cytokine-
Chapter 1: Introduction
8
dependent cell proliferation and motility, seizure activity, immune responses,
cardiovascular responses and insulin receptor-mediated glucose metabolism [73].
Thus, it is suggested that several DGK isozymes can act as potential drug targets for
cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes
[53, 73, 74].
DGKs are also found in other eukaryotic organisms, such as Drosophila melanogaster
[75], Caenorhabditis elegans [76], Arabidopsis thaliana [77] and yeast [78, 79] as well
as in Gram-positive bacteria encoded by the dgkB gene [80]. While DGKs from all
these different organisms that are typically water soluble do have at least one
conserved catalytic domain, there is a prokaryotic membrane integral DGK encoded by
the dgkA gene, which does not contain any of the canonical sequence features
(catalytic domain and cysteine-rich C1 domains) [56, 81-85]. It does not share
considerable homology with other known kinases and it does not feature any typical
kinase sequence motifs, such as P-loop (Gly-X-X-X-X-Gly-Lys-Thr/Ser) or any other
structural or functional motif. These observations strongly suggest that the prokaryotic
DGK encoded by the dgkA gene is evolutionarily unrelated to the eukaryotic
counterparts, and that the proteins exhibiting DGK activity have evolved independently
in bacteria [56, 70].
The dgkA gene codes for integral membrane DGK present in the Gram-negative
bacterium Escherchia coli (E.coli) and undecaprenol kinase (UDPK) located in Gram-
positive bacteria, such as Staphylococcus aureus, Streptococcus mutants and Bacillus
subtilis [84]. Genes homologous to dgkA are widely spread in the eubacterial domain.
However, they are not omnipresent. Mycobacteria, for example, do not have a dgkA
homolog. In Eukaryota, dgkA-like genes are rarely found. They could only be
determined in the California poplar tree (Populus trichocarpa), the castor oil plant
(Ricinus communis), and the freshwater amoeba (Paulinella chromatophora). There,
the function of the protein, which is encoded by the rare dgkA-like gene, is unknown so
far. Given its shortage in eukaryotes, this gene is likely an evolutionary relict, possibly
emanated from endosymbiosis with cyanobacteria [86-88].
In this study, E.coli diacylglycerol kinase is the object of interest, denoted, hereafter, by
DGK. It is a unique enzyme, which exhibits a high stability [10, 89] as well as a
remarkable complexity in structure and function [5-7]. The high complexity in
combination with a convenient experimental handling has set DGK into focus as a
model system for investigations of membrane protein structure, function and folding
[10, 18, 81, 84, 89-92] as demonstrated below.
Chapter 1: Introduction
9
1.1.1 Escherichia coli diacylglycerol kinase
1.1.1.1 Location and function
DGK is a homotrimeric enzyme located within the inner membrane of E.coli, where it
catalyzes the ATP-dependent phosphorylation of DAG to PA at the
membrane/cytoplasm interface. In 1978, Raetz and Newman mapped the location of
the dgkA gene and used genetic and biochemical experiments demonstrating that
DGK’s major function is the participation in the membrane-derived oligosaccharide
(MDO) cycle [2, 54] as shown in Figure 1. Large quantities of MDOs are produced in
the periplasm of E. coli under conditions of environmental stress such as low
osmolarity [15, 16]. During the MDO biosynthetic pathway, phosphoglycerol is
transferred from phosphatidylglycerol (PG) in the outer leaflet of the plasma membrane
(PM) to the nascent MDOs, generating DAG as a byproduct. DAG is known to be
potentially membrane-disruptive because of its preference for non-bilayer lipid phases.
DGK recycles DAG, after it transverses the PM to reach the inner leaflet, into non-toxic
PA, which is a central intermediate of the glycerophospholipid biosynthesis in bacteria.
DGK thereby provides the basis for restoring PG that is consumed during the MDO
biosynthesis.
Figure 1. Physiological role of E.coli diacylglycerol kinase (DGK) in recycling during the
biosynthesis of membrane-derived oligosaccharides (MDOs) [1, 2] that are largely generated in
response to environmental stress, such as low osmolarity [15, 16]. DGK is located within the
Chapter 1: Introduction
10
inner membrane, where it catalyzes the ATP-dependent phosphorylation of potentially
membrane-disruptive diacylglycerol (DAG) to non-toxic phosphatic acid (PA), providing the
basis for restoring phosphatidylglycerol (PG), which is consumed in the MDO cycle. The cartoon
is based on the X-ray structure using the PDB ID 4UXX [6]. The figure is adapted from Van
Horn et al. [84].
The second major function of DGK is the participation in the lipopolysaccharide (LPS)
biosynthesis, which is the main constituent of the bacterial outer membrane [17]. One
step of this pathway is the transfer of phosphoethanolamine from the outer leaflet of the
PM to lipid A, an LPS biosynthetic intermediate. This process generates DAG as a
byproduct, which is then recycled by DGK [84].
1.1.1.2 Enzymology
DGK belongs to the first integral membrane enzymes, which were subject of detailed
enzyme kinetic studies. First, it was solubilized in Cutscum [93], known to be a harsh
detergent. Over the past 40 years, most kinetic studies on DGK have been performed
in mixed detergent-lipid micelles, e.g. in Triton X-100/ lipid mixed micelles [94]. At that
time, DGK was purified into organic solvents, which is now known to cause protein
unfolding leading to low specific activities (<1 U/mg) [95]. Bell and co-workers
overcame this problem by solubilizing DGK in octylglucoside (OG), using
OG/phospholipid mixed micelles for kinetic experiments, which offered a clearly
increased activity of ~28 U/mg [96, 97]. With these early studies, Bell and co-workers
could demonstrate that DGK requires next to Mg2+ complexed to ATP (MgATP) a free
second Mg2+ ion for activation with the preference for Mg2+ as divalent ATP counterion
[98]. Additionally, it could be shown that the enzyme features an absolute requirement
for a lipid co-factor in micelles [97]. Cardiolipin turned out to be a particularly effective
activator in OG-micelles [97]. Furthermore, Bell and co-workers found out that DGK
does not feature lipid substrate specificity regarding its lipid substrate especially
concerning the fatty acyl chains [97]. This promiscuity for the lipid substrate is in
contrast to the detected specificity with respect to the nucleotide substrate. DGK
strongly favours ATP as the phosphate donor over other nucleotides [18, 99]. It could
be shown that ADP is a very weak phosphoryl donor, whereas adenosine
tetraphosphate features a remarkably reduced affinity relative to either ADP or ATP.
The ribose and adenine moiety specificities of DGK were tested. Measured kcat values
of DGK for guanosine triphosphate (GTP) and inosine triphosphate (ITP) were
decreased significantly compared to ATP, suggesting a purine selectivity.
Chapter 1: Introduction
11
Steady state kinetic studies were carried out by Badola and Sanders [18]. They were
consistent with a random equilibrium mechanism, in which ATP and DAG binding to the
enzyme can occur in both the presence and absence of the other substrate.
Additionally, these data suggest a direct, in-line phosphoryl transfer from ATP to DAG.
This is supported by the fact that no enzyme-phosphate covalent intermediate could be
detected. Furthermore, it could be demonstrated that the bisubstrate analogue
adenosine 5’-tetraphosphoryl-3-O-(1,2-dihexanoyl)-sn-glycerol inhibits the enzyme. It is
a better inhibitor than the bisubstrate itself. In addition, it was shown that DGK features
a higher affinity for ATP than for DAG: The KM was determined to be 1.2 ± 0.5 mM and
5.0 ± 2.2 mol% for ATP and DAG, respectively [18]. The substrate cooperativity factor,
α, was calculated to be 0.48 ± 0.17, implying a modest degree of positive
heteroallostery between the two substrates, which means that the binding of one
substrate enhances the affinity for the other substrate [18].
While DGK’s kcat/KM,ATP is with 104 M-1s-1 modest in comparison to the efficiency of
various water-soluble enzymes, DGK nevertheless seems to be an evolutionarily
optimized biocatalyst in a way that its reaction rate approaches the substrate diffusion-
controlled limit. Under physiological conditions, binding of ATP to DGK is unlikely the
rate-limiting step. Rather, the transbilayer diffusion of DAG appears to limit the rate of
DGK’s reaction in vivo. DAG has to perform a flip-flop through the inner membrane
from its site of production on the outer leaflet to the cytoplasmic site on the inner leaflet,
where DGK’s putative active site is located. However, it cannot be excluded that a
transporter or permease elevates the rate of DAG transbilayer diffusion. Evidence for
this assumption is given with the flip-flop rate of ~50 s-1 measured in lipid vesicles,
which is for a hydrophobic molecule as DAG rather rapid [100, 101]. The flip-flop rate is
remarkably similar to DGK’s kcat of ~26 s-1. Furthermore, DGK’s KM for DAG is with
~5.0 mol% on the same order as the concentration of DAG in the E. coli inner
membrane under conditions of an active MDO cycle [18]. Consequently, DGK seems to
be able to catalyze phosphorylation of DAG under physiological conditions at a rate
that is on the same order as the rate by which DAG can diffuse to its active site. Thus,
DGK was argued to satisfy Knowles’ classic definition of a “perfect enzyme” [102],
since its chemical step seems to be able to keep up with the rate of substrate diffusion
to the active site.
The overexpression of DGK with an N-terminal His6-tag by Bowie and co-workers [9]
provided the facile purification of DGK into either detergent micelles or into several
model membrane systems, such as bicelles or amphipols. DGK featured a maximal
activity of ~110 U/mg in mixed n-decyl-β-maltopyranoside (DM)/phospholipid micelles
at 30°C, which is in the same range as the activity in lipid vesicles of optimal
Chapter 1: Introduction
12
composition. Similar activity was determined for DGK in 3-([3-
cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO)-1,2-
ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) or CHAPSO-1,2-
dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) bicelles [103]. High activity was
also observed for DGK in lyso-myristoyl-phosphatidylcholine (LMPC) detergent
micelles in the absence of lipids [8], in amphipols [104], and in the lipidic cubic
mesophase formed by monoacylglycerols [105]. Kinetic studies demonstrated that
DGK is more active, when it is reconstituted into lipid vesicles composed exclusively of
phosphatidylcholine (PC) than in vesicles that also contain 20 mol% of anionic
phospholipids, or in lipid vesicles emulating the plasma membrane of E. coli [106].
Previous work in this lab applied time-resolved 31P MAS NMR for monitoring
simultaneously ATP hydrolysis taking place in the aqueous phase and DAG
phosphorylation proceeding in the membrane phase [107]. The enzymatic activity of
DGK was investigated with different lipid substrates, 1,2-dibutyrylglycerol (DBG, chain
length n=4, water soluble) and 1,2-dioctanoyl-sn-glycerol (DOG, chain length n=8,
amphiphilic), as well as ATP analogues. It was found out that DGK has a high basal
ATPase activity using DBG as substrate. Only ~70% of all hydrolyzed ATP molecules
were used for DBG phosphorylation. The ATPase activity could be demonstrated to
increase by a rising concentration of DBG, showing a strong, positive cooperativity.
DOG, a lipid substrate with longer acyl chains, was, in contrast, phosphorylated more
efficiently. Furthermore, this study showed that the transition state analogue ADP*VO4,
a complex of ADP with orthovanadate (VO4), decreased the ATP hydrolysis rate to
35% and uncoupled the ATP hydrolysis reaction from the DAG phosphorylation [107].
Using the ATP analogue ATPγS, it was found that DGK is able to transfer the
thiophosphoryl group of ATPγS to DBG, implying a certain plasticity of the active site
[107]. The rate of ATPγS hydrolysis was observed to be faster than of ATP, but no
further stimulation by the addition of lipid substrate could be determined. DGK was
observed to follow a random-equilibrium mechanism, which is in good agreement with
studies in detergent micelles [18].
1.1.1.3 Stability/ folding and misfolding
DGK has been demonstrated to be highly stable in native membranes, being resistant
to irreversible activation upon incubation for a few minutes at 100°C [93, 108]. Even in
detergent micelles, it was observed to be quite stable: In decylmaltoside (DM) micelles
at 70°C and pH 6.5, the t1/2 for irreversible inactivation was determined to be on the
Chapter 1: Introduction
13
order of several hours [8]. This high stability is reflected by a general tolerance of wild-
type DGK to mutations [109]. Lau and Bowie developed a method for quantitating the
thermodynamic stability of DGK under micellar conditions [9]. Folded DGK in DM
micelles was treated with sodium dodecylsulfate (SDS), which is known to be a harsh
denaturing agent. The obtained data offered for the transmembrane domain an
impressive unfolding free energy of 16 kcal/mol and for the cytoplasmic domain
6 kcal/mol, indicating that even the cytosolic domain exhibits a respectable stability [9].
However, it is reported that DGK is much less stable in detergent micelles than in
native membranes [99, 108]. Misfolding of DGK can normally be avoided or corrected,
applying reconstitutive refolding. During this procedure, a detergent is utilized to
solubilize the protein and is then subsequently removed after mixing with lipid-
containing micelles [110]. It could be demonstrated that DGK refolds during the final
steps of detergent removal and vesicle formation. This procedure allows to produce
properly folded single-Cys mutants of DGK, which were applied for kinetic studies to
identify active site residues and to perform disulfide mapping of DGK’s oligomeric
interface [7, 91]. Studies with more than 20 mutants exhibited that the overall rate of
folding and insertion of DGK into lipid vesicles relates to folding efficiency: The risk of
becoming trapped in misfolded states is for mutants higher, which insert and/or fold
slowly [90, 111-113]. A strong correlation between protein stability and folding
efficiency could be demonstrated [90, 113]. Mainly mutations, which cause a
significantly reduced folding efficiency, are mutations that destabilize DGK [90, 113].
Next to these mutants, which are characterized by destabilization promoted-misfolding,
a set of DGK mutants has been observed, which do not seem to be highly destabilized,
but are prone to aggregation and tend to express only at low levels in E. coli. Notably,
these mutations are all present in or near the active site [7].
Booth and co-workers demonstrated that misfolding takes place before association with
vesicles and that it is proposed to cause aggregation [114]. Additionally, it was shown
that irreversible misfolding occurs at the level of monomer [90].
Furthermore, it could be demonstrated that the rate and efficiency of DGK’s association
with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers can be modulated by
changing the lipid composition. The folding rate and efficiency was increased in the
presence of anionic 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and decreased
with lyso-OPC [115], most likely induced by lowering membrane curvature stress
and/or the increased lateral pressure in the head group region.
Bowie and co-workers showed that about 10% of the purified mutants possessed a
higher stability in detergent micelles than wild-type DGK [10]. They combined several
mutations into a single DGK mutant, causing partly additivity of the stabilization
Chapter 1: Introduction
14
enhancements detected for the parent single-site mutants [89], leading to a
thermostable quadruple-mutant form of DGK, Δ4-DGK (I53C, I70L, M96L, V107D)
(Figure 2). Δ4-DGK showed a functional half-life at 80°C in OG micelles of 35 min,
which is in contrast to ≪1 minute for the wild type. Δ4-DGK was also used, next to Δ7-
DGK (A41C, C46A, I53C, I70L, M96L, V107D, C113A) (Figure 2), for crystallization
purposes [5, 6]. Both mutants were still fully active after 10 min at 95°C, whereas the
wild type was already inactivated [5].
Figure 2. Sequence alignment of wild-type DGK and the two thermostable mutants, Δ4- and
Δ7-DGK [5]. The mutations in Δ4- and Δ7-DGK are labelled green. The N-terminal tag is
highlighted orange.
1.1.1.4 Structure
With 43 kDa (121 residues per monomer), DGK is the smallest known kinase [84]. In
spite of its small size, it exhibits a notable complexity in structure and function [5-7, 84].
DGK forms a homotrimer [5, 7, 91], in which each monomer contains three
transmembrane helices (H1-3) (Figure 3). H1 is the shortest and preceded by an N-
terminal amphiphilic surface helix (SH) [5, 7]. H2 and H3 extend into the cytoplasm and
are connected by a cytosolic loop (CL), likely to be quite mobile. On the other side of
the membrane, H1 and H2 are linked by a short periplasmic loop (PL).
Chapter 1: Introduction
15
Figure 3. Topology plot of wild-type DGK. The plot was created based on the DGK X-ray
structure [5] and refined by the CSI values obtained from chemical shifts in this study (Table
S5). The membrane is indicated by two solid black lines as calculated in the PPM server [116].
The secondary structure elements of DGK are denoted as: CL, cytoplasmic loop; H1-3, helices
1-3; PL, periplasmic loop and SH, surface helix.
The trimer contains three active sites, which are centred around the
membrane/cytoplasm interface. Each one is a composite of two subunits according to
the shared site model [5, 7, 10].
So far, two 3D structures have been published for DGK shown in Figure 4: one
obtained by solution NMR in dodecylphosphocholine (DPC) micelles [7] and one by 3D
crystallization in lipidic cubic phases (LCP) composed of monoacylglycerols (MAGs),
which also act as lipid substrates [5]. The 3D structures are distinct from other kinases,
including the water soluble DGKs. The solution NMR structure is based on a backbone
assignment of 90% of wild-type DGK and was revealed by a variety of structural
restraints including paramagnetic relaxation enhancement (PRE)-derived long range
distances, residual dipolar coupling (RDC)-based orientational restraints, and distance
restraints from biochemical disulfide mapping [7]. The structure shows a three-fold
symmetry, which is created around a left-handed parallel three-helix bundle built by
helix 2 (H2) from each of the three subunits [7]. This structural core represents the
centre of three overlapping four-helix bundles. Each four-helix bundle involves helices
from all three subunits. The SH, which is N-terminal to H1, is suggested to point away
from the protein core into the bulk solvent. Its first 25 N-terminal residues are observed
to be motional disordered. A key feature of the solution NMR structure is domain
swapping: H3 of one subunit interacts with H1 and H2 from the adjacent subunit in a
domain swapped fashion. The interface between each four-helix bundle is a membrane
immersed cavity that is described by the authors as portico. Each portico is bound by
H1 and H3 (“pillars”) and is covered by the loop linking H2 and H3 (“cornice”). The
Chapter 1: Introduction
16
portico comprises the majority of DGK’s highly conserved residues. Each active site is
formed by H2 of one subunit and H1 and H3 of the adjacent subunit according the
composite shared site model [7]. For the crystal structure, the lipidic cubic phase (LCP,
in meso) method was used [105]. A crystal structure for wild-type DGK and the
thermostable mutants Δ4-DGK (I53C, I70L, M96L, V107D) and Δ7-DGK (A41C, C46A,
I53C, I70L, M96L, V107D, C113A) was obtained with a resolution of 3.70 Å, 3.10 Å and
2.05 Å, respectively [5]. The crystal structure of all three constructs presents a trimer
with layered packing [117]. Δ4-DGK features the most complete model and is used for
the structure description. As the solution NMR structure, the X-ray structure shows an
approximate three-fold symmetry axis that passes through the centre of the trimer
normal to the membrane plane. The core of the trimer is formed by helix 2 (H2) from
each of the three subunits, creating a parallel three-helix bundle. Extending away from
the core, H1 and H3 of each subunit build the sides of an equilateral triangle with H1 at
the apex position. Viewed from the cytosol, the surface helix (SH), angles away from
the trimer core, tangenting H3 from an adjacent subunit. It is suggested to reside on the
cytosolic side of the membrane, anchoring the protein at the membrane interface. Each
active site is formed by the polar/apolar regions of H1–H3 of one subunit and the
surface helix of an adjacent subunit [5], leading to an unusual catalytic site architecture
of the composite shared site model [5]. Accordingly, the crystal structures for DGK
deviate substantially from the solution NMR version. In the solution NMR model, H3 is
domain swapped and contacts H1 of the adjacent subunit. This is based on the cross-
linking data, which indicate that residue 33, located in the cytosolic region of H1, is
linked to the residues 96 and 97 of H3 of another subunit. These two disulfide cross-
linking distance restraints are not compatible with the crystal structure, for which the
pairs of residues are significantly above the cut-off distance of 12 Å. As a result of the
different quaternary structures, the architecture and chemistry of the active sites
deviate notably between these two models. Though they are consistent with the shared
sites model, but for different reasons. The highest discrepancy correlates with domain
swapping, which is present in the solution but not in the crystal structure. This is
accompanied by the fact that the active site is ill-defined for the solution NMR structure,
whereas it is nearly complete for the X-ray structure. This is related to the SH, which is
assumed to be part of the ATP binding site [84]. In the X-ray structure, the SH is nearly
complete and well-defined, at least for two subunits, residing at the cytosolic part of the
membrane. In the solution NMR structure, it is motional disordered over its first 25 N-
terminal residues, pointing into the bulk solvent. If we take both structures as correct,
then the monitored differences occur presumably due to the manner, in which the
proteins are present at the time of structure determination. In one case, the protein is
Chapter 1: Introduction
17
embedded in a DPC micelle at pH 7.8 and 45°C. In the other case, it is incorporated in
the lattice of a LCP crystal composed of MAGs at pH 5.6 and -173°C. Thus, reasons
for these differences could be i.e. the shorter chain length, the very high curvature and
the small hydrophobic thickness of DPC micelles. Additionally, detergents are known to
be rather poor mimics of the lipid bilayer, in which membrane proteins have been
optimized for folding [118]. Thus, the observed motional disorder over the first 25 N-
terminal residues might be a consequence of destabilization of DGK by DPC micelles.
Also the fact that the solution NMR structure is a backbone-only model without side
chain restraints has to be considered. Whereas the structure of DGK in 3D crystals
might be affected by protein-protein crystal contacts and by properties of the lipid cubic
phases.
Figure 4. Comparison of the solution NMR (PDB 2KDC, wild-type DGK) and crystal structure of
DGK (PDB 3ZE5, Δ4-DGK). A view of the crystal (right) and solution NMR (left) structure from
the cytoplasm (a) and the membrane (b) plane.
Chapter 1: Introduction
18
The mentioned discrepancies between the two structures highlight the need for
studying DGK directly in detergent-free lipid bilayers by MAS NMR, which simulate the
physiological environment of the protein to a high extent. So far, a secondary structure
of a thermostable mutant of DGK with multiple mutations embedded into E.coli total
lipids was published by Yang and co-workers using MAS NMR [14]. Figure 5 illustrates
that the overall secondary structure and topology of DGK obtained by ssNMR is in
agreement with the data from solution NMR and X-ray studies. All three structures
show a high α-helical content. However, few notable differences exist, most likely
reflecting the impact of the environment, in which the membrane protein was
embedded (DPC micelles/ LCP consisting of MAGs/ E.coli lipid bilayers). In contrast to
the ssNMR data, the solution NMR structure features two small distortions in Y16
within SH and I70/L70 of H2. In addition, there are small deviations around the
interhelical turn (T) between H1 and SH and around the periplasmic loop (PL) between
H1 and H2 as well as the cytoplasmic loop (CL) between H2 and H3, which are 3, 1
and 6 residues longer in DPC micelles compared to E.coli lipid bilayers. Furthermore,
H/D exchange studies by ssNMR [14] demonstrated that there are residues in the SH
that are not water-accessible, indicating that these residues must be shielded from the
solvent either by close contact with the cytoplasmic surface of the membrane and/or
through close protein–protein contacts. This is contrary to the solution NMR structure,
which suggests that the SH points away from the protein core into the bulk solvent, a
situation in which water accessibility would be expected for all residues of the SH.
Another difference is detected for H2, for which the H/D exchange data suggest that it
is completely membrane embedded, whereas more than 10 residues are solvent
exposed at the cytosolic side in DPC micelles, although this helix has a similar length in
both preparations. One reason could be the shorter chain length and the high curvature
of the DPC micelles (30–40 Å diameters [119], which may interfere with the
approximate diameter of the DGK trimer (~100 Å) [7], which is in contrast to almost
planar lipid bilayers. Additionally, the N-terminal disorder caused by dynamics might be
a consequence of destabilization of DGK by DPC micelles, as mentioned above [118].
Generally, the ssNMR-derived secondary structure agrees better with the crystal
structure (PDB 3ZE5, Δ4-DGK), though the X-ray structure features asymmetries in the
secondary structure between the three subunits concerning the flexible regions, PL and
CL. The length of T, PL and CL is comparable. In the X-ray structure, T is only one
residue shorter. Deviations concerning PL are small as well. It is one residue shorter in
subunit A and C and one residue longer in subunit B. The comparatively highest
difference occurs with respect to the position of CL, which is shifted from residues 81–
85 in the ssNMR structure to residues 87–91 (subunit A), 86–91 (subunit B), and 83–87
Chapter 1: Introduction
19
(subunit C) of the X-ray structure. In addition, fewer residues of H2 and H3 are solvent
exposed in E.coli lipids. The structure and topology of DGK in 3D crystals might be
affected by protein–protein crystal contacts and by the properties of the LCPs. The
influence of the hydrophobic environment on the structure of membrane proteins has
already been extensively investigated by comparing the structures of the single-TM
protein influenza virus AM2 in three different solubilization environments [20, 21, 120-
122]. In terms of DGK, it should be noted that the variations in the secondary structures
in different environments all arise in catalytically critical regions, such as the SH,
cytosolic regions of H2 and H3 and the CL. The conformational plasticity of these
regions in different environments is likely a requirement for the catalytic activity of DGK.
However, further factors, such as mutations, different pH and temperatures may be
additional sources for structure perturbations as well. Summing up, the X-ray structure
of DGK clearly features more similarities to the solid state NMR structure concerning
secondary structure and topology than the solution NMR structure.
Figure 5. Comparison of the DGK secondary structures obtained from solution NMR (PDB
2KDC, wild-type DGK, chain A), solid state NMR [14], and X-ray crystallography (PDB 3ZE5,
Δ4-DGK, chain A): Rectangles symbolize α-helical regions, whereas solid lines reflect
deviations from helicity. Residues that were not resolved are illustrated by dashed lines. The
differences between the secondary structures are highlighted in green. Both the ssNMR and the
X-ray studies used a thermostabilized mutant, whereas the wild type was used for solution NMR
structure determination.
1.1.1.5 Mapping of the active site
So far, several studies were carried out using different techniques to map the active
site. They could demonstrate that the DGK trimer contains three active sites, which are
centred around the membrane/cytoplasm interface. Each one is a composite of two
subunits according the shared site model [5, 7, 10].
Bowie and co-workers identified five likely active-site mutants, such as A14Q, N72S,
E76L, K94L, and D95N (Table 1, Row 1) [10]. Mixtures of either A14Q or E76L with
N72S or K94L were observed to exhibit much greater activity than the mutant proteins
by themselves, indicating that Ala14 and Glu76 may be located on one half of the
active site, while Asn72 and Lys94 are on another half-site.
Chapter 1: Introduction
20
Sanders and co-workers characterized the active site by extensive cysteine scanning
mutational studies (Table 1, Row 2) [7]. Next to these mutational studies, they carried
out solution NMR based titration experiments. Here, wild-type DGK was titrated with
Mg*AMP-PCP or dibutyrylglycerol (DBG) at concentrations of 0 mM, 2 mM, 4 mM, 8
mM and 16 mM. Additionally, DGK was titrated by both substrates (16 mM and 40 mM,
respectively). Backbone chemical shift perturbations (CSPs) were determined as a
function of increased substrate concentration (Table 1, Row 8 and 9). All indications
were found to point to key residues on the cytosolic side of H2 and H3 and to a lesser
extent on H1. The structure and functional studies also illuminate the conclusion of
previous mutagenesis that DGK’s active site must lie at the interface between subunits
[10], involving H1 and H3 from one subunit and H2 from an adjacent subunit [7, 10].
Caffrey and co-workers, mapped the active site by solving the X-ray structure of Δ4-
DGK co-crystallized with the non-hydrolysable ATP analogue, adenylylmethylenedi-
phosphonate (AMP-PCP) (PDB 4UXX) [6]. Here, Δ4-DGK was crystallized in the LCP
composed of MAGs, which double as lipid substrates. Additionally, it was co-
crystallized with AMP-PCP, using a concentration of 10 mM. As divalent counterion,
zinc was used. In the complex, one active site contains two Zn atoms, one AMP-PCP
molecule and two lipid substrates, whereas the other two active sites are nucleotide-
free. They demonstrated that the active site is formed by the polar/apolar regions of
H1–H3 of one subunit and the surface helix of an adjacent subunit [5], leading to a
catalytic site architecture of the composite shared site model [5]. Next to crystallization
studies, Caffrey and co-workers investigated the impact of several residues on the
kinase activity by site-specific mutations monitored both by a coupled enzyme assay
and molecular dynamics simulations (MDS) [6] (Table 1, Row 3). Several residues,
which were identified by mutational studies [6, 7, 10] to have an impact on catalysis,
were found to be proximal (≤ 5 Å) to the nucleotide and lipid substrates in the X-ray
structure (Table 1, Row 4 and 5) [6]. In the following, the observations by Li et al. [6]
concerning possible directly interacting residues are described in detail (Figure 6):
Zn*AMP-PCP was found to be nearly completely stretched and localized by
electrostatic, hydrogen bonding and hydrophobic interactions along its length on the
comparatively flat, cytosol-exposed surface of H2 and H3 of one subunit. The two zinc
atoms bound to AMP-PCP were observed to be coordinated by the phosphates of
AMP-PCP on one side and Glu28 and Glu76 on the other side. The β- and γ-
phosphate groups, in turn, are assumed to be additionally coordinated by Arg9 and
Asn72, respectively. The two hydroxyls of the ribose in AMP-PCP were determined to
be tightly hydrogen bonded to the side-chain carboxyl group of Asp95, which was
found to interact with Gly91 via an H-bond. Additionally, the ribose of AMP-PCP was
Chapter 1: Introduction
21
observed to be hold in place by hydrophobic interactions with the methylenes of Lys94.
Furthermore, the ε-amino group of Lys94 was determined to interact with the N7 of the
purine and with the α-phosphate of AMP-PCP. Lys94 itself was found to be kept in
place by a salt bridge with Asp80. The purine ring of AMP-PCP was observed to be
oriented by hydrogen bonds between N1 and N6 of adenine with His87 and Glu85,
respectively, both located in the CL. The tyrosyl ring of Tyr86, which is present in the
CL between Glu85 and His87, is assumed to cover the adenyl of AMP-PCP. It is
suggested to lock adenine firmly against DGK by π-π stacking. The active site includes
additionally two lipid substrates (MAGs). MAG1 has been modelled into the electron
density map with its headgroup deep in the protein, located at the membrane/cytosol
interface with its reactive 1-OH next to the γ-phosphate of the nucleotide. The two
entities were found to be ~4 Å apart, which is consistent with a direct, in-line
phosphoryl transfer mechanism, excluding the formation of an enzyme phosphate
intermediate with subsequent phosphotransfer to the lipid substrate. This is in
agreement with kinetic and biochemical data mentioned above (1.1.1.2 Enzymology)
[18, 107]. MAG2 was observed to appear in the putative lipid substrate-binding pocket,
with its headgroup ~4 Å from MAG1. Their acyl chains were determined to extend into
the membrane along the hydrophobic surface of the protein. They were monitored to
reside within or close to a three-walled hydrophobic pocket created by the
transmembrane regions of H1-3. The electron density for MAGs in the complex was
observed to be variable and partly discontinuous, reflecting acyl chain flexibility. One of
the two other active sites in the complex, which lack nucleotide, was found to contain
two MAGs orientated similar to those in the active site with Zn*AMP-PCP. The other
active site without nucleotide was observed to contain just one, distant MAG, since its
SH is not visible in density until Ser17, leading to an active site wide open. The 1-OH of
MAG1 was monitored to be close to the side chain carboxyl group of Glu69, whereas
the 2-OH of MAG1 was found to be proximal to Ser98. The carbonyl oxygen of the
ester linkage in MAG1 was detected to be close to Ser17, allowing hydrogen-bonding.
However, it should be noted that the glycerol headgroup of MAG1 could not be oriented
unambiguously in the active site, based on the available electron density maps, making
the contribution of Glu69, Ser98 and Ser17 concerning lipid substrate interaction
assailable.
Chapter 1: Introduction
22
Figure 6. Substrate-binding sites determined in the X-ray structure (Δ4-DGK, PDB 4UXX) [6].
(a) Structure-based and possible interactions with the non-hydrolysable ATP analogue
adenylylmethylenediphosphonate (AMP-PCP, blue) and its two counterions (Zn, orange). The
figure is adapted and modified from Li et al. [6]. (b) Possible interactions of Ser17, Glu69 and
Ser98 with the lipid substrate monoacylglycerol (MAG, yellow).
Data from molecular dynamics (MD) simulations by Jia and co-workers are in good
agreement with the X-ray structure concerning the impact of Arg9, Glu28, Asn72,
Glu76, Lys94 and Asp95 during nucleotide binding and Arg9, Ser17, Glu69 and Ser98
according lipid substrate binding (Table 1, Row 6 and 7) [11]. Additionally, Jia and co-
workers found evidence for Lys12 to form hydrogen bonds with β- and γ-phosphate of
ATP. It could been shown that Lys12 leads to subsequent protonation of the β-
phosphate oxygen. Together with Arg9, it is reported to support the stabilization of the
transition state by dispersion of negative charges on γ-phosphate [11]. Furthermore,
Jia and co-workers could demonstrate that the phosphoryl transfer reaction applies a
dissociative mechanism [11].
Some of the mentioned identified active site residues were found to closely correlate
with the degree of residue conservation observed among homologs [5, 7, 10] (Table 1,
bold residues).
Chapter 1: Introduction
23
Table 1. Mapping of the active site through the identification of functionally relevant residues by
mutational studies, X-ray crystallization, MD simulations and solution NMR
Mutational studies1
X-ray structure (LCPs) [6]
MD simulation (POPE/POPG bilayer) [11]
solution NMR (DPC micelles) [7]
reduced activity when mutated
close proximity to the respective substrate
directly interacting with the respective
substrate
significant backbone CSPs
2
DM micelles
[10]
DPC micelles
[7]
LCPs [6]
AMP-PCP binding
lipid binding (MAG)
ATP binding
lipid binding (DOG)
AMP-PCP binding
lipid binding (DBG)
T8 T8
R9 R9 R9 R9 R9 R9
K12
A13 A13
A14
S17 S17
S17
S17
G20 G20
N27
E28 E28 E28
E28
A30 A30
F31
R32
Q33
Q33
E34 E34
E34
(indirect) E34 E34
G35
D51
I59
S60
V62
M63
V65
M66
E69 E69 E69
E69
E69 E69 E69
I70
N72 N72 N72 N72
N72
N72 N72
S73 S73
I75
E76 E76 E76 E76
E76
D80 D80
D80 (indirect)
D80 (indirect)
R81
Chapter 1: Introduction
24
G83 G83
G83
E85
Y86
H87
L89
S90 S90
G91
(indirect) G91
A93
A93
K94 K94 K94 K94
K94
K94
D95 D95 D95 D95
D95
D95
G97 G97
S98
S98
S98
S98
A99
A99
A100 A100
T112
C113
1 DGK mutants that exhibit ≤25% of wild type activity in the respective environment.
2 CSPs represent only the backbone. They are not determined for the side chains.
bold residues: >99% sequence identity across homologues [6]
italic residues: A direct interaction with the respective substrate is possible due to the X-ray
structure, but is not mentioned anywhere.
indirect: structure-based interaction with a catalytically important residues (D80->K94, G91-
>D95)
yellow labelling: All (dark yellow) or most (light yellow) of the mentioned studies agree
concerning the catalytic impact of this residue
1.1.1.6 Proposed catalytic mechanism
Based on the X-ray complex (PDB 4UXX) mentioned above, Caffrey and co-workers
suggested that Glu69 initiates the reaction by abstracting a proton from the primary
hydroxyl, since its side chain carboxyl is in hydrogen-bonding distance to the 1-OH of
MAG1, [6]. This is consistent with MD simulation (MDS) studies by Jia and co-workers
[11]. Additionally, a structure of nucleotide-free Δ7-DGK was obtained by serial
femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) at room
temperature (RT), monitoring two conformations for the following three critical residues:
Glu34, Glu69 and Glu76. In MD simulations, the two alternative conformers of the side
chains were observed to convert into one another by protonation or deprotonation of
the side chain carboxyl group [6]. Based on these observations from the X-ray
structures in combination with mutational studies [6, 7, 10] and MD simulations [6],
Caffrey and co-workers proposed following catalytic mechanism for DGK [6]:
Chapter 1: Introduction
25
In the nucleotide-free state, the catalytic site is hydrated. Glu34 is predicted to have a
pKa of 7.53 and is, thus, present in its protonated state at a neutral pH, forming a
hydrogen bond with the deprotonated Glu69. This was found to be a stable
configuration in the MD simulations. The coordination of the substrates to the active
site causes then a conformational change of Glu34, which allows its deprotonation by
water. This induces destabilizing electrostatic interactions between deprotonated Glu34
and Glu69. Due to the hydrophobic nature of the catalytic site, deprotonation of Glu34
will increase the pKa of Glu69, making it a stronger base for proton abstraction of the
lipid substrate. Thus, Caffrey and co-workers hypothesised that deprotonation of Glu34
promotes the deprotonation of the lipid substrate by Glu69, leading to a formation of a
nucleophilic alkoxide. The alkoxide, in turn, induces a nucleophilic attack on the γ-
phosphate of the nucleotide, which causes the formation of a pentahedral intermediate,
stabilized by Asn72 and/or Arg9. MD simulations could show that protonation of Glu69
induces a side-chain switch. This alternate conformation was found to extend deeper
into the membrane and to have a predicted pKa value of 8.82. Additionally, protonated
Glu69 presumably interacts with deprotonated Glu34 via a stabilizing hydrogen-bond.
Thus, the hydrogen-bonding is now inversed compared to the nucleotide-free state.
After the pentahedral intermediate is collapsed, which causes a breaking of the β-γ
linkage, the ADP and phosphorylated lipid substrate are formed.
This is followed by the diffusion of the products from the active site. The release of the
phosphorylated lipid, which is relatively bulky and negatively charged, from the active
site is expected to occur via the opening between SH and H1. This may be facilitated
electrostatically by Glu69 and Glu76 in H2, creating a push, and by Arg9 and Lys12 in
the SH, inducing a pull. In addition, Glu69 is deprotonated by Glu34. After product
release, the active site is reset for another round of catalysis: Water molecules diffuse
into the active site, hydrating it and Glu34 returns into its nucleotide-free state
conformation. Density functional theory (DFT) simulations on MDS-sampled
configurations, monitoring close MAG1 contacts, suggest that 1-OH proton abstraction
by Glu69 is kinetically more likely than phosphate cleavage.
26
Chapter 1: Introduction
27
1.2 Aim of thesis
Despite many years of research including solution NMR and X-ray crystallization
studies, important long-standing questions regarding DGK’s catalytic mechanism
remain unsolved. It is not clear yet, if the DGK trimer adopts a symmetric or
asymmetric conformation. Additionally, it is unknown whether the three active sites of
DGK are in same or different states during catalysis and whether DGK undergoes a
substantial conformational change prior to the actual phosphoryl transfer. Taking into
account that the DGK trimer exhibits a remarkable stability and that each active site is
built by components of two protomers based on the composite shared site model, the
question arises whether specific long-range intra- and interprotomer interactions exist.
In this study, these questions will be addressed by multidimensional high field as well
as dynamic nuclear polarization (DNP)-enhanced 13C,15N MAS NMR.
Preparing membrane protein samples for MAS NMR is still a challenge. In order to
answer these questions by MAS NMR defined above, it is required to achieve high
amounts of pure, active, stable and homogeneously reconstituted protein, which
provides well-resolved MAS NMR spectra. This task will be accomplished by a
stepwise optimization of the preparation protocol, defining the DGK construct,
detergent, lipid composition, reconstitution method and protein-to-lipid ratio (Chapter
3).
For the investigation of structural and dynamical changes defining the catalytic
mechanism of a protein, the assignment of its backbone and side chains is mandatory.
However, the assignment of membrane proteins by MAS NMR is still a highly
demanding task, challenging from sample preparation over experiment planning and
performance until data analysis. Thus, a NMR strategy has to be established, which
helps to diminish obstacles and enables a nearly complete assignment of wild-type
DGK in lipid bilayers (chapter 4). In detail, an appropriate isotope labelling strategy and
assignment procedure have to be defined. A strategy for improvements of the
sensitivity and resolution has to be found and an automatic assignment algorithm will
be tested to make the assignment faster and more reliable.
In order to highlight changes in structure and dynamics within the catalytic hotspots of
DGK, the apo state of DGK will be compared with the substrate bound states. On the
basis of the assignment, perturbations in peak position and intensity of the substrate
bound states have to be analysed in 3D and 2D heteronuclear correlation spectra.
Chapter 1: Introduction
28
Therefore, nucleotide and DAG-bound states of DGK have to be established for NMR
analysis (chapter 5).
In order to understand DGK’s remarkable stability and the cross-talk between its
subunits, key intra- and interprotomer contacts have to be identified. For the detection
of intraprotomer contacts, 13C-13C DARR and 2D NCOCX spectra with long mixing
times will be recorded using high field MAS NMR. For the detection of interprotomer
contacts, mixed labelled trimers have to be created, which contain 13C and 15N residue
contacts between adjacent protomers. These could then be visualized by DNP-
enhanced TEDOR experiments. For this purpose, a procedure has to be established
for the production of mixed labelled DGK trimers. Firstly, conditions have to be tested,
in which DGK trimers can be disassembled into monomers or dimers. If this is
accomplished, the next step will be to initiate a reassembling of the monomers back to
active trimers. The third step will be to find conditions for a sufficient signal
enhancement for DNP-enhanced TEDOR experiments, allowing the identification of
interprotomer contacts. Finally, the detected contacts have to be assigned. In addition,
it has to be found out, if these contacts are involved in nucleotide binding (chapter 6).
If this schedule is successful, not only a deeper understanding of DGK’s mechanism
can be obtained, it would also lead to an optimized and reproducible protocol for the
sample preparation for high field and DNP-based NMR experiments. Additionally, the
assignment of DGK would provide a valuable basis for all kinds of experiments in
future. All in all, this would be highly advantageous for subsequent research on DGK.
Chapter 1: Introduction
29
1.3 Solid state nuclear magnetic resonance (ssNMR) spectroscopy
1.3.1 Theoretical background
1.3.1.1 Interactions in ssNMR
Each nuclear spin interacts with its environment. These interactions are specific and
provide information about the local structure and dynamic of a membrane protein. They
can be provoked by external and internal fields. Internal interactions are shielding
interactions (chemical shift, CS), dipolar couplings (D), J-couplings (J) that are also
referred to as scalar couplings, and quadrupolar interactions (Q). The intrinsic
Hamiltonian (Hint) is defined as a sum of these interactions:
�̂�𝑖𝑛𝑡 = �̂�𝐶𝑆 + �̂�𝐷 + �̂�𝐽 + �̂�𝑄 (1)
While J-couplings are isotropic, quadrupolar interactions, chemical shielding and
dipolar couplings are anisotropic. Quadrupolar interactions occur only in nuclei with
spin quantum numbers >½. Since only nuclei featuring a spin quantum number of ½
are of interest in this study, quadrupolar interactions are not further discussed.
Chemical shielding and dipolar couplings are described in more detail in the following.
1.3.1.1.1 Chemical shift and dipolar coupling
The chemical shift characterizes the local, magnetic micro environment of a nuclear
spin. It is affected by diamagnetic and paramagnetic influences of the electron shell,
ring currents in aromatic residues, the solvent effect e.g. from surrounding buffers or
lipid bilayers, and distinct couplings between nucleus and electron within the molecule.
Soluble molecules tumble isotropically, leading to an isotropic chemical shift (δiso),
whereas membrane proteins in lipid bilayers are constrained in their mobility. Thus,
they usually adopt different orientations with respect to the magnetic field B0. This
results in a so-called powder spectrum. The dependence of the chemical shift on the
orientation of the molecule is defined as chemical shift anisotropy (CSA), which is
determined by a second rank tensor with three parameters: δxx, δyy, δzz.
The isotropic chemical shift (δiso) can be obtained as the mean value of these three
parameters:
𝛿𝑖𝑠𝑜 =
1
3 (𝛿𝑥𝑥 + 𝛿𝑦𝑦 + 𝛿𝑧𝑧) (2)
Chapter 1: Introduction
30
|𝛿𝑍𝑍 − 𝛿𝑖𝑠𝑜| ≥ |𝛿𝑋𝑋 − 𝛿𝑖𝑠𝑜| ≥ |𝛿𝑌𝑌 − 𝛿𝑖𝑠𝑜| (3)
Whereas, the anisotropic chemical shift (δaniso) characterizes the width of the
anisotropic peak:
𝛿𝑎𝑛𝑖𝑠𝑜 = 𝛿𝑍𝑍 − 𝛿𝑖𝑠𝑜 (4)
Additionally, the asymmetry of the chemical shift (η) describes the shape of the peak or
the symmetry of a spectrum:
𝜂 = 𝛿𝑌𝑌 − 𝛿𝑋𝑋
𝛿𝑎𝑛𝑖𝑠𝑜 (5)
The asymmetry factor (η) leads to a value between 0 and 1. In the case of an axial
symmetry, the value is zero. In the case of asymmetry, the value is one. In a static
sample, η ≠ 0 and anisotropic interactions are prevalent. By rotating the sample around
an axis with a rate larger that δaniso, the anisotropic interactions are cancelled and the
asymmetry parameter η = 0. The chemical shift δ is characterized by isotropic and
anisotropic contributions:
𝛿(𝛼, 𝛽) = 𝛿𝑖𝑠𝑜 +1
2 𝛿𝑎𝑛𝑖𝑠𝑜(3𝑐𝑜𝑠2𝛽 − 1 − 𝜂𝑠𝑖𝑛2𝛽𝑐𝑜𝑠2𝛼) (6)
It is dependent from the Euler angles α and β, describing the orientation towards B0.
The Hamiltonian for the chemical shift can be defined as:
�̂�𝐶𝑆 = �̂�𝛿𝑖𝑠𝑜+ �̂�𝛿𝑎𝑛𝑖𝑠𝑜
(7)
Homo- and heteronuclear dipolar coupling also add to peak broadening. The
Hamiltonians for the homo (equation 8, 10)- and heteronuclear (equation 9, 10) dipolar
couplings can be written as:
�̂�𝐼𝑆 = 𝑑𝐼𝑆 ∙ (3 ∙ 𝐼𝑍𝑆𝑍 − 𝐼 ∙ 𝑆) (8)
�̂�𝐼𝑆 = 𝑑𝐼𝑆 ∙ 2 ∙ 𝐼𝑍 ∙ 𝑆𝑍 (9)
with the dipolar coupling constant (dIS) defined as:
𝑑𝐼𝑆(𝑟𝐼𝑆, 𝛽𝐼𝑆) = −𝛾𝐼𝛾𝑆𝜇0ħ
8𝜋2𝑟𝐼𝑆3 ∙ (3𝑐𝑜𝑠2𝛽𝐼𝑆 − 1) (10)
Chapter 1: Introduction
31
The dipolar coupling constant (dIS) characterizes the direct interaction through space
between two spins. It is determined by the distance between those spins (r-3) as well as
the angle of their internuclear vector and the magnetic field (βIS). γI and γS are the
gyromagnetic ratios of the coupling spins. Dipolar couplings occur in ssNMR powder
spectra with the so-called PAKE pattern. The two signals correspond to energy
differences, depending on the parallel or antiparallel alignment of the I spin with respect
to the S spin. The IzSz term in equation 9 gives positive (parallel spins) and negative
(antiparallel spins) energies. The two maxima of the PAKE doublet relates to the
situation when the internuclear vector is perpendicular to the magnetic field (βIS = 90°).
This situation is the most frequent one, which explains the higher intensity. The
distance between the two maxima of the PAKE doublet correlates with the dipolar
coupling constant (dIS). The "feet" of the PAKE doublet correspond to the situation
when the internuclear vector is parallel to the magnetic field. There is only one possible
orientation of the dipolar vector, explaining the lower intensity (βIS = 0°).
1.3.1.2 Magic angle spinning (MAS)
Chemical shift anisotropy (CSA) and dipolar couplings cause severe line broadening,
leading to substantial signal overlap and thus to ambiguous data. In order to obtain
well-resolved spectra with a reasonable small line width, one makes use of the term
3cos2β-1, of which both interactions depend on. In detail, the sample is spun around
the angle β = 54,74° with respect to the external magnetic field B0. According equation
6 and 10, respectively, CSA becomes zero and dipolar couplings disappear.
Additionally, the asymmetry parameter η in the term ηsin2βcos2α is cancelled out by
sample rotation. This method is called magic angle spinning (MAS) (Figure 7). In order
to reduce dipolar coupling effectively by MAS, the spinning rate of the sample has to be
faster than the coupling between the spins. Slow rates generate visible spinning
sidebands, which appear in addition to the isotropic signal with the distance of the
spinning frequency.
Chapter 1: Introduction
32
Figure 7. Impact of magic angle spinning (MAS) at 54.74° on solid state NMR spectra. (a)
Depiction of a MAS rotor that is tilted in the magic angle β = 54.74° with respect to the magnetic
field B0. 15
N (b)- and 1H (c)-NMR spectra of the microcrystalline tri-peptide N-formyl-Met-Leu-
Phe-OH under static (blue) and MAS (red) conditions. In the static 15
N-NMR spectrum, the
isotropic (δiso) and the anisotropic (δaniso) chemical shift as well as the CSA parameters δxx, δyy
and δzz are labelled accordingly. Comparing the 15
N- and 1H-NMR spectra under MAS of
25 kHz, it becomes obvious that higher spinning speeds are needed to obtain well-resolved 1H-
NMR spectra (see chapter 4, outlook), whereas the 15
N-NMR spectrum features already a good
resolution at 25 kHz. The figures are adapted from the lecture script “Solid state NMR”,
prepared by Prof. Clemens Glaubitz, Goethe University Frankfurt am Main, summer semester
2015.
In addition to MAS, proton dipolar couplings can be suppressed by applying decoupling
sequences during mixing and acquisition time, which all together cause well-resolved
spectra.
For some studies, information about the distance of the nuclei and/or the bond angle
are relevant, which can be extracted from the dipolar coupling constant. In a rotational
resonance experiment, the spinning frequency relates to a multiple of the isotropic
chemical shift difference between the interacting nuclei. This way, the coupling is
restored for one particular spin interaction.
1.3.1.3 Cross polarization
Solution NMR is typically based on the detection of 1H nuclei. They offer a high
detection sensitivity due to a natural abundance of more than 99.9% and a high
gyromagnetic ratio. In MAS ssNMR, the linewidths of protons remain broad at
moderate MAS frequencies (10–20 kHz), because of the strong inter-proton dipolar
couplings. Hence, 13C and 15N nuclei are usually detected. They have smaller
gyromagnetic ratios than protons, leading to weaker bulk magnetizations and thus to
weaker signal intensities. To elevate the signals from these nuclei, cross polarization
(CP) is applied according to the Hartmann-Hahn condition, in which magnetization is
Chapter 1: Introduction
33
transferred from highly abundant protons (I spin) to dilute 13C or 15N (S spins), when
both spin systems are in contact. The exchange of magnetization is induced by the
simultaneous application of two continuous radiofrequency (RF) fields, B1, on the
respective resonance frequencies of I and S spin. The nutation frequency (ω1) of the
two spin systems (I, S) depends on their gyromagnetic ratio (γ) as well as the strength
of the RF field (B1).
𝜔1𝐼 = 𝛾𝐼𝐵1
𝐼 or 𝜔1𝑆 = 𝛾𝑆𝐵1
𝑆 (11)
If the nutation frequency (ω1) is equal for the I and S spin, a dipolar interaction between
the two spin systems is introduced, allowing the polarization transfer:
Static 𝜔1𝐼 = 𝜔1
𝑆 (12)
𝛾𝐼𝐵1𝐼 = 𝛾𝑆𝐵1
𝑆 (13)
MAS 𝜔1𝐼 = 𝜔1
𝑆 ± 𝜂𝜔𝑟𝑜𝑡 (14)
The CP pulse sequence (Figure 8) starts with a 1H 90°pulse that tilts the proton
magnetization from the z-axis into the xy-plane. Then, a spin lock field on both nuclei
channels is applied with a distinct contact time (tCP). During the CP time, typically
between 100 μs and 10 ms, the magnetization of the X nuclei increases, depending on
the strength of the dipolar coupling between 1H and X. The magnetization builds up
until a steady-state equilibrium is reached. At the same time, the magnetization at 1H
and X decreases due to spin-lattice relaxation in the rotating frame. The signal
enhancement under CP conditions is limited by the quotient of the gyromagnetic ratio
of the two spin systems (e.g. γ1H/γ13C ~ 4). After the CP step, the FID of the X spins is
detected, while proton decoupling is applied. Although CP experiments are performed
under MAS, strong 1H-X dipolar couplings cannot be completely removed. Therefore,
heteronuclear decoupling is used to exclude the couplings during signal acquisition.
This is carried out by either continuous RF irradiation in the proton channel (continuous
wave (CW) decoupling) or by decoupling pulse sequences, such as the two pulse
phase modulation (TPPM).
Chapter 1: Introduction
34
Figure 8. Pulse sequence of a cross polarization (CP) experiment according the Hartmann-
Hahn condition, in which magnetization is transferred from highly abundant I spins to dilute S
spins.
1.3.1.4 Multidimensional NMR experiments
For large and complex systems as membrane proteins, 1D spectra are usually not
sufficient to resolve unique signals due to high spectral crowding. This obstacle can be
resolved by the application of multidimensional spectra. With 2D and 3D NMR
experiments, further information about spin correlations and a better resolution of
signals in the additional spectral dimension(s) can be obtained. The basic 2D
experiment consists of a preparation, evolution (t1), mixing and detection period (t2).
During the preparation period, the spins are tilted into the xy plane by 90° pulses or a
cross polarization step, generating transverse magnetization, which then evolves
during the evolution period (t1). During this time, relaxation takes place under the
influence of various nuclear spin interactions. Thereafter, the mixing period follows,
during which a second pulse flips the y component onto z axis. The idea of the mixing
step is to allow spin communication (e.g. via cross-relaxation, exchange or spin
couplings) for a fixed period, under the application of specific pulse sequences. Then,
the magnetization is prepared for detection. During the detection period, the signal is
recorded as a free induction decay or FID at regular time intervals as a function of t2
(S(t)).
In 2D NMR spectroscopy, the signal is detected as a function of two time increments, t1
and t2. The resulting data are Fourier transformed twice to obtain a spectrum, which is
a function of two frequency variables. In detail, the t1 time is elongated successively by
a distinct increment from zero to an upper limit. The signal amplitude, which is recorded
during t2, depends on the t1 time, oscillating as a cosine function. This oscillation of the
signal amplitude in the indirect dimension provides a second FID. Thus, 2D
experiments are assembled 1D experiments that evolve into the second dimension via
Chapter 1: Introduction
35
incrementing the t1 time. A drawback of multidimensional NMR experiments is the
extended experimental time. Data as a function of t1 is time consuming to record, since
for each t1-increment the whole pulse sequence has to be executed.
1.3.2 High-field MAS NMR
1.3.2.1 Dipolar coupling based experiments for the detection of immobile
residues
Immobile domains, usually transmembrane regions, are molecular segments with
smaller amplitude motions. They are not sufficient to fully average anisotropic
interactions (such as HH, HC, or HN dipole couplings) and can be observed by dipolar
coupling based cross-polarization (CP) as described above.
1.3.2.1.1 Homonuclear correlation experiments based on proton driven spin
diffusion
One conventional 2D experiment in solid state NMR is based on proton driven spin
diffusion (PDSD). It is typically conducted to evaluate structural homogeneity,
resolution and secondary structure of the protein sample (as shown in chapter 3). Spin
diffusion is a homonuclear transfer of magnetization between 13C- or 15N-nuclei through
the spin network of heteronuclear coupled protons. In this study, only a 13C-based
PDSD was carried out. The pulse sequence is shown in Figure 9. The first step, the
preparation period, involves a CP transfer from 1H to 13C. Thus, a magnetization on the
13C nuclei is generated in the x,y-plane. During the evolution period, t1, the 13C spins
evolve under the influence of the Hamilton operator of the chemical shift, while proton
decoupling is applied. During this time, no transfer between spins occurs. The evolution
period is terminated with a 90° pulse on the 13C spins, rotating them at the z-axis. This
is followed by a mixing step, in which the spin diffusion between 13C nuclei takes place
through space by flip-flop interactions. The mixing time typically lasts from 10 ms to 3 s.
During this time, the protons are not decoupled. The exchange through spin diffusion is
principally related to the distance between two spins by a factor of r-6. Thus, the length
of the mixing time defines the distance of the magnetization transfer. With short mixing
times (10 - 20 ms), the magnetization is mostly transferred between neighbouring
atoms, causing peaks within one residue. Longer mixing times (100 ms – 3 s) enable
more distant interactions, leading to inter-residue connectivities. After the mixing, a
second 90° pulse turns the magnetization from the z-axis into the transversal x,y-plane,
Chapter 1: Introduction
36
where it is detected. During acquisition the heteronuclear decoupling is applied. Since
the PDSD experiment is homonuclear, a diagonal crossing the 2D spectrum is
observable. The diagonal accords to the 1D spectrum. All off-diagonal peaks are called
cross peaks and correspond to the chemical shifts of those nuclei that undergo spin
diffusion.
Figure 9. Pulse sequence of a 2D 13
C-13
C PDSD experiment. During the preparation time, the
magnetization is transferred from 1H to
13C via CP step. This is followed by the t1 period, when
the 13
C chemical shift evolves, while 1H nuclei are decoupled. Thereafter, the magnetization is
transferred back on the z-axis through a 90° pulse on the 13
C spins and the mixing step takes
place, in which the proton driven spin diffusion between 13
C nuclei occur through space by flip-
flop interactions. For detection, the 13
C spins are transferred back in the x,y-plane and the FID is
recorded under 1H decoupling.
In a PDSD experiment, the proton decoupling is switched off during the mixing time to
reintroduce the dipolar coupling between 13C and 1H nuclei. Through homogenous line
broadening based on dipolar 1H-13C and 1H-1H coupling, a 13C spin pair can overlap
with different chemical shifts, allowing proton driven spin diffusion between the 13C
nuclei. However, at higher MAS rates, the heteronuclear dipolar 1H-13C coupling is
scaled down. Dipolar assisted rotational resonance (DARR) can be applied, in which
the 1H-13C dipolar coupling is recovered by CW irradiation on 1H, whereupon the 1H RF
field intensity satisfies the condition of rotary-resonance: ω1H = ωMAS. Applying this
experiment with long mixing times, higher magnetic fields and/or higher MAS rates
enables a more efficient magnetization transfer. This in turn leads to stronger cross
peaks as compared to a PDSD spectrum.
Chapter 1: Introduction
37
1.3.2.1.2 Heteronuclear correlation experiments for the sequential assignment
The sequential assignment process includes spin system identification, assignment of
the spin system to the amino acid type, linking of the spin systems, and mapping them
to the protein amino acid sequence. This is achieved by the analysis of three dipolar
coupling based heteronuclear 3D correlation experiments, NCACX, NCOCX, and
CONCA [123, 124]. Here, C-N connections are needed. They are gained by a
heteronuclear band-selective cross-polarization (CP) transfer between the 15N amide
and the 13Cα (NCA transfer) or the 13CO (NCO/CON transfer). The NCA transfer is
intra-residue, since it links 15N[i] with 13C[i], whereas the NCO/CON transfer is inter-
residue, as it connects the 15N[i] with 13CO[i-1] through the peptide bond.
1.3.2.1.2.1 3D NCACX and NCOCX
In the first CP step, the magnetization is transferred from 1H[i] to 15N[i] under the
Hartmann-Hahn matching condition. Then, the evolution of the 15N[i] chemical shift
takes place. Afterwards, the magnetization is selectively transferred to 13Cα[i] or 13CO[i-
1] during a specific second CP step. The 15N/13C CP steps are very sensitive to the
matching conditions for the spin lock fields, reliant on RF field amplitude, chemical shift
and MAS frequency [125]. The matching condition is gained with the MAS rate ωr:
ωI,eff – ωS,eff = n * ωr I = 13C, S = 15N (15)
The 15N frequency is set on resonance to the amide backbone (~ 120 ppm) and the 15N
RF amplitude (~ 2.5 * ωr) is chosen accordingly. Concurrently, the 13C frequency is set
for the favoured transfer to Cα (~ 55 ppm) or CO (~ 170 ppm) and the matching
condition is calculated with respect to the 13C amplitude (~ 1.5 * ωr). For the 15N-13C
transfer, spin lock fields are optimized to improve the efficiency and to compensate RF
inhomogeneity. 13C, 15N amplitudes, frequencies, CP spin lock fields and decoupling
are optimized in incremental steps to obtain the best conditions for each sample. After
the double cross polarization (DCP), evolution on 13Cα/13CO takes place, which is
followed by a non-specific 13C-13C transfer step that transfers the magnetization to any
other proximate 13C nuclei (CX) through a DARR period [126, 127]. Longer DARR
mixing times enable more distant interactions, leading to inter-residue connectivities,
which can be useful in the assignment process. Summing up, the chemical shift is
evolved on 15N and 13Cα/13CO and then detected on 13Cx, leading to a 3D spectrum.
The pulse sequence of the NCACX and NCOCX experiment and the respective
polarization transfer pathways are illustrated in Figure 10.
Chapter 1: Introduction
38
Figure 10. (a) Pulse sequence of the NCACX and NCOCX experiment. Both are 15
N-13
C
correlation transfer experiments with a subsequent 13
C-13
C mixing step. During the preparation
period, a broad-band 1H
15N-CP step is used to generate
15N polarization that evolves during t1
under proton decoupling. For the 15
N-13
C transfer, optimized spin lock fields on the 15
N and 13
C
channel are applied under proton decoupling. The 13
C off-set is centered in the Cα region for
NCA and in the CO region for NCO. After the double cross polarization (DCP), evolution on 13
Cα/13
CO takes place under proton decoupling during t2. Subsequently, a DARR step follows,
which transfers the magnetization to any other proximate 13
C nuclei. Therefore, two 90° pulses
at an off-set of 100 ppm were applied for excitation and reconversion of longitudinal
magnetization. The detection of 13
C magnetization (t3) represents the final step, during which
the protons are decoupled. (b) Polarization transfer pathway for the NCACX (left) and NCOCX
(right) pulse sequence, schematically illustrated for a di-peptide. The selected off-set
frequencies on Cα or CO enable a magnetization transfer within the same amino acid [i] along
the side chain, resulting in cross peaks in the NCACX spectrum, or along the side chain of the
previous amino acid [i-1], leading to cross peaks in the NCOCX spectrum, respectively (black
arrows). Additional through-space dipolar-assisted pathways are possible as well (grey arrows).
1.3.2.1.2.2 3D CONCA
Magnetization is generated on 1H and transferred to 13C via the first CP step. 13C
chemical shift evolves during t1. Band selective 13CO[i-1] to 15N[i] is obtained via spin
lock on 15N and ramping through the specific CP matching on the 13C channel. The
magnetization then evolves on 15N during t2, followed by a transfer to 13Cα[i] and
Chapter 1: Introduction
39
detection. The pulse sequence of the CONCA experiment and the respective
polarization transfer pathway are shown in Figure 11.
Figure 11. (a) Pulse sequence of the CONCA experiment. It is a 15
N-13
C correlation transfer
experiment accomplished by three CP steps. During the preparation period, a selective 1H
13C-
CP step is used to generate 13
C polarization that evolves during t1 under proton decoupling.
This is followed by a second selective CP step, which transfers magnetization from 13
CO[i-1] to 15
N[i]. The 13
C off-set is centered in the CO region for CON. The magnetization then evolves on 15
N[i] during t2 under proton decoupling. Subsequently, the third CP step takes place,
transferring magnetization from 15
N[i] to 13
Cα[i]. The 13
C off-set is centered in the Cα region for
NCA. The detection of 13
C magnetization (t3) represents the final step, during which the protons
are decoupled. (b) Polarization transfer pathway for the CONCA pulse sequence, schematically
illustrated for a di-peptide. The selected off-set frequencies on CO and later Cα enable a
magnetization transfer from CO of the previous amino acid [i-1] via N towards Cα (black arrow)
and sometimes Cβ (grey arrow) of the following amino acid [i], leading to cross peaks in the
CONCA spectrum (black arrows).
1.3.2.2 Scalar coupling based experiments for the detection and tentative
assignment of highly mobile residues
Highly mobile regions, usually termini or loops, feature fast and large amplitude
fluctuations on time scales of <10-5 s. They can be selected by a refocused INEPT
(insensitive nuclei enhanced by polarization transfer) [128] step based on scalar
couplings, leading to solution state-like spectra due to efficient molecular averaging of
anisotropic interactions.
1.3.2.2.1 2D 1H-13C/15N HETCOR
The magnetization is transferred from 1H to attached 13C or 15N nuclei through a
refocused INEPT (insensitive nuclei enhanced by polarization transfer) step [128]
Chapter 1: Introduction
40
based on J-couplings. After the initial 90º 1H pulse, 1H chemical shift evolution during
the variable t1 period takes place. The evolution delay is fixed to achieve antiphase 1H
magnetization with respect to 13C via JHC (JHC ~200 Hz) or 15N via (JHN ~90 Hz). The
magnetization is transferred to 13C/15N by applying simultaneous 90º 1H and 13C/15N
pulses. The 13C/15N chemical shift evolution during the variable t2 period takes place
and is then detected on 13C/15N nuclei.
1.3.2.2.2 2D 13C-13C TOBSY
The magnetization is transferred from 1H to attached 13C nuclei through a refocused
INEPT step based on J-couplings as described above. This is followed by isotropic 13C
mixing using TOBSY (through-bond correlation spectroscopy). The 13C-13C correlation
is established through carbon-carbon J-couplings (JCC ~35-53 Hz). The detection takes
place on 13C. The pulse sequence of the TOBSY experiment is illustrated in Figure 12.
Figure 12. Pulse sequence of the 13
C-13
C TOBSY experiment. The magnetization is transferred
from 1H to attached
13C nuclei through a refocused INEPT step based on J-couplings: After the
initial 90º 1H pulse,
1H chemical shift evolution during the variable t1 period takes place. The
evolution delay is fixed to achieve antiphase 1H magnetization with respect to
13C via JHC (JHC
~200 Hz). The magnetization is transferred to 13
C by applying simultaneous 90º 1H and
13C
pulses. The 13
C chemical shift evolution during the variable t2 period takes place. This is
followed by isotropic 13
C mixing using TOBSY. The 13
C-13
C correlation is established through
carbon-carbon J-couplings (JCC ~35-53 Hz). The detection of 13
C magnetization represents the
final step (t3), during which the protons are decoupled.
1.3.3 Dynamic nuclear polarization (DNP)-enhanced MAS NMR
Though conventional MAS NMR is capable to answer a large number of biological
questions, the inherent low sensitivity can limit its applicability for certain issues due to
Chapter 1: Introduction
41
very long measurement times. Especially the detection of long-range interprotomer
contacts exhibts a challenge for conventional MAS NMR [129].
DNP has turned out to be perfect to overcome the drawback of low sensitivity. It
increases the sensitivity by a microwave-driven magnetization transfer from unpaired
electrons. This process relies on the fact that electrons feature a significantly higher
magnetization compared to nuclei due to a much higher gyromagnetic ratio of the
electron spins. During microwave irradiation, the transitions of the electron spin energy
become saturated, leading to a transfer of the electron polarization to proximate
nuclear spins, which in turn causes an almost homogeneous nuclear hyperpolarization.
It elevates the sensitivity in NMR spectra by up to ∼102 or lowers the acquisition time in
multidimensional experiments by up to ∼104 [130]. The theoretical enhancement (ε) for
protons is defined as the ratio of the gyromagnetic ratio (γ) of the electron (e-) and
proton (1H):
휀𝑚𝑎𝑥 ≈𝛾𝑒−
𝛾1𝐻 ≈ 660 (16)
Nuclear enhancement can be caused by different mechanisms: the Overhauser effect
(OE), the solid effect (SE), the cross effect (CE) and thermal mixing (TM) [131]. The
Overhauser effect (OE) is connected with relaxation-driven processes. It takes place,
when the interaction between electron and nucleus is time/motion-dependent. Thus, it
is primarily important for signal enhancement of solution NMR samples [132]. Thermal
mixing (TM) is closely related to the cross effect, with the difference that more dipolar
coupled electrons are used in the sample. Additionally, it is mediated by interactions at
low temperatures (T<10 K). Therefore, the conditions for both the OE and the TM are
not optimal during MAS-DNP for biological samples. Mainly the cross effect (CE) and to
some extent the solid effect (SE) contribute to signal enhancement. The SE takes
place in electron-nuclear spin interactions, which are motion independent, as in frozen
solutions or solids. It is a two spin process, including one electron and one nucleus. If
the electron spin system is irradiated at:
ω = ωe ± ωn, (17)
with ωe as electron and ωn as nuclear Larmor frequency, a simultaneous flipping of
electron and nuclear spins occurs. This results in reallocation in populations among the
electron-nucleus sublevels, leading to enhancement. The signal enhancement due to
SE is inversely proportional to the magnetic field. The cross effect (CE) is a three-spin
Chapter 1: Introduction
42
process, including an electron-electron dipole and one nucleus. It takes place under
following condition [132]:
ωn = ωe1 – ωe2, (18)
with ωe1 and ωe1 as the frequencies of the dipole and ωn as nuclear Larmor frequency.
In this study, signal enhancement is obtained by the CE using a biradical agent,
serving as a source for unpaired electrons (Figure 13). One such biradical is AMUPol
(Figure 13a) containing two linked nitroxide radicals [133]. It has been widely used for
biological samples and could been shown to be optimal for membrane proteins [134]. It
is used in this study as well. It is water soluble due to its PEG-ylated linker and can be
dissolved in a mixture containing water, glycerol (cryoprotectant) and D2O (slows
proton relaxation). Maximal enhancements can be achieved at low concentrations of
10-20 mM [134].
Figure 13. The expected low number of interacting spin pairs make dynamic nuclear
polarization for signal enhancement essential, which is obtained by the three spin cross effect
using the biradical AMUPol as a source for unpaired electrons. (a) Reconstituted mixed labelled
DGK doped with AMUPol [133] is depicted. (b) It is subjected to continuous wave microwave
irradiation, resulting in polarization transfer from electrons via protons to the sites of interest.
1.3.3.1 13C-15N TEDOR experiments for the dectection of interprotomer contacts
Specific interprotomer contacts can be detected by 15N−13C transferred echo double
resonance (TEDOR) spectroscopy [135, 136]. In the TEDOR experiment [135], the
heteronuclear 13C-15N coupling is re-introduced during MAS. It builds up on the
Chapter 1: Introduction
43
rotational echo double resonance (REDOR) experiment [137] that is used to obtain 13C-
15N distances, depending on their dipolar coupling [138]. The extent of dipolar
interactions alters with the angle of the internuclear vector towards the external
magnetic field, B0. During MAS, a single rotor period features positive and negative
values for heteronuclear coupling, depending on the orientation. Thus, in MAS NMR,
the average value of dipolar coupling for each rotor period is zero. In order to re-
introduce 13C-15N interactions, the REDOR pulse sequence includes a mixing period
with rotor synchronized 180° pulses in the 15N channel, which are applied after the 1H-
13C CP step. Thereby, the 15N magnetization is each flipped by 180°, changing the sign
of the Hamiltonian of the 13C-15N coupling. This way, the heteronuclear interaction is
not averaged to zero during MAS. Two 180° pulses per rotor period effectively rebuild
~70% of the coupling [139]. In the middle of the mixing time (tmix/2), a 180° pulse in the
13C channel is irradiated to refocus the 13C magnetization. In absence of 15N 180°
pulses, a 13C reference signal (S0) is detected, which decreases with elongated mixing
times due to homogeneous factors. In order to monitor inter-nuclear distances with
REDOR, the experiments are performed with increasing numbers of 15N 180° pulses.
These experiments are compared to the respective reference spectra (S/S0). The 13C
signal intensities (S/S0) are charted against the conducted mixing times, resulting in a
curve that can be fitted to gain the 13C-15N dipolar coupling [140]. TEDOR experiments
provide 1D or 2D spectra with all 13C-15N correlations revealed at once. The pulse
sequence (Figure 14) starts with a CP step from 1H to 13C nuclei in the preparation
period, followed by a first mixing time involving the REDOR sequence (tmix/2), which re-
introduces heteronuclear dipolar couplings. Then, one 90° pulse is irradiated each in
the 13C and 15N channel. These two pulses are applied time-delayed, separated by a z-
filter period (Δ). They result in a coherence transfer to 15N spins that evolve during the
evolution period (t1). Then, a pair of 90° pulses is irradiated in the 13C and 15N channel,
resulting in a transfer of the spin coherence back to 13C. Thereafter, a second mixing
step represented by the REDOR sequence takes place, followed by another z-filter
period (Δ) enclosed by two 90° pulses in the 13C channel. Finally, the FID detection
period (t2) occurs on the 13C spins. The z-filter periods are necessitated to compensate
13C-13C J-couplings, which affect the detection of weak 13C-15N dipolar couplings.
These J-couplings can cause wrong cross peaks and phase twisted 2D signals [136].
2D Fourier transformation leads to 2D spectra with cross peaks appearing at 13C and
15N frequencies of the interacting spins. The cross peak intensity depends on the
heteronuclear distance, thus on the degree of dipolar coupling. Additionally, it depends
on the length of the used mixing time (tmix), which relates to the number of 180° pulses
in the 15N channel during tmix/4 (l0). Short mixing times disclose short distance
Chapter 1: Introduction
44
correlations, such as between covalently bound 13C and 15N nuclei e.g. in peptide
bonds of proteins. While longer mixing times lead to long-ranging distances.
Figure 14. TEDOR pulse sequence [136]. The sequence starts with a CP-step, transferring
magnetization from 1H to
13C nuclei. This is followed by two REDOR-steps (tmix/2) to reintroduce
heteronuclear dipolar couplings, which are enclosed by 90° pulses. L0 describes the number of
180° pulses during tmix/4. The evolution time (t1) takes place in-between the two REDOR steps.
The detection of 13
C magnetization (t2) represents the final step, during which the protons are
decoupled.
Chapter 2: Materials and Methods
45
2 Materials and Methods
The chemicals, materials and equipment that were used in this study are listed in Table
S1, Table S2, Table S3.
2.1 Constructs and cells
The synthetic gene coding for wild-type diacylglycerol kinase (UniProtKB accession
code: P0ABN1) was cloned into the plasmid vector pSD005, a derivative of pTrcHisB.
The plasmid vector pSD005 was a generous gift from Prof. Dr. C. R. Sanders
(Vanderbilt University, Nashville, USA) and has been already used before in this lab.
The vector contains an ampicillin resistance sequence. The synthetic DGK gene is
localized between the unique cleavage sites NcoI and HindIII and is expressed from
the strong isopropyl-D-galactosidase (IPTG) inducible promoter Ptrc. The encoded
protein incorporates an N-terminal leader sequence containing a hexahistidine-tag for
purification. The gene product has a size of 14.3 kDa. Next to the DGK wild-type
variant, a quadruple mutant (Δ4-DGK) that has been used in this lab before, was
applied for comparison, including following mutations: I53C, I70L, M96L and V107D
[89]. The sequences for wtDGK and Δ4-DGK are shown in Figure 15.
Figure 15. Sequence alignment of wild-type DGK and the thermostable mutant, Δ4-DGK [89].
The mutations in Δ4-DGK are labelled green. The N-terminal tag is highlighted orange.
The DH5α E.coli strain (New England Biolabs, Frankfurt, Germany) featuring a high
copy number was used for transformation after PCR-based site-directed mutagenesis,
Chapter 2: Materials and Methods
46
whereas the T7 Express E.coli strain (NewEngland Biolabs, Frankfurt am Main,
Germany) was applied for protein expression.
2.2 Molecular Cloning
All single-site mutations were introduced to the wtDGK template vector by polymerase
chain reaction (PCR) amplification with overlapping mutagenic primers. Therefore,
primers were designed, which are complementary to the parental DNA and contain the
desired mutation. In Table 2 all designed primer sequences used in this study are
illustrated:
Table 2. Primer sequences for single-site mutations
mutation primer sequence
R9A 5'- CC GGT TTC ACC GCT ATC ATC AAA G -3'
5'- GC TTT GAT GAT AGC GGT GAA ACC -3'
R22A 5'- GG AAA GGC CTG GCT GCT GCT TGG ATC -3'
5'- CCA AGC AGC AGC CAG GCC TTT C -3'
R32A 5'- GCT GCA TTC GCT CAG GAA GGT GTT GC -3'
5'- C ACC TTC CTG AGC GAA TGC AGC TTC -3'
R55A 5'- GCT ATC ACC GCT GTT CTG CTG ATC -3'
5'- CAG CAG AAC AGC GGT GAT AGC GTC -3'
R81A 5'- C GAA GCT GTT GTT GAC GCT ATC GGA TCC GAA TAC CAC -3'
5' - G GTA TTC GGA TCC GAT AGC GTC AAC AAC AGC TTC GAT A - 3'
R92A 5'- C CAC GAA CTG AGC GGC GCC GCT AAA GAC ATG GG -3'
5'- CC CAT GTC TTT AGC GGC GCC GCT CAG TTC GTG G -3'
The primers bind to the complementary sequence on the template DNA around the
mutation site, from where the DNA polymerase begins to extend the single strand
primer complimentary to the template. The elongation of the primer generates a
plasmid containing the desired mutation. The following reaction mixture (Table 3) and
program (Table 4) was used for the PCR-based site-directed mutagenesis.
Chapter 2: Materials and Methods
47
Table 3. Components of PCR reaction mixture
Components amount
template DNA (50 ng/μl) 1 μl
5x Phusion/ GC buffer 10 μl
forward primer (0.1 μg/µl) 1 μl
reverse primer (0.1 μg/µl) 1 μl
dNTPs (10 mM) 1 μl
Phusion polymerase 0.5 μl
100% DMSO (optional) 1.5 μl (3%)
ddH2O 34 μl
Total 50 μl
Table 4. Standard PCR program used for mutagenesis of DGK
PCR-steps Temperature time cycles
pre-heating 98°C 30 s 1
denaturation 98°C 10 s
30 annealing 55°C 30 s
elongation 72°C 5 min
final elongation step 68°C 10 min 1
The annealing temperature was adapted to the melting temperature of the respective
primer. Additionally, the number of cycles was optimized for each mutagenesis.
The next step was accomplished, in order to exclude non-mutated parental from newly
mutated DNA. DNA isolated from nearly all E. coli strains is dam-methylated, whereas
the newly amplified DNA including the desired mutation contains no methyl-groups.
Therefore, the DNA was treated with DpnI endonuclease (target sequence: 5’-
Gm6ATC-3’), which is specific for methylated and hemimethylated DNA and digests
the non-mutated parental DNA. In detail, 1 µl DpnI were added to the DNA, followed by
an incubation at 37°C for 1 h.
In order to obtain high-quality, pure plasmid DNA for routine molecular biology
applications, a PCR purification kit (Macherey-Nagel NucleoSpin Plasmid) was used
according the instructions of the manufacturer. Thereafter, the mutated plasmid was
transformed into competent DH5α cells.
Chapter 2: Materials and Methods
48
2.2.1 Transformation
For the transformation, plasmid DNA and cell suspension were thawed on ice. 2 μl
DNA (~100 ng μl-1) were added to 50 μl cells and incubated on ice for 30 min. Cells
incubated with 2 μl water acted as referee. After a heat shock at 42°C for 90 s was
applied, the cells were kept on ice for 5 min. Subsequently, the cells were resuspended
in 200 μl sterile LB medium and incubated for 1 h at 37°C and 550 rpm. The cell
suspension was plated on LB agar plates containing 0.1 mg/ml ampicillin and
incubated overnight at 37°C. The following day, sterile LB medium with 0.1 mg/ml
ampicillin was inoculated with a bacterial colony from the LB agar plate and incubated
overnight at 37°C and 220 rpm.
The plasmid DNA was isolated from 5 ml bacterial culture using a DNA extraction kit
(Macherey-Nagel NucleoSpin Plasmid) according to the manufacturer instructions. The
DNA was eluted with ddH2O and the plasmid concentration was determined at the
spectrophotometer (Thermo Scientific NanoDrop 1000). Sequences of wtDGK and all
single-site mutant constructs were verified at Eurofins MWG Operon.
2.2.2 Glycerol stocks
For the production of bacterial glycerol stocks, 100 ml sterile LB medium with
0.1 mg/ml ampicillin were inoculated with a bacterial colony and incubated overnight at
37°C and 220 rpm. 500 µl of the bacterial culture were added to the glycerol stock
(Roth), which was then inverted 10x. Afterwards, the supernatant was completely
removed. Subsequently, the glycerol stock beads with the attached E. coli cells were
shock-frozen immediately in liquid nitrogen and kept at -80°C.
2.3 Protein expression and purification
2.3.1 Samples for high field MAS NMR
For expression of DGK in E.coli T7 express cells, pre-cultures were prepared.
Therefore, 100 ml sterile LB medium with 0.1 mg/ml ampicillin were inoculated with a
bacterial colony from the LB agar plate or with one bead from the glycerol stock and
incubated overnight for ~20 h at 27°C and 220 rpm. Main cultures were inoculated with
1% of the pre-culture and grown in M9 minimal medium with [U-13C]glucose and
[15N]ammonium chloride. The salt components of the used M9 minimal medium are
listed according to the respective labelling Table 5a, Table 6a, Table 7a. They were
Chapter 2: Materials and Methods
49
dissolved in ddH2O and the pH was adjusted to 7.0. In order to reduce spectral overlap,
unlabelled amino acids were added to the M9 medium to suppress isotope labelling of
these residues. In this study, a reverse labelled sample was prepared, in which
isoleucine (Ile), leucine (Leu) and valine (Val) were unlabelled (U-13C,15N-DGK-I,L,V)
(Table 7). For expression, 0.5 l of M9 medium was poured into a 2.5 l expression flask
and autoclaved at 121°C for 20 min. Before inoculation, autoclaved or filter-sterilized
solutions were added as listed in Table 5b, Table 6b, Table 7b.
Table 5. Composition of M9 minimal medium for the expression of unlabelled DGK
Components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
NH4Cl 0.5 g
added before inoculation:
b 1 M CaCl2 50 µl
autoclaving at 121°C for 20 min 1 M MgSO4 x 7 H2O 500 µl
40% glucose solution 5 ml
vitamin mix (see below) 1 ml filter sterilization via 0.2 µm filter
ampicillin (100 mg/ml) 500 µl
Table 6. Composition of M9 minimal medium for the expression of U-13
C,15
N-DGK
Components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
added before inoculation:
b 1 M CaCl2 50 µl autoclaving at 121°C for 20 min
1 M MgSO4 x 7 H2O 500 µl 15
NH4Cl 0.5 g
filter sterilization via 0.2 µm filter U-13
C-glucose 2 g
vitamin mix (see below) 1 ml
ampicillin (100 mg/ml) 500 µl
Chapter 2: Materials and Methods
50
Table 7. Composition of M9 minimal medium for the expression of U-13
C,15
N-DGK-I,L,V
Components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min
KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
isoleucine (unlabelled) 0.12 g
leucine (unlabelled) 0.12 g
valine (unlabelled) 0.12 g
added before inoculation:
b 1 M CaCl2 50 µl autoclaving at 121°C for 20 min
1 M MgSO4 x 7 H2O 500 µl 15
NH4Cl 0.5 g
filter sterilization via 0.2 µm filter U-13
C-glucose 2 g
vitamin mix (see below) 1 ml
ampicillin (100 mg/ml) 500 µl
To prepare the vitamin mix, 1.5 g of vitamin tablets (Centrum, Pfizer Consumer
Healthcare GmbH, Berlin, Germany) were pulverized and dissolved in 20 ml sterile,
deionized H2O by vortexing. The suspension was centrifuged at 8000 rpm for 20 min at
4°C. The supernatant was filter-sterilized with a 0.2 μm filter and used for the
expression medium.
For inoculation of the M9 medium, 10 ml pre-culture were pelleted by centrifugation for
10 min at 6000 rpm. The supernatant was discarded and the pelleted cells were
washed with sterile LB medium. This was followed by a second centrifugation step.
Then, the pellet was resuspended in 10 ml of the respective M9 medium and the cell
suspension was added to the expression flasks. E.coli cells were grown until an
OD600 = 0.6 - 0.8 was reached, whereupon protein expression was induced by the
addition of 500 µl of IPTG (200 mg/l). Cells were harvested after 16 h of protein
expression at 27°C and 220 rpm. The cell harvesting was performed by centrifugation
at 6000 rpm for 15 min at 4°C. The supernatant was discarded and the pelleted cells
were washed with buffer A (300 mM NaCl, 50 mM HEPES, pH 7.5). This was followed
by a second centrifugation step. Then, the pellet was resuspended in 30 ml of buffer A
and stored at -80°C.
The cell suspension was thawed. One tablet of “complete protease inhibitor”, 3% (w/v)
OG, a spatula tip of DNase I and MgCl2 were added. The solubilization of wild-type
Chapter 2: Materials and Methods
51
DGK was performed by stirring the cell suspension for 2 h at 4°C, whereas for Δ4-DGK
an extended incubation time of ~16 h at 4 °C was necessary.
The solubilized protein, which remains in the supernatant, was separated from the non-
solubilized material by centrifugation at 10 000 rpm for 30 min at 4°C.
Then, DGK was purified by immobilized metal ion affinity chromatography (IMAC) using
Ni-NTA as resin. In detail, 1.5 ml Ni-NTA and 0.75 ml imidazole (2 M) were added to
the supernatant and incubated for 1 h at 4°C under gentle stirring. Due to its N-terminal
hexahistidine-tag, the protein binds to Ni-NTA. The bound protein was washed with
75 ml buffer A containing 1.5% (w/v) OG and 50 mM imidazole. Then, the detergent
was exchanged by washing with 50 ml of buffer A containing 0.05% DDM. The protein
was finally eluted with buffer A containing 400 mM imidazole and 0.05% DDM until an
OD280<0.05 was reached. The protein concentration was determined by absorption
spectroscopy at 280 nm using the law of Lambert-Beer:
𝐴280 = 휀 ∙ 𝑐 ∙ 𝑙 (19)
A280 = absorbance at 280 nm, c = concentration of the sample (mg/ml), l = pathlength
(0.3 cm), ε = extinction coefficient (DGK: 30 480 M-1 cm-1)
2.3.2 Mixed labelled samples for DNP-enhanced MAS NMR
The expression and purification of mixed labelled DGK was in general performed as
described above with the difference that 13C- and 15N-labelled samples were expressed
separately to create mixed samples that only exhibit interprotomer and no
intraprotomer 13C−15N contacts (Table 8, Table 9). For the selectively labelled sample,
15N-labelled arginine and lysine (15Nh1/2-Arg, 15Nz-Lys) were added to M9 minimal
medium (Table 10). 12C-enriched glucose (99.5%) was used in 15N-labelled samples
instead of normal glucose to suppress 13C natural abundance within a protomer.
Chapter 2: Materials and Methods
52
Table 8. Composition of M9 minimal medium for the expression of U-13
C-DGK
components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
NH4Cl 0.5 g
added before inoculation:
b 1 M CaCl2 50 µl autoclaving at 121°C for 20 min
1 M MgSO4 x 7 H2O 500 µl
U-13
C-glucose 2 g filter sterilization via 0.2 µm filter
vitamin mix 1 ml
ampicillin (100 mg/ml) 500 µl
Table 9. Composition of M9 minimal medium for the expression of U-12
C,15
N-DGK
components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
added before inoculation:
b 1 M CaCl2 50 µl autoclaving at 121°C for 20 min
1 M MgSO4 x 7 H2O 500 µl
U-12
C-glucose 2 g
filter sterilization via 0.2 µm filter 15
NH4Cl 0.5 g
vitamin mix 1 ml
ampicillin (100 mg/ml) 500 µl
Table 10. Composition of M9 minimal medium for the expression of U-12
C,15
NArg,Lys-DGK
components amount for 0.5 l way of sterilization
a Na2HPO4 (anhydrous) 3 g
autoclaving at 121°C for 20 min KH2PO4 (anhydrous) 1.5 g
NaCl 0.5 g
NH4Cl 0.5 g
added before inoculation:
b 1 M CaCl2 50 µl autoclaving at 121°C for 20 min
1 M MgSO4 x 7 H2O 500 µl
U-12
C-glucose 2 g filter sterilization via 0.2 µm filter
15Nh1/2-arginine 0.20 g
Chapter 2: Materials and Methods
53
15Nz-lysine 0.21 g
vitamin mix 1 ml
ampicillin (100 mg/ml) 500 µl
In order to create mixed labelled complexes, DGK trimers had to be disassembled into
monomers, which was performed by SDS after the purification step. Differently labelled
monomers were then mixed. Subsequently, SDS had to be removed to allow
reassembling of the monomers to mixed labelled trimers, which was carried out by
washing with buffer A including DDM. In order to perform the washing step, the protein
containing a hexahistidine-tag was immobilized by binding to the Ni-NTA resin.
Therefore, the imidazole, which was used to elute DGK from the Ni-NTA resin during
the previously performed IMAC step, had to be removed from the protein solution. This
was carried out via a PD-10 desalting column, which was used according to the
instructions of the manufacturer. Therefore, in turn, it was necessary to concentrate the
protein solution to a small volume, which was performed via an Amicon centrifugal filter
with a molecular weight cutoff of 10 kDa according to the instruction manual.
Afterwards, the protein concentration was determined by absorption spectroscopy at
280 nm as described above. Then, the desalted protein solution was diluted to a
concentration of 0.2 mg/ml and was laced with 2% SDS, in order to disassemble the
DGK trimers. A low concentration of 0.2 mg/ml had to be used for disruption, since
DGK is more stable at higher protein concentrations [141]. Differently labelled protein,
for instance U-13C-DGK and U-12C,15N-DGK, were mixed in a 1:1 ratio. The protein was
incubated in SDS overnight at room temperature under gentle stirring. Additionally Ni-
NTA was added, to which the protein binds due to its histidine-tag. The next day, the
bound DGK monomers were washed with 300 ml buffer A containing containing 0.05%
DDM to remove SDS and to regain trimeric DGK. Then, the protein was eluted with
buffer A containing 400 mM imidazole and 0.05% (w/v) DDM until an OD280<0.05 was
reached and the protein concentration was determined. Using this approach, samples
were prepared, in which DGK consists of U13C- and U15N,12C-protomers ([CN]-DGK) or
U13C- and U15Nh1/2-Arg,15Nz-Lys,12C-protomers ([CN(Arg,Lys)]-DGK).
2.4 Protein reconstitution
2.4.1 Liposome preparation
The lipids DMPC and DMPA were weighted, dissolved in chloroform/ methanol (2:1)
and mixed in a 90mol%-to-10mol%-ratio, respectively. The solvent was evaporated
under a stream of nitrogen and the lipids were then further dried overnight by vacuum
Chapter 2: Materials and Methods
54
rotary evaporation with a pressure of 40 mbar. The next day, the lipids were rehydrated
with lipid buffer (50 mM HEPES, 300 mM NaCl, 1 mM EDTA, pH 8) at a concentration
of 0.045 mmol/ml. For the preparation of liposomes, DDM was added to enable a
homogeneous insertion of DGK during the reconstitution. In order to produce small
multilamellar liposomes, 6 - 8 freeze-thaw cycles were carried out by the application of
liquid nitrogen and sonication.
2.4.2 Reconstitution via BioBeads
The protein was mixed with the pre-softened liposomes and incubated for 1 h at 22°C.
Detergent removal was done with autoclaved SM-2 BioBeads. In detail, 4-times 80 mg
BioBeads/ml were added and incubated primarily overnight at 4°C and subsequently 3-
times for each 2 h at 22°C. Finally, the BioBeads were removed by filtration.
For all NMR experiments, reconstituted DGK was washed 6-7 times with NMR buffer
(20 mM HEPES, 3 mM MgCl2, pH 7.2).
2.5 Sample characterization
2.5.1 SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for
the qualitative characterization of the sample purity. 13 μl sample were mixed with 7 μl
loading buffer (RunBlue LDS Sample Buffer 4x: 40% glycerol, 4% LDS, 0.8 M
triethanolamine-chloride pH 7.6, 4% Ficoll-400, 0.025% Phenol Red, 0.025%
Coomassie Brilliant Blue G250, 2 mM EDTA) containing, additionally, a concentration
of 10% β-mercaptoethanol. A total of 15 μl of each sample and the protein marker
(AppliChem Protein-Marker III 6.5-200 kDa) were loaded on precast gels from
ExpedeonTM (RunBlue SDS Gel 4-20%, 20 μl loading wells). The gel was fixed in the
SDS-PAGE apparatus. The inner cathode chamber was filled with cathode buffer
(0.1 M Tris, 0.1 M Tricine, 0.1% SDS, pH 8.25) and the outer anode chamber was filled
with anode buffer (0.2 M Tris, pH 8.9). Electrophoresis took place at 180 V for ~30 min
until the dye front reached the base of the gel. The, the gels were stained with 0.025%
Coomasie Brilliant Blue G250 for 30 min and subsequently destained in 50% methanol,
40% acetic acid and 10% ddH2O.
Chapter 2: Materials and Methods
55
2.5.2 SEC
In order to characterize the homogeneity of the protein sample, size exclusion
chromatography (SEC) was carried out, during which the proteins are separated
according to their hydrodynamic volume. This way aggregates or co-purified proteins
can be separated.
For SEC analysis of DGK, the ÄKTA-Explorer System with a Superdex 75 10/300 GL
column with a separation range between 3-70 kDa (GE Healthcare) was used at room
temperature. The column had a bed volume of 24 ml. Before loading the protein
sample, the column was washed with 50 ml sterile filtered and degassed ddH2O. Then,
it was equilibrated with 50 ml filtered and degassed SEC buffer (100 mM NaCl, 50 mM
HEPES, pH 7.2) with the respective detergent. The washing steps were performed at
slow flow rates of 0.2 ml min-1. The protein sample was filtered with Pall Life Sciences
500 μl tubes (0.2 μm filter pore size), before it was loaded onto the column. The, the
sample was filled into the 500 µl sample loop via a syringe. The run was performed at
higher flow rates of 0.5 ml min-1. During the run, the absorption at 280 nm was detected
against the elution volume. After the run was completed, the column was washed with
ddH2O and afterwards with 20% ethanol.
2.5.3 BN-PAGE
Blue native-polyacrylamide gel electrophoresis (BN-PAGE) was used to assess the
oligomeric state of DGK. In contrast to the SDS-PAGE, which has a denaturing effect
on proteins, the native state of the proteins is retained during the BN-PAGE by using
Coomassie Brilliant Blue G250 as the charge shift molecule. Coomassie Brilliant Blue
G250 binds to the hydrophobic regions of the protein, replacing the detergent micelle
[142]. Thus, different oligomeric states of a protein can be determined by BN-PAGE.
Samples were prepared as described in Table 11 with BN-PAGE loading buffer 4x
(200 mM BisTris, 64 mM HCl, 200 mM NaCl, 40% (w/v) glycerol, 0.004% (w/v)
Ponceau S, pH 7.2) and 5% Coomassie Brilliant Blue G250 solution. The concentration
of Coomassie Brilliant Blue G250 was adjusted to one quarter of the detergent
concentration in the sample.
Table 11. Sample preparation for BN-PAGE
Component volume
protein in detergent micelles x µl (3.5-4 µg protein)
BN-PAGE loading buffer 4x 6.25 µl
Chapter 2: Materials and Methods
56
10% DDM solution 0.75 µl
5% Coomassie Brilliant Blue G250 solution 0.1-1 µl
ddH2O adjusted to 25 µl
The samples were loaded onto Precast NativePAGE Novex Bis-Tris 4-10% gels from
InvitrogenTM, which were fixed in the BN-PAGE apparatus. The outer anode chamber
was filled with BN-PAGE running buffer (50 mM BisTris, 50 mM Tricine, pH 6.8) and
the inner cathode chamber was filled with dark blue BN-PAGE cathode buffer (50 mM
BisTris, 50 mM Tricine, 0.02% Coomassie Brilliant Blue G250, pH 6.8). Afterwards,
25 µl of each sample and 5 µl of the protein marker (NativeMarkTM unstained protein
standard from InvitrogenTM: 20-1200 kDa) were loaded onto the gel. The
electrophoresis was then carried out at 150 V for ~2 h at room temperature until the
dye front reached the base of the gel. Finally, the gel was incubated in a fixing solution
(40% ethanol and 10% acetic acid), microwaved for ~45 s and shaken at room
temperature for 15 min. The same procedure was repeated with destaining solution
(8% acetic acid). Subsequently, the gel was incubated in the destaining solution until
the bands became clearly visible for analysis.
2.5.4 LILBID-MS
Laser induced liquid bead ion desorption-mass spectrometry (LILBID-MS) was used to
probe the oligomeric state of DGK in different detergents, verifying the results obtained
by BN-PAGE. All LILBID-MS measurements on DGK were carried out by Oliver Peetz
of the research group of Prof. Dr. Nina Morgner (Institute of Physical and Theoretical
Chemistry, Goethe University Frankfurt am Main). Since LILBID is highly tolerant
concerning salts and detergents, it enables the investigation of biomolecular complexes
in native-like environments, such as membrane proteins. LILBID-MS is soft enough to
prevent fragmentation. However, fragmentation can be induced on demand by
elevating the laser intensity, which allows the determination of subunit compositions.
During LILBID, microdroplets of an aqueous solution containing buffer, salt and further
additive components (e.g. detergent) next to the analyte (e.g. membrane protein) are
injected into the vacuum and irradiated one-by-one by mid-IR laser pulses. For the
LILBID-MS experiments on DGK in different detergents, the protein was transferred
into a salt-free buffer containing 50 mM ammonium acetate and the respective
detergent. This was carried out via a PD-10 desalting column according to the
instructions of the manufacturer. In detail, samples containing 20 µM DGK in 50 mM
ammonium acetate buffer with 0.025% DDM and samples containing 4 µM DGK in
Chapter 2: Materials and Methods
57
50 mM ammonium acetate buffer with 0.1% SDS were prepared. The LILBID-MS
measurements were performed using previously published standard settings [143]. A
piezo-driven droplet dispenser (MD-K-130 by Microdrop Technologies GmbH,
Norderstedt, Germany) generated droplets of 50 μm diameter at a repetition rate of
10 Hz. The droplets were transferred into a two-stage differential vacuum chamber.
Beginning at ~0.3 bar, they were transferred into high vacuum (10-5 mbar) via apertures
that reduce pressure. There, the droplets were irradiated one-by-one by mid-IR laser
pulses produced by a home-build Nd:Yag pumped LiNbO3 optical parametric oscillator
(OPO) [144, 145]. The IR pulses were tuned to the absorption wavelength of water at
2.94 µm. Consequently, the IR radiation directly excite the symmetric and asymmetric
stretch vibration of H2O. At this frequency, the penetration depth of the IR beam in H2O
is just ~1 µm. The excited stretch vibrations were shown to relax in bulk water within a
few hundreds of femtoseconds [146], leading to a production of heat. Thus, by applying
a pulse, causing an absorption of multiple photons, a high temperature is generated.
Due to the fulminating strong temperature increase, an elevated pressure is induced,
leading to a supercritical state, in which the droplets expand explosively and the
analyte ion is released into the gas phase. The ions are accelerated by a pulsed
electric field and mass-analyzed by a reflectron time-of-flight (TOF) mass spectrometer.
The mass spectra were recorded with a 8-bit digitiser card (Aquiris). The hardware was
controlled by a home-written software using LabView that enables the timing of the
measurements and data accumulation. The software Massign was used for data
processing, including signal calibration, smoothing, and background subtraction [147].
2.5.5 Sucrose density gradient centrifugation
A sucrose gradient (40% - 10%) was carried out to verify a homogenous reconstitution
of the protein into liposomes. Therefore, 1 ml of each solution, starting with 40%, was
carefully layered into a 5 ml centrifuge tube and left to equilibrate for 1 h at room
temperature. Then, 0.2 ml of a 0.4 mg ml-1 solution of empty liposomes or
proteoliposomes were carefully layered on top. The centrifugation was performed at
33 000 rpm in a SW50.1 rotor overnight at 4°C.
2.5.6 Coupled activity assay
The activity of reconstituted DGK was determined using a coupled enzyme assay [18].
The formation of ADP during the catalytic reaction of DGK was measured in a two-step
Chapter 2: Materials and Methods
58
process. In the first step, the produced ADP and phosphoenolpyruvate were converted
to ATP and pyruvate by pyruvate kinase (PK). In the second step, pyruvate was
converted to lactate by lactate dehydrogenase (LDH), oxidizing NADH to NAD+. The
decrease of NADH absorbance was monitored at 340 nm.
The measurement was done in a Tecan Reader Infinite M200 at 30°C. The assay
buffer (100 µl per measurement), provided on a 96well plate, was based on 25 mM
PIPES at pH 6.8 and contained 1 mM phosphoenolpyruvate, 0.5 mM NADH, 3 mM
MgATP, 15 mM MgCl2, 50 mM LiCl and 0.1 mM EDTA. 5 µl of an enzyme mix of
pyruvate kinase (PK) (18.2 units) and lactate dehydrogenase (LDH) (7.5 units) was
added to the assay buffer. Everything was preincubated at 30°C until the remaining
ADP was dissipated. The reaction was then initiated by the addition of 1-2 µl of protein.
The activity was stimulated by 2.63 µl of the water soluble lipid substrate analogue 1,2-
dibutyrylglycerol (DBG) (100 mM). The synthesis of DBG was carried out by Andreas
Jakob of the research group of Prof. Dr. Alexander Heckel (Institute of Organic
Chemistry and Chemical Biology, Goethe University Frankfurt am Main).
Concentrations of the different constituents were adapted so that the reaction catalyzed
by DGK was rate-limiting. The activity was calculated from the linear decrease of the
NADH absorption over time.
2.6 Preparing substrate-bound states of DGK
In order to saturate DGK with nucleotide substrate, reconstituted DGK was incubated
with a 14-fold molar excess of the ATP analogue adenylylmethylenediphosphonate
(AMP-PCP) and a 28-fold molar excess of MgCl2 overnight at 4°C. AMP-PCP and
MgCl2 were dissolved in 20 mM HEPES (pH 7.2). The accessibility of the nucleotide to
all active sites of DGK was enhanced by a 5 min sonication step in water bath (followed
by an incubation on ice to prevent over-heating) prior to the incubation time at 4°C. In
order to saturate DGK with the lipid substrate in a 10-fold molar excess, it was
reconstituted in a molar protein-to-lipid ratio of 1:50 into liposomes consisting of
80 mol% DMPC/DMPA (9:1) and 20 mol% 1,2-dioctanoyl-sn-glycerol (DOG, n=8). For
the production of DGK saturated with both the nucleotide and the lipid substrate, it was
firstly reconstituted into 80 mol% DMPC/DMPA (9:1) and 20 mol% DOG (n=8) in a
molar protein-to-lipid ratio of 1:50 and then incubated with a 14-fold molar excess of
AMP-PCP and a 28-fold molar excess of MgCl2 overnight at 4°C.
Chapter 2: Materials and Methods
59
2.7 MAS NMR
2.7.1 MAS NMR at high field
For the NMR experiments at high field, all proteoliposome samples were sedimented
by ultracentrifugation at 55 000 rpm for 1 h at 4°C and packed into a 3.2 thin wall rotor.
Approximately 18 mg of DGK could be loaded into the thin wall rotor. The NMR
experiments were then carried out on a Bruker wide bore Avance III solid state NMR
spectrometer with a 1H frequency of 850.32 MHz. A sample spinning rate of 15.2 kHz
was applied in each case. All samples were adjusted to a temperature of 275 K and
pH 7.2. The NMR time of dipolar coupling based experiments was reduced by
paramagnetic doping with 2 mM Gd3+-DOTA [12] in combination with an E-free 3.2 mm
triple-resonance HCN MAS probehead, which enabled using a recycle delay of 0.8 s.
Thereby, ~3x of the measurement time could be saved compared to the standard
probehead (recycle delay of 2.5 s). The E-free probehead was custom-built by Bruker.
2.7.1.1 Manual resonance assignment
NMR resonance assignment is based on multidimensional spectra that correlate
nuclear spins, leading to cross peaks. These nuclear correlation experiments are
chosen to complement each other, linking spin systems and mapping them to the
protein amino acid sequence, causing a network of peaks.
For the sequential assignment of the immobile domains of DGK, a combination of
dipolar coupling based 3D experiments (NCACX, NCOCX, CONCA) [123, 124] was
carried out. 13C and 15N assignments were mainly performed using uniformly labelled
samples (U-13C,15N-DGK). Residual ambiguities were resolved by reverse labelling of
Isoleucine, Leucine and Valine (U-13C,15N-DGK-I,L,V). The experiments were either
performed with an E-free or standard 3.2 mm triple-resonance HCN MAS probehead
(Bruker). All experimental parameters are listed in Table S4.
For the tentative assignment of highly mobile residues of DGK, scalar coupling based
2D experiments (13C-13C TOBSY, 1H-13C HETCOR, 1H-15N HETCOR) [46, 148, 149]
were applied. Therefore, only the uniformly labelled sample (U-13C,15N-DGK) was used.
The experiments were performed with a standard 3.2 mm triple-resonance HCN MAS
probehead (Bruker). Typical 90° pulse lengths were 2.5 µs (1H), 4.5 µs (13C) and 6 µs
(15N). A recycle delay of 2 s and a SPINAL64 1H decoupling of 100 kHz were used. For
all heteronuclear transfer steps a refocused INEPT (insensitive nuclei enhanced by
polarization transfer) [128] was applied with scalar couplings of 200 Hz (HC) and 90 Hz
Chapter 2: Materials and Methods
60
(HN). For the 13C-13C homonuclear polarization transfer, TOBSY (through-bond
correlation spectroscopy) [148] was applied with a 3.75 ms P916 mixing sequence.
2.7.1.2 Substrate bound states
The binding of the substrate(s) to DGK was verified for each case (AMP-PCP,
DOG+ATP, DOG+AMP-PCP) by 31P-CP (cross polarization) and 31P-DP (direct
polarization) experiments. Therefore, the standard 3.2 mm double-resonance HX MAS
probehead (Bruker) was used. Typical 90° pulse lengths were 3 µs (1H) and 4 µs (31P).
A recycle delay of 3 s and a SPINAL64 1H decoupling [150] of 83.3 kHz were used.
The CP contact time was 5 ms. All CP spectra were recorded with 16 000 scans, while
the DP spectra were carried out with 2000-4000 scans.
Scalar and dipolar coupling based experiments of DGK in its apo state, saturated with
AMP-PCP, DOG and with AMP-PCP + DOG were carried out for the analysis of CSPs
and peak intensities during substrate(s) binding. The experiments were either
performed with an E-free or standard 3.2 mm triple-resonance HCN MAS probehead
(Bruker). All experimental parameters are listed in Table S4.
2.7.1.3 Data analysis
All spectra were processed in TopSpin 3.5.b.91. pl 7 (Bruker). For comparison, the
respective spectra were processed the same way. Data analysis of multi-dimensional
experiments and resonance assignment were performed using CCPN 2.4.2 [151].
For the first 2D NCA spectrum, 13C chemical shift referencing was carried out with
respect to DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) through alanine
(179.85 ppm). 15N chemical shifts were indirectly referenced to liquid NH3 at 0 ppm
through the 15N/13C gyromagnetic ratio of ~0.4. All other 2D and 3D spectra were
subsequently referenced to an isolated resonance in this spectrum (112TrpCa). 31P
chemical shift referencing was performed with respect to 10% phosphoric acid at
0 ppm through crystalline triethylphosphine (TEP) (58.62 ppm).
Concerning the analysis of chemical shift perturbations (CSPs) in 3D NCACX spectra,
weighted chemical shift changes were calculated according to [152]:
∆𝛿(𝐶𝑥, 𝐶𝛼, 𝑁) = √(∆𝛿𝐶𝑥)2 + (∆𝛿𝐶𝛼)2 + (∆𝛿𝑁
2.48)² (20)
Chapter 2: Materials and Methods
61
where ΔδCx and ΔδCα and ΔδN represent chemical shift perturbations in the 13Cx,
13Cα and 15N dimension, respectively. All CSPs ≥ 0.2 ppm were counted as significant.
2.7.2 Automatic resonance assignment by ssFLYA
The ssFLYA algorithm [13] is predicated on FLYA, an automated resonance
assignment algorithm for solution NMR [153] and integrated in the software package
CYANA [154, 155]. In this study, ssFLYA was conducted on DGK by Dr. Sina Kazemi
of the research group of Prof. Dr. Peter Güntert (Institute for Biophysical Chemistry,
Goethe University Frankfurt am Main). ssFLYA assigns the frequencies to the spins by
mapping the network of expected peaks with unknown positions to the unassigned
detected peaks with known position. As input, it used solely the sequence of DGK and
the unassigned peak lists from dipolar coupling based 3D experiments (NCACX,
NCOCX, CONCA) obtained with the uniform (U-13C,15N-DGK) and reverse (U-13C,15N-
DGK-I,L,V) labelled sample. Partial assignments like grouping chemical shifts to a spin
system or setting them to atom types (N, Ca, Cb, Cg, etc.) within one residue were not
necessary. Peak lists were gained by manual peak picking, guided by input information
gathered from the manual assignment. The N-terminal region (-9Met–14Ala) of the
protein sequence, which is known to be highly mobile and not detectable, was
excluded in the calculations. Identified spectral artifacts like folded peaks were
removed from the input peak lists as well. For the calculations including the peak lists
of the reverse (U-13C,15N-DGK-I,L,V) labelled sample, the residue types Ile, Leu and
Val were excluded.
The entire experimental data set was used simultaneously to obviate possible
entrapments, in which results of an already performed step stay fixed for subsequent
steps. Instead of dictating a specific assignment strategy, ssFLYA creates peaks that
are expected in a given spectrum by applying a set of rules for through-bond or
through-space polarization transfer. Then, it constructs an optimal mapping of the
expected peaks that are assigned by definition but with unknown positions with the
detected peaks that are initially unassigned but with known positions in the spectrum
[153, 156-158]. Expected peaks are predicated on covalent connections between
atoms. Most of these ssNMR experiments include a relatively unspecific 13C–13C
transfer, leading to additional signals from neighbouring carbons with a high probability
for directly bound ones. This effect was considered by adding covalent bond patterns
with lower detection probability. An evolutionary optimization procedure was applied
that functions with a population of individuals, each typifying an assignment solution for
Chapter 2: Materials and Methods
62
the protein. The search area of an expected peak was defined by BMRB chemical shift
statistics [159]. Detected peaks were assigned within a given tolerance. Just one
detected peak could be mapped by one expected peak. The first generation of
assignment solutions was obtained randomly, but subjected to these conditions. In
each generation a local optimization algorithm revoked little parts of a mapping and
reassigned the expected peaks for a defined number of iterations with 15 000 as
default. Then, the obtained different solutions of one generation were recombined into
a new generation. Individuals and particular parts of an individual that are conducive to
a new individual were defined by a scoring function (equation 21). The solution, which
developed this function to a maximum, represents the final assignment. The global
score for complete assignment solutions assessed four criteria of an assignment
solution: the distribution of chemical shift values with regard to the chemical shift
statistics, the assimilation of peaks assigned to the same atom, the totality of the
assignment, and a penalty for chemical shift degeneracy. The global score G was
calculated as following [13]:
𝐺 = ∑ [𝑤1(𝑎)𝑄1(𝑎) + ∑ 𝑤2(𝑎, 𝑛)𝑄2(𝑎, 𝑛)/𝑏(𝑛)]𝑛∈𝑁′𝑎𝑎∈𝐴
∑ [𝑤1(𝑎) + ∑ 𝑤2(𝑎, 𝑛)]𝑛∈𝑁𝑎𝑎∈𝐴0 (21)
A0 describes the set of all atoms for which expected peaks are present, whereas A ⊆
A0 represents the set of assigned atoms, Na the set of expected peaks for atom a, and
N’a ⊆ Na the subset of expected peaks, which were mapped to a detected peak. b(n)
relates to the ambiguity of the assignment and aligns the number of expected peaks
that were assigned to the same detected peak as expected peak n. Unassigned atoms
and unmapped peaks were conducive via the normalization by the denominator. The
weighting factors were set to w1(a) = 4 and w2(a, n) = 1 for all calculations in this
study. The measure of quality Q1(a) refers to the agreement of the average chemical
shift ϖ(a) in the chemical shift list of atom a with the respective chemical shift statistics.
Likewise, Q2(a,n) quantifies the agreement between the chemical shift of atom a
determined from the detected peak, mapped by the expected peak n, and the average
frequency of the atom in the assigned peaks of the respective spectrum [13, 153]. For
a perfect match, the measures of quality Q = 1. In all other cases Q < 1. If Q = 0 or Q =
-∞, the assignment was considered as insufficient and equates to no assignment.
Thus, the global score G is 1 for a theoretical perfect assignment, and G < 1 in all other
cases. To enhance and evaluate the accuracy of the assignment, 20 independent runs
of the algorithm were carried out with several random seeds. For each atom a
consensus chemical shift was generated from the values determined in the individual
Chapter 2: Materials and Methods
63
runs [13, 153, 160, 161]. The consensus chemical shift ϖ(a) for an atom a is the value,
which developed this function to a maximum [13]:
µ(𝜔) = 1
𝑚 ∑ 𝑒𝑥𝑝 (−
1
2(
𝜔 − 𝜛𝑗(𝑎)
휀(𝑎))
2
)𝑚
𝑗=1 (22)
where ϖj(a) is the chemical shift value determined for atom a in run j, and ε(a) is the
chemical shift tolerance, which was set to 0.55 ppm for all calculations. The maximum
value of this function, µ(ϖ(a)), is a measure of the consensus of the chemical shift
values gained in the individual runs. It can be determined without the knowledge of
reference assignments. If all chemical shift values are identical, then µ(ϖ(a)) = 1.
Assignments with µ(ϖ(a)) ≥ 0.8 were classified as “strong”, all others as ‘‘weak’’. Weak
assignments were considered as tentative and needed further verification, since 39-
72% turned out to be erroneous with respect to manual assignments of already
performed automatic assignments by ssFLYA [13]. The assignment calculation could
be performed within approximately 10 min, if 20 CPU cores were availabe.
2.7.3 DNP-enhanced MAS NMR
Reconstituted protein samples were doped with a polarizing agent in order to achieve
DNP signal enhancement. Two proteoliposome pellets, each ~20 µl, were covered with
∼20 μL of a 20 mM AMUPol [133] solution (60% D2O, 30% glycerol-d8, 10% H2O) and
incubated for 20 h at 4°C. The solution was completely removed before the sample
was packed into a 3.2 mm ZrO2 rotor.
DNP-enhanced MAS NMR spectra were recorded on a Bruker 400 DNP system
consisting of a 400 MHz WB Avance II NMR spectrometer, a 263 GHz Gyrotron as
microwave source, and a 3.2 mm HCN Cryo MAS probe. All experiments were
conducted with 8 kHz MAS, and the microwave power at the probe was 12.5 W. During
DNP experiments, the temperature was kept at 105 K. For all experiments, a
SPINAL64 1H decoupling [150] of 100 kHz was applied during acquisition. A recycle
delay of 2.2 s was used. 2D 15N−13C correlation spectra were acquired using the z-
filtered TEDOR sequence [136]. Typical pulse lengths of 2.5 µs (1H 90°), 4.0 µs
(13C 90°), 8.0 µs (13C 180°), 7.5 µs (15N 90°) and 15 µs (15N 180°) were applied. The
CP contact time was 1.1 ms. A mixing time of 6.25 ms (24 rotor cycles) was used for all
experiments. The 2D-spectra for [CC]DGK and [CN(Arg,Lys)]DGK were acquired with
1504 scans in the direct dimension and 60 increments of 125 µs each in the indirect
dimension. The FID acquisition time in the direct dimension was 10 ms. The 15N pulse
Chapter 2: Materials and Methods
64
carrier was set to 54 ppm and the 13C pulse offset was set to 174 ppm. The 2D-spectra
for [CN]DGK and [CN]DGK-RxA were acquired with 2880 scans in the direct dimension
and 25 increments of 250 µs each in the indirect dimension. The FID acquisition time in
the direct dimension was 10 ms. The 15N pulse carrier was set to 100 ppm and the
13C pulse offset was set to 174 ppm.
All spectra were processed in TopSpin 3.5.b.91. pl 7 (Bruker). For comparison, the
respective spectra were processed the same way. Data analysis of 2D experiments
was performed using CCPN 2.4.2 [151].
Chapter 3: Sample optimization
65
3 Sample optimization
3.1 Introduction
E. coli DGK serves as a model system for membrane proteins since decades starting
1965 [93]. One good reason is its convenient experimental handling. Consequently, its
molecular cloning, expression and purification are well established. It can be easily
expressed recombinantly in E.coli cells, solubilized in octylglucoside (OG) [96, 97] and
purified by immobilized metal ion affinity chromatography (IMAC) using a hexa-His
tagged form [9]. Additionally, DGK features an easily assayable function. Badola and
Sanders designed an assay system [18], which is based on central aspects of the
mixed micellar assay developed by Bell and co-workers [97]. It is combined with the
classic pyruvate kinase/lactic dehydrogenase reaction coupling system that was long
utilized in studies of water-soluble kinases, in which rate-limiting ADP production by
DGK is coupled to NADH oxidation [103, 110].
However, the preparation of a membrane protein sample for MAS NMR is generally
considered to be a challenge, since high amounts of pure, isotope labelled protein are
required due to the inherently low sensitivity of the technique. Furthermore, in terms of
membrane proteins, it is highly beneficial, if they are embedded into lipid bilayers, since
they are strongly affected by interactions with the surrounding membrane. Additionally,
the catalytic mechanism of numerous membrane proteins is substantially linked to the
lipid bilayer [11, 20, 21, 162]. Thus, the reconstitution step is of key importance for the
investigation of a native-like membrane protein. It is a process, in which a purified,
typically detergent solubilized membrane protein is incorporated into an artificial lipid
bilayer. In this connection, a homogenous reconstitution of the protein into lipid
membrane is necessary to obtain well-resolved MAS NMR spectra. Likewise important
is the stability of the reconstituted sample, since the measurements can take up to two
weeks.
Summing up, the general aim of membrane protein preparation for MAS NMR
experiments is the production of high amounts of pure, active, stable and
homogeneously reconstituted protein, which provides well-resolved MAS NMR spectra.
Previously in this lab, efforts have been made to prepare a DGK sample that features
good spectral resolution for structural and functional studies by MAS NMR [163]. In
detail, the thermostable quadruple mutant (I53C, I70L, M96L, V107D) of DGK was
used. As detergent environment and for lipid softening, dodecyl phosphocholine (DPC)
and as lipid components 67.3mol% DMPC/ 32.7mol% cholesterol were utilized. The
Chapter 3: Sample optimization
66
reconstitution step was performed by dialysis and the molar protein-to-lipid ratio was
1:80.
This chapter demonstrates a stepwise optimization of the whole preparation protocol,
leading not only to a more native-like sample, but also to high quality MAS NMR
spectra and a very efficient preparation process.
3.2 Results and Discussion
3.2.1 DGK construct: Quadruple mutant (Δ4-DGK) vs. wild-type DGK
Since the main aim of the sample optimization was to elaborate a most native-like
sample, wild-type DGK and its thermostable mutant Δ4-DGK (CLLD-DGK), used in
preliminary studies in this lab, were compared. The quadruple mutant was introduced
by Zhou and Bowie [89]. It features a high thermostability, proven to be advantageous
for crystallization purposes [5, 6]. However, the wild type has been shown to be itself
remarkable stable: In native membranes, it is resistant for a few minutes to irreversible
inactivation at 100°C [93, 108]. Even in detergent micelles, DGK features a high
stability. The half time for irreversible inactivation at 70°C and pH 6.5 is on the order of
hours [8, 164]. Lau and Bowie worked out a method for quantitating the thermodynamic
stability of DGK [9]. Denaturing sodium dodecylsulfate (SDS) was titrated to folded
DGK in decylmaltoside micelles. This way, an impressive unfolding free energy of 16
kcal/mol for the transmembrane domain and even 6 kcal/mol for the cytoplasmic
domain could be demonstrated. These data indicate that wild-type DGK should be
stable enough for functional studies by MAS NMR.
In this study, wild-type DGK and its thermostable mutant were compared according
their activity (Figure 16a). Using the coupled enzyme assay, it could be shown that the
wild type is 61% more active than the Δ4-mutant, indicating that the four inserted
mutations most likely lead to unfavorable structural alterations in the enzyme. This
remarkable difference clearly highlights the need to use wild-type DGK for structural
and functional studies. Also the high quality MAS NMR spectra of wtDGK, shown in
3.2.6, were convincing (Figure 21b). Thus, further optimization was done with the wild
type.
Chapter 3: Sample optimization
67
3.2.2 Type of detergent: DPC vs. DDM
The next step was to find a perfect detergent. After the expression, DGK has to be
solubilized by the transfer into detergent micelles to remove impurities and to enable
manipulations in solution. The purification on a Ni-NTA column allows the transfer of
the bound protein into different detergent environments by washing the column with
another detergent prior to elution. The type of detergent influences the stability and
activity of membrane proteins and affects the homogeneous incorporation into
liposomes [165, 166]. Not only the protein itself but also the preformed liposomes are
saturated with detergent to disrupt lipid-lipid interactions, resulting in a more permeable
bilayer for protein uptake.
In order to check the purity and stability of wtDGK in the respective detergent, size
exclusion chromatography (SEC) was carried out immediately after the IMAC step. The
chromatogram of DGK in dodecyl phosphocholine (DPC) features a small peak
indicating aggregation (Figure 16c2). In contrast, wtDGK eluted in n-dodecyl-β-d-
maltopyranoside (DDM) is stable over several weeks at 4°C without showing any
aggregates (Figure 16c1). Also the peak is clearly broader in DPC. This is reflected in
the activity as well. Reconstituted DGK shows a notable reduced activity in DPC
compared to DDM, which is decreased by 35% (Figure 16b). This can be explained by
the physico-chemical characteristics of the two detergents according to their head
group and hydrophobic chain. Non-ionic detergents, like DDM, bear an uncharged
hydrophilic glycosidic head group. They are known to be mild and comparably non-
denaturizing, since they break lipid-lipid and lipid-protein rather than protein-protein
interactions. DPC in contrast belongs to zwitterionic detergents, which are considered
to be harsher and more deactivating than non-ionic ones [167, 168]. Therefore, DDM is
the detergent of choice for all subsequent studies.
Chapter 3: Sample optimization
68
Figure 16. (a) Comparison of the activity of wt- (dark grey) and Δ4-DGK (red) reconstituted into
DMPC/DMPA. Both samples are prepared the same way. (b) Comparison of the activity of
reconstituted wtDGK, prepared in DDM (dark grey) and DPC (red). 100% activity corresponds to
the rate recorded with wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1
mg-1
.
All activity measurements were repeated three times. The activity was calculated as the mean
value. Error bars correspond to standard deviations. (c) Size exclusion chromatography (SEC)
of wtDGK in 0.05% DDM (c1) and 0.5% DPC (c2), performed after several weeks at 4°C or
immediately after the IMAC step, respectively.
3.2.3 Characterizing the yield, purity and oligomeric state of wtDGK in DDM
Protein expression and purification were performed as described in chapter 2. The
sample is characterized regarding yield, purity and oligomeric state. The yield for
wtDGK could be increased by 50%, from 20-30 mg of DGK per liter E. coli culture to
30-45 mg/L, most likely simply by an optimized handling of the sample. The inserted
mutations of the quadruple mutant seem not to play a role, since for both wild type and
Δ4-mutant similar yields were obtained. The purity of wtDGK in DDM micelles was
proved by SDS-PAGE, showing a pure protein sample after IMAC purification (Figure
17a). BN-PAGE and LILBID-MS analysis were used for a reliable assessment of the
oligomeric state. The BN-PAGE shows wtDGK exclusively in its trimeric form without
any aggregates observable (Figure 17b). LILBID-MS verifies the results from BN-
PAGE, demonstrating that wtDGK forms predominantly trimers in DDM micelles
(Figure 17c). LILBID-MS was carried out by Oliver Peetz of the research group of Prof.
Chapter 3: Sample optimization
69
Dr. Nina Morgner (Institute of Physical and Theoretical Chemistry, Goethe University
Frankfurt am Main).
Figure 17. Characterization of the purity and oligomeric state of wtDGK in DDM micelles. (a)
The SDS-PAGE verifies the purity of the protein solution after IMAC purification. (b) The BN-
PAGE offers a reliable assessment of the oligomeric state, clearly showing wtDGK as trimer in
DDM micelles. (c) The trimeric state of wtDGK in DDM micelles is confirmed by LILBID-MS: The
signals for the monomeric, dimeric and trimeric form of wtDGK are labelled by “1”, “2” and “3”,
respectively. They occur at charged states of −1 and −2. The LILBID mass spectrum was
recorded by Oliver Peetz of the research group of Prof. Dr. Nina Morgner (Institute of Physical
and Theoretical Chemistry, Goethe University Frankfurt am Main).
3.2.4 Reconstitution method: Dialysis vs. BioBeads
Since membrane proteins in general and DGK in particular are strongly affected by
interactions with the surrounding membrane [11, 20, 21, 162], DGK was reconstituted
into liposomes for its investigation by MAS NMR. Therefor, the detergent had to be
removed. For this purpose, different removal methods for detergents are available.
They take advantage of the features of the respective detergent, i.e. critical micellar
concentration (CMC), charge or aggregation number. Most important for the
reconstitution step is that the detergent has to be completely removed, since even
small impurities interfere with the NMR spectra. Furthermore, the membrane protein
should be distributed among the liposomes evenly to ensure well-resolved MAS NMR
spectra. Most common removal methods are dialysis and hydrophobic absorption via
BioBeads [167, 168]. During dialysis, the concentration of the detergent is diluted to
values below the CMC, leading to the decomposition of micelles to single detergent
monomers. These monomers can be easily eliminated by a concentration gradient over
Chapter 3: Sample optimization
70
a dialysis membrane with a certain cut-off. This process requires a detergent-free
buffer of about 1000-fold excess compared to the protein-liposome-detergent solution.
It is quite time-consuming, since it takes usually over one to two weeks. However, this
reconstitution method might be useful for certain proteins, which are prone to
aggregation during a fast detergent removal. Dialysis functions best with detergents of
a high CMC, with low molecular weight and small cross-sectional area. Non-ionic
detergents are difficult to eliminate by dialysis due to their low CMC. They can be
clearly better removed by hydrophobic absorption via BioBeads [165, 169], whereupon
amphiphilic detergents bind to the hydrophobic surface of the insoluble beads using
their hydrophobic tail. The beads are mixed with the solution containing the detergent,
incubated for one to two days under slow stirring and can then be easily removed by
centrifugation or filtration.
Since the type of detergent used in this study was altered from zwitterionic DPC to non-
ionic DDM, the method of reconstitution was changed from dialysis to hydrophobic
absorption via BioBeads. The sucrose gradient (40%-10%) in Figure 20 shows that
DGK could be homogenously reconstituted using Biobeads. Also the high quality MAS
NMR spectra of DGK reconstituted into liposomes via BioBeads were convincing
(Figure 21b). Additionally, the usage of BioBeads as reconstitution method instead of
dialysis had the positive side effect of reducing the expenditure of time remarkably from
two weeks to two days.
Table 12. Detergents used and compared in this dissertation. The classification, CMC and
concentration range are shown [163].
Detergent Classification CMC [mM] Conc.% [w/v]
DDM n-dodecyl-β-maltopyranoside non-ionic, alkyl maltoside 0.15-0.19 0.03-0.10
DPC dodecyl phosphocholine zwitterionic, alkylphosphocholine 1.10 0.20-0.60
3.2.5 Lipid composition, protein-to-lipid ratio and functional characterization
Another crucial point for the preparation of a membrane protein sample for MAS NMR
is the definition of the liposome composition. The model membrane should guarantee
both a perfect emulation of the native environment to maintain the functional native
state of the membrane protein and a high resolution as well as a high sensitivity of the
NMR spectra. The membrane affects the structure and activity of a membrane protein
through specific or unspecific interactions [21, 165, 166]. Biological membranes contain
a complex mixture of lipids, which are distinguished on the one side by their acyl chain,
Chapter 3: Sample optimization
71
according to length and double bonds, and on the other side by their head group,
concerning size and charge. Thus, membranes reveal particular physical properties
depending on the lipid composition, such as thickness, curvature, shape, lateral
pressure, hydration or dielectric constant. Attempts to create a proper model
membrane are guided by these properties along with the goal to maintain the lipid
composition as simple as possible particularly with regard to a better reproducibility.
Thus, synthetic lipids are typically chosen.
In this study, the liposome composition was changed from 67.3mol% DMPC/ 32.7mol%
cholesterol to 90mol% DMPC/ 10mol% DMPA. Both compositions have the zwitterionic
phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, di(C14:0)PC) in
common. Zwitterionic phospholipids are known to be the major lipid species in
membranes [170]. For preliminary work in this lab, cholesterol was used in addition to
modulate protein motions in the membrane [163]. However, it is rather uncommon as a
lipid component to mimic the E.coli membrane. It is known to be part of animal cell
membranes, where it is essential to maintain both membrane structural integrity and
fluidity. In this study, the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphate
(DMPA, di(C14:0)PA) was used instead of cholesterol. As zwitterionic lipids, anionic
ones are known to be part of all membranes. The presence of anionic lipids is
especially important for the binding of peripheral membrane proteins or protein
segments to the membrane surface. Whereupon positively charged residues interact
electrostatically with the anionic lipids in the bilayer. Furthermore, it has to be
highlighted that 90mol% DMPC in combination with 10mol% DMPA is a widely used
liposome composition. It has been already successfully used for green proteorhodopsin
(GPR) [134, 171], krokinobactereikastus rhodopsin 2 (KR2) [172] and the ATP-binding
cassette (ABC) transporter MsbA [173]. It was also applied for the structure
determination of Anabaena sensory rhodopsin (ASR) by MAS NMR [174]. Especially
the latter is a good example that the usage of 90mol% DMPC/ 10mol% DMPA can
support a high spectral resolution that cannot be taken for granted in MAS NMR.
The two liposome compositions were compared according their fingerprint and
resolution in 2D 13C-13C PDSD spectra. Figure 18 shows a similar fingerprint for DGK in
both lipid compositions, indicating a similar secondary structure. Nonetheless,
differences in the spectral resolution can be observed as well. The enlargement of the
representative region in the 2D 13C-13C PDSD spectra (Figure 18) displays clearly a
reduced resolution for 67.3mol% DMPC/ 32.7mol% cholesterol. For instance, the
selected peak features a line width in F1 dimension of 138 Hz and 406 Hz and in F2
dimension of 574 Hz and 1006 Hz for 90mol% DMPC/ 10mol% DMPA and 67.3mol%
DMPC/ 32.7mol% cholesterol, respectively. The reduced resolution implies a lesser
Chapter 3: Sample optimization
72
homogenous sample in 67.3mol% DMPC/ 32.7mol% cholesterol, which might reflect
the disordering effect of cholesterol for the lipid bilayer in the gel phase. In contrast,
DMPA has possibly a stabilizing effect, especially on the amphiphilic surface helix
(SH), which contains several positively charged residues (Arg9, Lys12, Lys19 and
Arg22) [5] that most likely interact electrostatically with the anionic lipids in the bilayer.
However, kinetic studies by Lee and co-workers indicated that product analogues of
DGK’s physiological forward reaction, such as DMPA, possibly bind to the active site
[106]. Pilot et al. demonstrated that 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA,
di(C18:1)PA) increases the Km of the lipid substrate 1,2-dihexanoylglycerol (DHG,
chain length n=6) [106]. This has been explained by a possible binding of the product
analogue to the active site in competition with the lipid substrate. However, the
superposition of the 2D 13C-13C PDSD spectra of DGK embedded into 90mol% DMPC/
10mol% DMPA and 67.3mol% DMPC/ 32.7mol% cholesterol shows the same
fingerprint with no chemical shift perturbations (CSPs) observable. Cholesterol, which
is anyway rather uncommon for E.coli membranes, is not reported to bind to the active
site of DGK. Thus, the same spectral fingerprint of the two liposome compositions
indicates that DMPA does not bind to DGK’s active site. If this would be the case, DGK
in complex with DMPA would have provided significant CSP’s compared to its pure apo
state. Based on the high sensitivity of NMR signals to the local environment, even
weak ligand binding can be analysed by chemical shift changes. In addition, NMR
features the advantage that changes can be investigated directly and specifically at an
atomic level.
The differences concerning the assumption of the binding of the product analogue to
the active site of DGK might arise from different experimental conditions. For the kinetic
studies by Lee and co-workers, DGK was reconstituted into 80mol% DOPC/ 20mol%
DOPA in a molar lipid-to-protein ratio of 6000:1. This implies a remarkable high molar
DOPA-to-protein ratio of 1200:1. In our study by MAS NMR, a clearly lower molar
DMPA-to-protein ratio of 5:1 was applied. Thus, a very high molar excess of DMPA
compared to DGK seems to be necessary to force its binding to the enzyme. Based on
our MAS NMR data, this is obviously not the case at a 5-fold molar excess of DMPA. In
addition, it has to be mentioned that Lee and co-workers carried out the activity
measurements on unsealed membrane fragments consisting of phospholipids and
detergent (cholate). They were obtained by dilution of the reconstition mixture into
buffer, decreasing the concentration of cholate below its critical micelle concentration.
In contrast, DGK was present in detergent-free liposomes during the MAS NMR
experiments.
Chapter 3: Sample optimization
73
Due to the high quality of the MAS NMR spectra, the widely and successfully used
90mol% DMPC/ 10mol% DMPA composition was used for all subsequent studies.
Figure 18. Superimposed 2D 13
C-13
C PDSD spectra of U-13
C,15
N-DGK embedded into 90mol%
DMPC/ 10mol% DMPA (black) or 67.3mol% DMPC/ 32.7mol% cholesterol (red), showing a
similar fingerprint. The enlargement of a representative region in the 2D 13
C-13
C PDSD spectra
displays clearly a reduced resolution for 67.3mol% DMPC/ 32.7mol% cholesterol (red). For
instance, the selected peak, P, features a line width in F1 dimension of 138 Hz and 406 Hz and
in F2 dimension of 574 Hz and 1006 Hz for 90mol% DMPC/ 10mol% DMPA (black) and
67.3mol% DMPC/ 32.7mol% cholesterol (red), respectively. The line widths were obtained from
CCPN analysis 2.4.1 [175].
In order to pack more protein into the rotor (volume ≤ 50 μl), which in turn elevates the
sensitivity, the molar protein-lipid ratio was increased stepwise from 1:80 to 1:20. The
starting point of 1:80 was given by previous studies in this lab [163]. However, an
increased protein-lipid ratio should be accompanied with a well-preserved activity of the
membrane protein, when it is incorporated into the liposomes. Thus, the activity was
checked for each sample. Figure 19 shows that a higher molar protein-lipid ratio up to
1:50 does not reduce the activity. If the ratio is further increased up to 1:20, the activity
decreases significantly, which indicates that most likely unfavorable protein-protein
interactions and adverse changes of the protein-lipid-interaction occur. Consequently,
the molar protein-lipid ratio of 1:50 was used for all subsequent studies, which allows to
pack 30% more protein into the rotor compared to the starting point of 1:80.
Chapter 3: Sample optimization
74
Figure 19. Comparison of the activity of wtDGK reconstituted into DMPC/DMPA with different
molar protein-to-lipid ratios increasing from 1:80 to 1:20. The ratio of 1:50 (grey) is used for all
subsequent studies. 100% activity corresponds to the rate recorded with wtDGK in 90mol%
DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1
mg-1
. Experiments were repeated three times.
The activity was calculated as the mean value. Error bars correspond to standard deviations.
Furthermore, a sucrose gradient (40%-10%) was performed to verify, if DGK is
homogenously reconstituted into the DMPC/DMPA liposomes, which is of key
importance for well-resolved MAS NMR spectra. The sucrose gradient clearly reveals a
homogenous size distribution of the proteoliposomes, since only one sharp band is
visible without any empty liposomes observable (Figure 20).
Figure 20. Sucrose density gradient (40%-10%) centrifugation of empty liposomes (left) and
wtDGK reconstituted in DMPC/DMPA in a molar protein-to-lipid ratio of 1:50 (right), revealing
homogeneous size distribution of proteoliposomes without any empty liposomes observable.
Chapter 3: Sample optimization
75
To verify the functionality of wtDGK in liposomes, we used a coupled enzyme assay as
described in chapter 2. An average activity of 90 (± 9.9) µmol min-1 mg-1 was obtained
for wtDGK in 90mol% DMPC/ 10mol% DMPA. The measured activities were highly
reproducible from sample to sample and are comparable to values published before [7,
107].
3.2.6 Evaluating the optimized DGK proteoliposomes for MAS NMR application
To further determine the quality of the proteoliposome sample, 1D 13C and 15N cross-
polarization (CP) MAS as well as 2D 13C–13C correlation spectra, namely proton driven
spin diffusion (PDSD) spectra, were carried out. In spite of spectral overlap
characteristically for α-helical proteins, a fine structure is clearly visible even in 1D
spectra, and some single resonances are resolved. For instance, a sharp band at
approximately 107.0 ppm in the 15N CP spectrum corresponds to the 15N glycine
signals (Figure 21a). The 2D 13C-13C PDSD was conducted with short carbon-carbon
mixing (20 ms) to yield one bond correlations between aliphatic atoms (Figure 21b).
The spectrum provides a fingerprint of the sample, evaluating structural homogeneity,
resolution and secondary structure. The high number of well resolved peaks
demonstrates a homogeneous sample preparation.
Figure 21. Evaluation of the proteoliposom sample by MAS NMR. 1D 13
C and 15
N cross
polarization (CP) spectra of U-13
C,15
N-wtDGK reconstituted into DMPC/DMPA, exhibit a good
Chapter 3: Sample optimization
76
spectral resolution (a). 2D 13
C-13
C PDSD spectrum of U-13
C,15
N-wtDGK reconstituted into
DMPC/DMPA. A mixing time of 20 ms was used to yield one bond correlations between
aliphatic atoms. The spectrum evaluates structural homogeneity, resolution and secondary
structure. The high number of well resolved peaks demonstrates a homogeneous sample
preparation (b).
3.3 Summary
The optimization resulted in a proteoliposome sample, which mimics physiological
conditions to a higher extent than before. The activity could be increased by using wild-
type DGK instead of the quadruple thermostable mutant and by the application of DDM
as detergent instead of DPC. The yield for DGK could be increased by 50%, from 20-
30 mg of DGK per liter E.coli culture to 30-45 mg/l. The purity of wtDGK in DDM
micelles was verified by SDS-PAGE and SEC. Additionally, BN-PAGE and LILBID-MS
showed that DGK appears exclusively in its trimeric form. Moreover, it could be proven
that DGK can be homogenously reconstituted into 90 mol% DMPC/ 10 mol% DMPA
using BioBeads, leading to a fully active protein and even better resolved spectra than
shown before in this lab. Using BioBeads instead of dialysis as reconstitution method
has the positive side effect of reducing the expenditure of time remarkably from two
weeks to two days. Furthermore, by increasing the molar protein-to-lipid ratio, 30%
more protein can be packed into the rotor, yielding to a higher sensitivity. Another
feature of the sample, which is worth to mention, is its long-term stability, being
especially advantageous for recording time-consuming 3D spectra regarding
assignment purposes. Hence, the new optimized preparation protocol is more efficient
and provides a more native-like sample, which delivers high quality MAS NMR spectra,
increasing the value of all subsequent studies. Especially, the good spectral resolution
provides the basis for an assignment of DGK, which is demonstrated in the following
chapter.
Chapter 4: Resonance assignment
77
4 Resonance assignment
4.1 Introduction
For the investigation of structural and dynamical changes defining the catalytic
mechanism of a protein, the assignment of its backbone and side chains is mandatory.
So far, an assignment of a thermostable mutant of DGK in E.coli total lipids was
published by Yang and co-workers using MAS ssNMR [14]. A comparison of the first
13C-13C correlation MAS ssNMR spectrum of wild-type DGK with the 13C chemical shifts
derived from the mutant (BMRB entry: 19754) [14] is shown in Figure 22. Upon first
inspection, it becomes obvious that a transfer of these assignments to the wild type
sample is not possible. The cross peaks from the mutant clearly do not match the
peaks from the wild type. Deviations are most likely caused by inserted mutations.
Additional sources might arise from different experimental conditions. Our experiments
were carried out on DGK reconstituted into DMPC/DMPA under a pH of 7.2 and full
hydration. The thermostable mutant was reconstituted into E.coli total lipids and
measured under a pH of 6.6 and 23 wt% H2O [14].
Figure 22. Comparison of MAS ssNMR data of the wild type with its thermostable mutant. 2D 13
C-13
C PDSD spectrum of U-13
C,15
N-wtDGK, recorded with a mixing time of 20 ms (left).
Enlargement of the selected region in the 2D 13
C-13
C PDSD spectrum (right). The spectrum is
compared with the assignment of the thermostable mutant of DGK gained by MAS NMR [14].
The comparison shows that a transfer of these assignments to the wild type sample is not
possible. The red cross peaks were obtained with CCPN analysis 2.4.1 [175].
Chapter 4: Resonance assignment
78
Thus, we carried out sequential assignment of wild-type DGK. The capability of magic
angle spinning (MAS) solid state NMR for the assignment of membrane proteins in
phospholipids could be demonstrated for the light-harvesting 2 protein, LH2 [176, 177];
sensory rhodopsin II from Natronomonas pharaonis, NpSRII [178]; and green
proteorhodopsin, GPR [124] as well. For few membrane proteins, even complete 3D
structures could be determined in phospholipids, including: the sensory rhodopsin from
Anabaena, ASR [174]; the human chemokine receptor, CXCR1 [179]; the M2 1H
channel from influenza virus [180, 181]; the bacterial inner membrane protein DsbB
[182, 183]; the Mycobacterium tuberculosis cell division protein, CrgA [184] and the
membrane-inserted form of the fd bacteriophage major coat protein [185] (Figure 23).
Figure 23. Examples of membrane protein structures determined in phospholipids by solid state
NMR. (a) Anabaena sensory rhodopsin, ASR: PDB 2m3g, with bound retinal (yellow) [174]. (b)
human chemokine receptor, CXCR1: PDB 2lnl [179]. (c) M2 1H channel from influenza virus:
PDB 2l0j [180, 181]. (d) Bacterial inner membrane protein DsbB: PDB 2leg [182, 183]. (e)
Mycobacterium cell division protein, CrgA: PDB 2mmu [184]. (f) Membrane-inserted form of the
fd bacteriophage coat protein: PDB 1mzt [185]. The figure is adapted from Marassi and Opella
[186].
Prescind from these outstanding achievements, the assignment of membrane proteins
by MAS ssNMR is still a highly challenging task. A first obstacle to overcome is to
produce sufficient amounts of purified, stable and active protein, which is
homogenously reconstituted in an appropriate lipid environment, as demonstrated in
the previous chapter 3. The second obstacle arises from spectral crowding. Membrane
proteins are generally built up of residues that are all constrained in nearly the same
secondary structure, yielding a narrow chemical shift dispersion. A third obstacle
occurs due to the low intrinsic sensitivity of the technique. The intensity of an NMR
signal is proportional to the population difference between the spin states, specified by
Boltzmann statistics. The differences in the population between the spin states are
minor, since they are separated by only weak energy differences in the radio-frequency
range. This turns solid state NMR spectroscopy into a relatively insensitive technique
Chapter 4: Resonance assignment
79
compared to other spectroscopic methods, requiring long signal accumulation times to
gain spectra with a sufficient signal-to-noise ratio. The fourth obstacle addresses the
data analysis for the achievement of a reliable assignment to large extent, which poses
a highly demanding and time-consuming task.
In this chapter, a strategy is presented, which helps to diminish these obstacles and
enables a nearly complete assignment of wild-type DGK in lipid bilayers.
4.1.1 Applied isotope labelling strategy
A repertory of isotope labelling techniques, ranging from uniform to site specific, were
developed for in vivo and in vitro expression systems. For uniform labelling, the
expression medium contains a sole carbon source, such as glucose or derivatives
(glycerol, acetate, pyruvate, succinate) and a sole nitrogen source, like ammonium
salts (chloride, nitrate, sulfate). It offers a maximum of isotope labelled sites in one
single sample. However, based on the presence of a dominating single type of
secondary structure (α-helix or β-strand) and the natural repetitiveness of hydrophobic
amino acids, the spectra suffer from spectral overlap, causing ambiguities in data
analysis. Spectral crowding can be, for instance, reduced by reverse labelling, which is
complementary to other labelling strategies, implying selective unlabelling of specific
amino acids. The expression medium, including 13C-glucose and 15NH4Cl, is
supplemented with unlabelled amino acids, containing 12C and 14N. The high presence
of the unlabelled amino acids leads to their direct incorporation into the protein
sequence during expression. The cells do not need to synthesize these amino acids
from the labelled precursors. This way, they are undetectable for NMR, resulting in a
significant decrease of spectral crowding. This labelling approach is rather inexpensive
compared to others. Additionally, same growth conditions as for uniform 13C/15N
labelling can be applied, simplifying its application. For the development of reverse
labelling, following aspects were taken into account: Based on the amino acid
sequence, a first selection was carried out on the relative abundance of each amino
acid type. Usually, it proves to be difficult to assign every amino acid of a type with high
abundance. Transmembrane segments are normally rife with hydrophobic amino acids,
for which spectral overlap is known. Thus, key hydrophobic residues are usually
chosen for unlabelling [178, 187]. In contrast, extramembrane loop regions typically
consisting of polar or charged amino acids are well resolved. Aromatic residues, which
are often located at the membrane interface, could be considered for reverse labelling
as well, since their resolution can be compromised by ring currents. One issue that has
Chapter 4: Resonance assignment
80
to be taken into account during reverse labelling or selective unlabelling, is scrambling.
During the amino acid biosynthesis in E. coli, metabolic conversions occur, in which
amino acids are transformed into one another. During these metabolic reactions
labelled nuclei could be transferred to amino acids that should not be labelled, leading
to unwanted signals. This issue can be overcome by supplementing the culture
medium with high amounts of all twenty amino acids, since biosynthetic enzymes are
regulated by feedback inhibition. Another solution is provided by specifically modified
host strains, in which scrambling of certain amino acids is controlled with defective
enzymes. One such strain is e.g. E.coli CT19 featuring a reduced transaminase activity
[188]. The easiest option to avoid or at least reduce scrambling is the use of amino
acids, which are end products of the bacterial metabolic cycle and therefore not
converted into other amino acids. These amino acids are Arg, Cys, His, Ile, Leu, Lys,
Met, Phe, Pro, Trp, Tyr and Val. The other amino acids (Ala, Asn, Asp, Glu, Gln, Gly,
Ser and Thr) should be avoided due to the risk of scrambling [189]. In this study,
uniform 13C,15N and reverse labelling was applied. For reverse labelling, the most
frequent hydrophobic amino acids: Ile, Leu and Val (Table 13), which are not subjected
to scrambling, were chosen.
Table 13. Amino acid composition of wild-type DGK. The numbers in brackets belong to the
His6-tag and linker. The most frequent hydrophobic amino acids: Ile, Leu and Val, which were
chosen for reverse labelling, are highlighted orange.
amino acid type number
Ala 18
Arg 6
Asn 4
Asp 4
Cys 2
Gln 1
Glu 6 (+1)
Gly 8 (+1)
His 2 (+6)
Ile 15
Leu 12 (+1)
Chapter 4: Resonance assignment
81
Lys 3
Met 3 (+ 1)
Phe 3
Ser 8
Thr 5
Trp 5
Tyr 2
Val 14
4.1.2 Applied strategy for improvements of the sensitivity and resolution
4.1.2.1 High magnetic fields
Improvements of the sensitivity can be already obtained by high magnetic fields, since
the population difference increases with the magnetic field strength (B0). In practice, the
signal-to-noise ratio of modern NMR spectrometers scales approximately with B01.5.
Thus, an enhancement in sensitivity of about 2 is gained, when a 18.7 T (800 MHz)
spectrometer is compared with a 11.7 T (500 MHz) instrument. With increasing field
strength, the spectral resolution improves as well, since line-broadening mechanisms
involving dipolar or scalar couplings only arise in higher-order perturbation terms.
Hence, a high magnetic field is a prerequisite for assignment studies. The assignment
of wtDGK, performed here, was carried out on a Bruker wide bore Avance III solid state
NMR spectrometer with a 1H frequency of 850 MHz.
4.1.2.2 Paramagnetic doping in combination with an E-free probehead
Further improvements can be achieved by the enhancement of the signal-to-noise ratio
per unit time. Nearly 90% of the measurement time is needed to restore the 1H
Boltzmann equilibrium after each cross-polarization (CP) step. The required recycle
delay matches 4–5-times the 1H longitudinal relaxation time (1H-T1), which is generally
needed to restrict the probehead duty cycle and to prevent surplus sample heating, as
well. One possible route for reducing 1H-T1 and thus enhancing the signal-to-noise
ratio per unit time is doping the sample with paramagnetic relaxation agents [190].
Paramagnetic agents feature unpaired electrons, which have a remarkable influence
on the NMR spectra due to the electronic magnetic moment, being about 650 times
Chapter 4: Resonance assignment
82
higher compared to protons. Random variations of electron spin-nuclear spin
interactions cause paramagnetic relaxation enhancement (PRE), allowing faster data
acquisition schemes. For the use in protein NMR, the unpaired electrons must reside in
chemically non-reactive compounds that are stable in aqueous solution, e.g. in a
chelate complex. Previously in this lab, Ullrich et al. could demonstrate that Gd3+-DOTA
is the most potent relaxation agent for membrane proteins so far [12]. Gd3+ contains
seven unpaired electrons. It does not cause pseudo-contact shifts (PCS), since its
magnetic susceptibility tensor is isotropic, but the size of the isotropic tensor
component and its slow electronic relaxation rates cause large paramagnetic relaxation
enhancement (PRE). PRE effects by Gd3+ are larger than those reported for nitroxides
and comparable to Cu2+ and Mn2+ (reviewed in [191]).
However, higher probehead duty cycles could cause sample damage due to an
increased sample heating. Paramagnetic doping in combination with fast MAS (≥ 40
kHz) [192-194] or sample deuteration [195] with low power decoupling would represent
possible solutions. In this study, sample heating was avoided by using a MAS
probehead with reduced E-field [196], which was specifically designed for
measurements on a Bruker wide bore Avance III solid state NMR spectrometer with a
1H frequency of 850 MHz. It allows utilizing protonated samples, high power
decoupling, moderate sample spinning rates and MAS rotors of conventional size.
4.1.3 Applied assignment procedure
DGK consists of transmembrane α-helices, connected by a cytosolic and a periplasmic
loop, and an amphiphilic surface helix. This architecture results in very different
molecular dynamic time scales and amplitudes within the protein [149]. Motions at
different time scales and amplitudes are crucial for the function of membrane proteins,
which are embedded in lipid bilayers and exposed to water at the water-membrane
interface. Immobile domains, usually transmembrane regions, are molecular segments
with smaller amplitude motions. They are not sufficient to fully average anisotropic
interactions (such as HH, HC, or HN dipole couplings) and can be observed by dipolar
coupling based cross-polarization (CP). In this study, the sequential assignment of
immobile domains was performed by dipolar coupling based 3D heteronuclear
correlation experiments: NCACX, NCOCX and CONCA. In contrast, highly mobile
regions, usually termini or loops, feature fast and large amplitude fluctuations on time
scales of <10-5 s. They can be selected by a refocused INEPT (insensitive nuclei
enhanced by polarization transfer) [128] step based on scalar couplings, leading to
Chapter 4: Resonance assignment
83
solution-state-like spectra due to efficient molecular averaging of anisotropic
interactions. Thus, for the completion of the assignment of wtDGK, scalar coupling
based 2D experiments, 1H-13C/15N HETCOR and 13C-13C TOBSY, were carried out to
detect highly mobile residues. All dipolar coupling based 3D and scalar coupling based
2D experiments are explained in detail in chapter 1 under the bullet 1.3.2.
4.1.3.1 Sequential assignment of immobile domains
The sequential assignment process includes spin system identification, assignment of
the spin system to the amino acid type, linking of the spin systems, and mapping them
to the protein amino acid sequence. For the identification of the spin systems, it has to
be taken into account that DGK contains a total of 121 residues, of which 41 are not
labelled in the reverse labelled DGK-I,L,V sample (15 isoleucines, 12 leucines, and 14
valines). Thus, a maximum of 121 and 80 spin systems in the uniformly and reverse
labelled sample, respectively, are expected. However, it has to be considered as well
that not all of them might be visible due to higher dynamics. Therefore, scalar coupling
based experiments represent a proper solution.
For the identification of the amino acid type, the BMRB database [159] was used. The
amino acid type was largely assigned through the measurement of the chemical shifts
of the side-chain carbon atoms. Here, glycines represent very good starting points for
the assignment procedure, since they have a characteristic Ca chemical shift of ~45
ppm. The amino acids Ala, Ser and Thr can be easily identified as well due to their
specific Cb chemical shift. For these residues (with short or unique side chains), short-
range one- and two-bond correlations, are sufficient to identify the amino acid type.
With respect to the average Ca and Cb chemical shift range, the other amino acids can
be distinguished into 4 groups:
Group 1 (Ca ~57 ppm, Cb ~30 ppm): Lys, Arg, Gln, Glu, Met, His, Trp, Cys (reduced)
Group 2 (Ca ~57 ppm, Cb ~40 ppm): Asp, Asn, Phe, Tyr, Leu, Cys (oxidized)
Group 3 (Ca ~60 ppm, Cb ~40 ppm): Ile (very specific side chain pattern)
Group 4 (Ca ~65 ppm, Cb ~30 ppm): Val
Their unambiguous identification requires the detection of three- or four-bond carbon–
carbon connectivities.
Chapter 4: Resonance assignment
84
In this study, an 3D NCACX experiment with a DARR mixing time of 50 ms was used to
establish these long-range intra-residue correlations, enabling the identification of as
many amino acids by type as possible. For the 3D NCOCX experiment, even a DARR
mixing time of 100 ms was used to enable long-range inter-residue correlations. For
the linking of the spin systems and mapping them to the protein amino acid sequence,
the 3D NCOCX and 3D CONCA experiment are used, establishing inter-residue
correlations between nitrogen atoms and carbon atoms of the preceding residue. The
NCO/CON connects the 15N[i] with 13CO[i-1] through the peptide bond. The 3D CONCA
experiment is essential in the spectral assignment strategy, because it allows matching
cross-peaks in 3D NCACX and 3D NCOCX experiments based on at least two shifts,
greatly increasing the reliability of the matching.
4.1.3.1.1 Automatic assignment of immobile domains by ssFLYA
Manual resonance assignment is generally considered as very arduous. It requires a
considerable amount of time by an experienced spectroscopist. Therefore, automatic
assignment algorithms were developed to fasten the assignment procedure.
Additionally, they can be used as a verification of the already performed assignments.
However, manual resonance assignment persist the standard in solution NMR and the
almost exclusive procedure in solid state NMR due to features or imperfections of
experimental NMR spectra, such as broad linewidth, signal overlap, low sensitivity and
spectral artefacts. These imperfections are usually more pronounced in ssNMR
spectra, increasing difficulties concerning an automated assignment. Only very few
automated algorithms have been developed for solid state NMR resonance
assignment. One algorithm [197] is based on the AutoAssign package for solution NMR
[198] and was used to assign the backbone resonances of GB1 with peak lists from 3D
NCACX, CAN(CO)CA and 4D CANCOCX experiments as input. Another approach
[199, 200] enables the assignment of backbone and side chain resonances by
analysing random combinations of spectra with random dimensions. For the shown
examples, the algorithm uses input peak lists from NCACX, NCOCA and CONCA
spectra. However, this approach holds the drawback that signals must be grouped
together to a spin system and assigned to atom types (e.g., N, Ca, Cb, Cg).
Additionally, possible assignments of the amino acid type must be predefined before
running the algorithm. In this study, ssFLYA, a generally applicable algorithm for
automated backbone and side-chain resonance assignment of protein solid state NMR
spectra [13], is applied. It is predicated on FLYA, an automated resonance assignment
Chapter 4: Resonance assignment
85
algorithm for solution NMR [153] and integrated in the software package CYANA [154,
155]. The ssFLYA algorithm uses solely the protein sequence and unassigned peak
lists from any combination of multidimensional ssNMR spectra. So far, only
microcrystals, such as ubiquitin and the Ure2 prion C-terminal domain as well as
amyloids like HET-s(218–289) and a-synuclein have been successfully assigned by
ssFLYA. In this study, we tested its principal applicability for membrane proteins and
used it for validation of the manually obtained assignments of DGK. For this purpose,
ssFLYA used the dipolar coupling based 3D experiments (NCACX, NCOCX, CONCA).
4.2 Results and Discussion
4.2.1 Spectral resolution and isotope labelling
With the help of the high magnetic field (1H frequency of 850 MHz) and an optimized
sample preparation (chapter 3), well-resolved NMR-spectra of high signal-to-noise ratio
were observed by MAS ssNMR. Thus, the 13C and 15N assignments were mainly
carried out using uniformly labelled samples (U-13C,15N-wtDGK). Here, 15N and 13Ca
signals in the 2D NCA spectra exhibit comparable good average linewidths of
approximately 105 and 185 Hz, respectively (Figure 24). Residual ambiguities could be
resolved by reverse labelling of isoleucine, leucine and valine (U-13C,15N-wtDGK-I,L,V).
Compared to the uniform 13C,15N-labelled sample, which offers a maximum of isotope
labelled sites with 132 labelled residues, the reverse labelled sample contains only 90
labelled residues. Thus, the number of signals in the spectrum of the reverse labelled
sample should be theoretically reduced by 32%, which would be valid for the case that
all labelled residues are visible in the spectrum. Figure 24 shows that in the spectrum
of the reverse labelled sample, the peaks for Ile, Leu and Val are clearly missing
compared to the uniform labelled sample. Apart from that, the two spectra do not
remarkably differ from each other. Overall, this indicates a successful reverse labelling.
Chapter 4: Resonance assignment
86
Figure 24. Comparison of the 2D NCA spectra of uniform labelled U-13
C,15
N-wtDGK (black) and
reverse labelled U-13
C,15
N-wtDGK-I,L,V (green). The 15
N and 13
Ca signals of the uniform
labelled U-13
C,15
N-wtDGK exhibit already comparable good average linewidths of approximately
105 and 185 Hz, respectively. To resolve residual ambiguities, Ile, Leu and Val are specifically
unlabelled in the reverse labelled sample. The inscriptions for Ile, Leu and Val are labelled in
red and for the other amino acids in black. In the 2D NCA of the reverse labelled sample, the
peaks for Ile, Leu and Val are clearly missing as expected. Apart from that, the two NCA spectra
do not remarkably differ from each other.
4.2.2 Paramagnetic doping in combination with an E-free probehead
The obstacle of low intrinsic sensitivity of MAS ssNMR, leading to long signal
accumulation times to gain spectra with sufficient signal-to-noise ratio, could be
diminished by paramagnetic doping with Gd3+-DOTA (Ullrich, Holper et al. 2014) and
an E-free probehead. The E-free probehead enables short recycle delays of 0.8 s,
saving ~3x of the measurement time compared to the standard probehead with a
recycle delay of 2.5 s (Figure 25).
Chapter 4: Resonance assignment
87
Figure 25. 15
N CP spectra of U-13
C,15
N-DGK. The spectra were recorded with an E-free (green)
and with a standard (black) 3.2 mm triple-resonance HCN MAS probehead (Bruker). In both
cases 128 scans were applied. The E-free probehead enables to use a recycle delay of 0.8 s,
saving ~3x of the measurement time compared to the standard probehead with a recycle delay
of 2.5 s. The E-free probehead was custom-built and is still under development.
Next to paramagnetic doping in combination with the E-free probehead, different
strategies for a faster signal build-up are available, such as optimum control pulse
sequences [201] and non-uniform sampling [202-204].
4.2.3 Sequential assignment of immobile domains
Sequential assignment of wild-type DGK was carried out using a combination of dipolar
coupling based 3D experiments, NCACX, NCOCX and CONCA. A representative
sequential walk linking I26 to A29 is shown in Figure 26. By analyzing the three
heteronuclear 3D correlation experiments, we are able to identify, assign and to link the
spin systems, which allow to map them to the protein amino acid sequence. Figure 26
shows 2D planes extracted from the 3D NCACX and NCOCX spectra with a DARR
mixing time of 50 ms and 100 ms, respectively, enabling the detection of long-range
intra- or inter-residue correlations of three- or four-bond carbon-carbon connectivities.
Furthermore, 2D planes of the 3D CONCA experiment are shown permitting the
matching of cross-peaks in the NCACX and NCOCX spectra based on two shifts. Thus,
the CONCA spectrum considerably increases the reliability of the matching. Each set of
three spectra stands for a Cx[i−1]–N[i]–Cx[i] spin system. For example, the N27
NCACX peaks are linked to the I26-N27 CONCA peak through the same N and Ca and
Chapter 4: Resonance assignment
88
the I26 NCOCX peaks are connected to the I26–N27 CONCA peak via the same N and
CO, thus generating a Cx[i−1]–N[i]–Cx[i] system. It is associated with the prior system
through all carbon shifts of I26 that are visible in both NCACX and NCOCX spectra. In
addition to CO[i−1]–N[i]–CA[i] correlations in the CONCA spectrum, we also detected
CO[i−1]–N[i]–Cb[i] correlations. Although these correlations are not necessary for
building spin systems and for the linking process, they provide a quality control in
validating assignments. So-called shuffled peaks in the NCACX spectrum serve as a
validation as well. They originate from N[i]-Ca[i]-CO[i-1]-Cx[i-1] or N[i]-Ca[i]-CO[i]-
Ca[i+1]-Cx[i+1], such as 108AlaN–108AlaCa-107ValCO-107ValCa-107ValCb-
107ValCg1-107ValCg2 or 97GlyN–97GlyCa-97GlyCO-98SerCa-98SerCb-98SerCO,
among others. These peaks can be easily distinguished, since they are much weaker
than those emanating from the one- or two-bond N[i]-Ca[i]-Cb[i] transfers within the
same residue. In addition, they often originate from amino acids with short side chains,
such as glycines, alanines and serines. Shuffled peaks are also visible in the NCOCX
spectrum. They are from the N[i]-CO[i-1]-Ca[i]-Cx[i] type as it is e.g. observable in the
2D plane for E28, which includes, among the peaks for E28, the peaks for Ca and Cb
of A29 as well (Figure 26).
Chapter 4: Resonance assignment
89
Figure 26. Resonance assignment of U-13
C,15
N-wtDGK based on a set of 3D NCACX, NCOCX
and CONCA spectra. A representative sequential walk from I26 to A29 is shown. Each set of
three spectra represents a Cx[i−1]–N[i]–Cx[i] spin system. For example, the N27 NCACX peaks
are connected to the I26-N27 CONCA peak via the same N and Ca. The I26 NCOCX peaks are
linked to the I26–N27 CONCA peak through the same N and CO, resulting in a Cx[i−1]–N[i]–
Cx[i] system, which is linked with the preceding system through all carbon shifts of I26 that are
visible in both NCACX and NCOCX spectra. The assignments are depicted by lines.
The backbone and most side chain carbon and nitrogen resonances could be assigned
for ~82% of the residues (= 99 residues), from which 73 residues (~74% of the
assigned residues) are completely assigned. Assigned resonances are highlighted in a
2D NCA spectrum (Figure 27).
Chapter 4: Resonance assignment
90
Figure 27. 2D NCA spectrum of U-13
C,15
N-DGK with assigned peaks labelled.
4.2.3.1 Automatic assignment of immobile domains by ssFLYA
For validation, ssFLYA, a generally applicable algorithm for the automatic assignment
of protein solid state NMR spectra [13], was applied. ssFLYA was performed by Dr.
Sina Kazemi of the research group of Prof. Dr. Peter Güntert (Institute for Biophysical
Chemistry, Goethe University Frankfurt am Main). So far, it has been used for
microcrystals and amyloids [13]. In this study, its principal applicability for demanding
systems as membrane proteins could be demonstrated for the first time. Overall, 91.5%
of the backbone and 89.1% of all (backbone + side chains) assignments could be
confirmed by ssFLYA, verifying the manually obtained assignments. A full overview of
the assignment is provided in Figure 28. However, few incorrect assignments with
respect to the manual assignment appeared as well. They occur for residues close to
the termini or loops, which are known to be dynamic, yielding very weak signals (G15-
W18, S84, E88) or are probably not observable in the spectra at all (E85-H87, S118-
G121). Remote side chain atoms feature less intense signals and thus lead to incorrect
assignments as well. Further incorrect assignments could originate from spectral
Chapter 4: Resonance assignment
91
artifacts or missing peaks due to overlapped regions. Thus, for the automatic
assignment of DGK by ssFLYA, careful manual peak picking with knowledge about the
spectra is a prerequisite. The percentage of strong assignments depended on the
quality of the input peak lists. Known spectral artifacts like folded peaks had to be
removed from the input peak lists, since the algorithm could not distinguish between
folded peaks and correct ones, leading to a disturbed calculation and hence to overall
incorrect assignments. Especially, the peaks for R32, R81, R92 and K94, arising from a
magnetization transfer starting from the side chains, which are additionally folded in,
led to incorrect assignments by ssFLYA. However, ssFLYA features the big advantage
of accelerating the time-consuming assignment process. It is able to perform an
assignment calculation within approximately 10 min, if 20 CPU cores are availabe.
Thus, an implementation of ssFLYA into the manual assignment procedure lead to a
faster and more reliable assignment.
Figure 28. Automated resonance assignment by ssFLYA confirms 91.5% of the backbone and
89.1% of all (backbone + side chains) assignments obtained manually. Assignments are
classified as strong, if ≥ 80% of the individual chemical shift values from 20 independent runs of
the algorithm differ by less than 0.55 ppm from the consensus value (strong colors). Other
assignments by ssFLYA are graded as weak (light colors). From other studies by ssFLYA, they
are known to be erroneous for 39 – 72% [13]. Each assignment for an atom is symbolized by a
colored rectangle: green - assignment by ssFLYA agrees with the manual reference assignment
within a tolerance of 0.55 ppm; red - assignment does not match with the reference; blue -
assigned by ssFLYA, but not manually; black – assigned manually, but not by ssFLYA. The
second row illustrates backbone assignments for N, Ca, and CO. The third to eighth row
represent the side chain assignments. For branched side chains, the relevant row is subdivided
into an upper part for one branch and a lower part for the other branch. ssFLYA was performed
by Dr. Sina Kazemi of the research group of Prof. Dr. Peter Güntert (Institute for Biophysical
Chemistry, Goethe University Frankfurt am Main). He also kindly provided this figure.
Chapter 4: Resonance assignment
92
4.2.4 Tentative assignment of highly mobile regions
Since not all residues could be detected in CP-based experiments, scalar coupling
based spectra were recorded as well. Comparable to the data reported for the
thermostable DGK mutant [149], some mobile residues could so be monitored. 1H-15N
HETCOR, 1H-13C HETCOR and 13C-13C TOBSY spectra (Figure 29) were recorded,
showing well-resolved peaks of “solution-state”-like quality due to motions on the
submicrosecond time scale. Most of the peaks could be assigned to types of amino
acids based on the BMRB database [205], such as Ala, Arg, Asn, Gly, His, Ile, Leu,
Lys, Phe and Thr. Hence, these peaks could be tentatively assigned to the two termini
and the cytosolic loop between helix 2 and 3, where residues occur that could not be
detected by dipolar coupling based experiments. Peaks for arginine and lysine could be
assigned unambiguously to Arg9 and Lys12 by exclusion, as all other arginines and
lysines are already assigned by experiments based on dipolar coupling.
Chapter 4: Resonance assignment
93
Figure 29. 2D scalar coupling based 1H-
15N HETCOR (a),
1H-
13C HETCOR (b) and
13C-
13C
TOBSY (c) of U-13
C,15
N-DGK with tentative assignments. All residues, which could not be
detected and assigned by dipolar coupling based experiments are considered as possible
candidates for detection by experiments based on scalar coupling. INEPT and TOBSY were
applied for 1H-
15N or
1H-
13C heteronuclear polarization and
13C-
13C homonuclear mixing,
respectively. Peaks for Arg9 and Lys12 are labelled green, as they could be assigned
unambiguously. Peaks for the aromatic rings were folded in the indirect dimension to save
measurement time. Amino acids that refer to the His-tag are labelled by ‘tag’.
Chapter 4: Resonance assignment
94
4.2.5 Summary of the assignment
With the careful optimization of the sample preparation (chapter 3) and the used NMR
strategy, 84% of the residues (= 101 residues) located in transmembrane and
extramembranous regions could be assigned by dipolar and scalar coupling based
experiments. The assigned chemical shifts have been deposited to BioMagResBank
(BMRB entry 27570). All assignments are plotted in Figure 30a and the assigned
residues are mapped on the topology of DGK (Figure 30b). Additionally, they are
itemized in Table S5. The residues of the termini and the cytosolic loop were not or
only tentatively assigned, since they were too mobile for dipolar- but not mobile enough
for scalar coupling based experiments.
Figure 30. Resonance assignment of DGK. (a) Each assignment for an atom is symbolized by a
blue rectangle: The second row illustrates backbone assignments for N, Ca, and CO. The third
to eighth row represent the side chain assignments. For branched side chains, the relevant row
is subdivided into an upper part for one branch and a lower part for the other branch. This figure
was kindly provided by Dr. Sina Kazemi of the research group of Prof. Dr. Peter Güntert
(Institute for Biophysical Chemistry, Goethe University Frankfurt am Main). (b) The assigned
residues are mapped on the topology plot of DGK. The plot was created with respect to the X-
Chapter 4: Resonance assignment
95
ray structure of DGK (PDB 3ZE5) [5] and refined by CSI values obtained from chemical shifts
(Table S5). The membrane is depicted by two solid black lines. 84% residues of DGK were
assigned by dipolar and scalar coupling based experiments.
4.2.6 Secondary structure analysis
Based on the resonance assignment, the secondary structure of DGK was calculated
by chemical shift index (CSI) analysis. Figure 31a displays the CSI Δδ as a function of
the residue number, in which Δδ stands for the deviation of the experimentally
determined MAS NMR chemical shifts (exp) for Ca and Cb from their random coil
standard chemical shifts (rc) according to the following equation [206]: Δδ =[δCa(exp)-
δCa(rc)] - [δCb(exp)-δCb(rc)]. For Gly residues and residues without any assignment of
Cb, only Ca secondary shifts were considered. Strongly positive (≥ 1.5 ppm) values of
the CSI indicate an α-helical structure, whereas negative or near-zero values imply
deviations from helicity. Figure 31a shows the comparison of the secondary structures
of wild-type DGK, its thermostable mutant [14] determined both by MAS NMR and the
crystal structure [6] of wtDGK (PDB 3ZE4, chain A). All three secondary structures
exhibit substantial similarities, particularly regarding the high α-helical content.
However, there are some differences, too. In contrast to both MAS NMR secondary
structures, the crystal structure features small deviations around the flexible regions:
the interhelical turn (T) between helix 1 (H1) and the surface helix (SH), the periplasmic
loop (PL) between helix 1 (H1) and helix 2 (H2), as well as the cytoplasmic loop (CL)
between helix 2 (H2) and helix 3 (H3). In subunit A of the crystal structure, the position
of T and PL is slightly displaced upstream by two residues in comparison with the MAS
NMR structures. Furthermore, T is one residue longer and PL one residue shorter in
the X-ray structure than in the MAS NMR structures. Regarding the position and/or
length of the CL, all three structures differ from each other. CL is shifted from the
residues 83-87 in the MAS NMR structure of wtDGK to the residues 81–85 of the
thermostable mutant and to the residues 83–90 (subunit A) of the X-ray structure.
However, it has to be endorsed that the positions and lengths of the non-helical
structures even vary between the three different subunits A, B and C within the X-ray
structure itself [5, 14]. Especially the CL ranges from residue 83 to 90 (subunit A), 86 to
91 (subunit B) and 82-87 (subunit C) within one trimer.
Chapter 4: Resonance assignment
96
Figure 31. Secondary structure analysis based on the chemical shifts. The chemical shift index
(CSI) Δδ is derived from the difference between the experimentally determined MAS NMR
chemical shifts (exp) for Ca and Cb and their random coil standard chemical shifts (rc)
according to Δδ =[δCa(exp)-δCa(rc)] - [δCb(exp)-δCb(rc)] [206]. For Gly residues and residues
without any assignment of Cb, only Ca secondary shifts were used. Strongly positive
(≥ 1.5 ppm) values of the CSI imply an α-helical structure, whereas negative or near-zero values
indicate deviations from helicity. (a) The secondary structure of wild-type DGK determined by
MAS NMR is compared with the MAS NMR structure of the thermostable mutant [14] and the X-
ray structure of wtDGK (PDB 3ZE4, chain A) [6]. Rectangles represent α-helical regions
involving the surface helix (SH) and the three transmembrane helices (H1-3), whereas solid
lines symbolize deviations from helicity including the interhelical turn (T), the periplasmic (PL) as
well as the cytoplasmic loop (CL). Residues that were not resolved by ssNMR or by X-ray
crystallography are depicted by dashed lines. Disparities between the three secondary
structures are highlighted in green. (b) The 2D NCACX spectrum of U-13
C,15
N-DGK shows all
assigned glycines. The regions for helical and random coil (rc) secondary structure are
coloured. Gly83 and Gly91 are labelled bold, since the DGK X-ray structure exhibits
asymmetries for both residues: Both were observed within a helical and a random coil structure
[5]. These asymmetries are not detectable by MAS NMR.
4.2.7 DGK forms a symmetric trimer in its apo state
During the assignment procedure, no systematic peak doublets or triplets were
observed, indicating that the DGK trimer adopts a symmetric conformation in its apo
state. This is already to some extent visible in the 2D NCA spectrum, e.g. for well-
resolved peaks, such as Gly20, Asn27, Ala29, Glu88, Ala99 and Trp112 (Figure 27).
This is opposite to the X-ray structure of nucleotide-free DGK, possessing asymmetries
in the secondary structure between the three subunits as already mentioned above [5,
Chapter 4: Resonance assignment
97
14]. The DGK X-ray structure [5] features asymmetries for instance for Gly83 and
Gly91, which were determined both within a helical and a random coil structure in the
DGK trimer. By MAS NMR, no hints for more than one peak for Gly83 and Gly91 are
visible (Figure 31b). The deviations regarding the conformational symmetry of DGK
between MAS NMR and X-ray data might occur due to different experimental
conditions. In this connection, particularly crystal packing may be a potential source for
structural asymmetries [207]. Additionally, radiation damage such as decarboxylation of
glutamates and aspartates could lead to the loss of salt bridges [208]. This in turn may
lead to partial instabilities and thus partial slow movements within the protein molecule
despite the frozen state [208], causing conformational inhomogeneities. A possible salt
bridge, which might be partly lost, would be between Arg81 and Glu88 as shown in
chapter 6 (Figure 42a), whereupon Glu88 is supposed to be in the cytosolic loop [5]. In
general, a high mobility of the loop regions, which is reflected by higher B-factors and
chain displacements [5] may cause ambiguities regarding the definition of loop position
and length.
4.3 Outlook
4.3.1 Further labelling strategies
For an unambiguous assignment of larger regions, suffering from spectral overlap,
and/or the determination of distance restraints, position-specific labelling might be a
proper solution. It is based on [1,3-13C]-glycerol or [2-13C]-glycerol, which replace
glucose in the expression medium [209]. Amino acids that originate from the glycolytic
and pentose phosphate pathways (Ala, Cys, Gly, His, Leu, Phe, Ser, Trp, Tyr, Val)
have all sites either 13C or 12C labelled in almost all cases according the “all-or-nothing”
principle. The remaining amino acids are synthesized from precursors that participate
in the citric acid pathway and cause non-random mixtures of isotopomers (mixed
labelling). This way, amino acids can be allocated to sub-groups, which show similar
labelling patterns. The assignment strategy uses these characteristic labelling patterns
for the different amino acid types to identify spin systems in the spectra of [1,3-13C]-
glycerol, [2-13C]-glycerol and [U-13C]-labelled samples. These spin systems are then
linked and mapped to the amino acid sequence of the respective protein. [1,3-13C]- and
[2-13C]-glycerol labelling reduces signal overlap and improves spectral line width. The
applicability of this assignment strategy has been demonstrated for the microcrystalline
chicken α-spectrin SH3 domain (62 residues) [209], the αB-crystallin (175 residues)
[210] and the outer membrane protein G, OmpG (281 residues) [211]. Other extensive
Chapter 4: Resonance assignment
98
selective labelling schemes utilized [1,4-13C], [2,3-13C], and [1,2,3,4-13C] succinic acid
based samples [212, 213]. Fractional [U-13C]-glucose labelling [214, 215], labelling with
[1-13C]-glucose [216] or [2-13C]-glucose [217] represent alternative labelling approaches
as well. They might be suitable for proteins with specific amino acid type compositions.
For a detailed look on certain residues, still suffering from signal overlap, selective
labelling (including labelling of unique pairs) could be used. Here, single isotope
labelled amino acid(s) are added to the growth medium, which are directly incorporated
in the protein during expression in E.coli. One major drawback arises from amino acid
scrambling. This decreases the labelling efficiency on the target site and leads to
additional signals in the spectrum, complicating data analysis. Here, as mentioned
above, amino acids, which are end products of the bacterial metabolic cycle and
therefore not converted into other amino acids, have to be used to reduce scrambling
to a minimum. This restricts the applicability of selective labelling on following amino
acids: Arg, Cys, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, Tyr and Val.
In order to still apply selective labelling on the other amino acids without the risk of
scrambling, E.coli based cell-free expression could be tested as an alternative to in
vivo expression [218, 219]. It allows almost any amino acid labelling scheme due to the
lack of a cellular metabolism as a source of amino acid scrambling [220]. Furthermore,
this in vitro system exhibits an attractive option, since high yields can be obtained while
only low amounts of expensive isotope labelled amino acids are needed. Additionally,
experimental parameters such as pH, redox potential or co-factor dependence can be
better controlled compared to in shake flask expression [218, 219]. Another advantage
of this method is its highly timesaving character: The cell-free produced protein
typically needs no further purification as compared to in vivo expression [220].
However, protein expression is a highly complex, evolutionarily optimized procedure.
Thus, there exist a risk for certain systems that only little amount can be obtained by
cell-free expression or the optimization process takes too much time. In these cases,
1H detection in combination with ultra-fast MAS might be an answer. It allows the
investigation of sub-milligram amounts of protein (0.7 mm probe, ~0.5 mg sample) as
explained in the following section.
4.3.2 Perspective: 1H detection in combination with ultra-fast MAS
Since molecular tumbling is suppressed in membrane proteins, their NMR spectra are
broadened by strong anisotropic interactions. Moderate magic angle spinning (MAS)
[221, 222] in combination with high power decoupling [223] is applied to average out
Chapter 4: Resonance assignment
99
these interactions and to re-establish high resolution for low gamma nuclei like 13C or
15N. As demonstrated in this study, this approach is very successful and commonly
used. However, protons would offer a much greater detection sensitivity than those of
13C or 15N, since they feature a natural abundance of more than 99.9% and a high
gyromagnetic ratio. Unfortunately, the linewidths of protons remain immensely broad at
moderate MAS frequencies (10–20 kHz), because of the strong inter-proton dipolar
couplings. Remarkable engineering progress [224-228] and improvements of sample
preparations [229-232] have facilitated 1H-detection in many systems with limited
mobility, and have turned it to an important technique for increasing sensitivity and
resolution these days. 1H detection in ssNMR is enabled by very fast spinning rates
(>40 kHz) and high magnetic fields, in order to diminish homogeneous line broadening
by suppressing the large network of strong homonuclear 1H-1H dipolar couplings [181,
233-242]. Higher spectral resolution can be obtained partly by proton dilution strategies
as well, such as perdeuteration and back-protonation at the exchangeable sites [231].
Admittedly, perdeuteration might be problematic during protein expression, because of
anemic growth in deuterium oxide, which sometimes is even incompatible with protein
expression, like for example in mammalian cells. When feasible, it allows reintroduction
of 1H species only at sites that are exchangeable and accessible to solvent. This
excludes the large hydrophobic transmembrane regions from analyses by 1H-detected
experiments [243]. For certain systems, unfolding and refolding of membrane proteins,
would be a solution. This allows the incorporation of 1H species in transmembrane
regions. However, such protocols are rare and not applicable to all proteins [181, 234,
241, 244]. In order to overcome this obstacle at least in part, membrane proteins are
expressed in H2O in the presence of deuterated 13C glucose, leading to 1H/2H species,
which are homogeneously distributed in both water-accessible and inaccessible
regions [245]. But this labelling strategy holds the drawback of poorly resolved 13C
resonances from side-chain moieties, which are essential for structure determination.
Consequently, extensive deuteration may not always be a realizable approach.
However, this obstacle can be solved by radiofrequency probes with spinning rates
greater than 100 kHz [228], permitting NMR studies of fully protonated samples. With
increasing MAS, the proton linewidths in proteins decrease linearly, enhancing
resolution and sensitivity. With MAS rates >100 kHz, proton linewidths are sufficiently
narrow to resolve most resonances in a two-dimensional spectrum, involving a proton
dimension for detection. Increasing the spinning frequency to even higher values would
narrow the resonance lines further and would allow additional improvements in
spectroscopic methods and achievable resolution. It is assumed that at 250 kHz MAS
the homonuclear dipolar couplings are effectively averaged out for fully protonated
Chapter 4: Resonance assignment
100
proteins and solution-state like spectra can be obtained [240]. 1H detection in
combination with fast MAS allow high resolution solid state NMR of membrane proteins
as well. A number of structural studies could demonstrate its applicability for both β-
barrel and α-helical membrane proteins in lipids and 2D crystal preparations [234, 241,
246-251]. Here, the work of Pintacuda and co-workers is outstanding [251]. They
demonstrated the applicability of 1H detection in combination with ultra-fast MAS at
100 kHz on the fully protonated hepta-helical membrane protein proteorhodopsin (PR)
in native-like lipids (DMPC/DMPA). Their approach provides 1H-based sequential
assignments and the identification of long-range interhelical 1H−1H contacts between
the side chains in transmembrane regions. Thus, this work represents a step towards
structure determination of membrane proteins by 1H detected ssNMR. Additionally,
ultra-fast MAS allows the investigation of sub-milligram amounts of protein (0.7 mm
probe, ~0.5 mg sample), which is especially beneficial for systems, suffering from low
yield. An additional benefit of fast spinning is the applicability of low-power pulse
sequences improving the electrical stability of the probeheads, as well as greatly
reducing rf heating of the sample.
For U-13C,15N-wtDGK in phospholipids (DMPC/DMPA), very first 1H detected
experiments were carried out as well. 2D 1H-15N correlation experiments (2D hNH)
[252] were performed on a Bruker 600 MHz spectrometer at ~4°C and a MAS rate of
111 kHz (Bruker 0.7 mm rotor, ~0.5 mg sample). They were conducted by Dr. Venita
Decker at Bruker BioSpin GmbH in Rheinstetten. The 2D hNH experiment is
comparable to a solution NMR HSQC (Heteronuclear Single Quantum Correlation)
experiment, but with the difference that it is based on CP. The initial 1H magnetization
is transferred to 15N via a HN-CP step. The 15N signal evolves under 1H decoupling.
Using an H/C/N triple channel probe, 13C-decoupling can be used as well. Water
suppression is carried out with a MISSISSIPPI scheme [253], which is applied on-
resonance with the water peak. The 15N magnetization is transferred back to 1H via a
NH-CP step, which is followed by 1H-detection under low power decoupling on 15N, and
possibly 13C. Hence, the 2D hNH experiment correlates the amide proton to its
nitrogen. Figure 32 demonstrates its principle applicability. It is noteworthy that with
only ~0.5 mg sample a comparatively high signal intensity can be obtained within
~12 h. This clearly highlights 1H detection at ultra-fast MAS as an important technique
for increasing sensitivity. For few, well-resolved peaks, the assignments from 13C/15N
detected experiments (Table S5) performed under moderate MAS (15.2 kHz) could be
transferred. However, further optimization steps, ranging from rotor packing to
appropriate pulse optimizations, are still needed to gain a higher signal-to-noise ratio
e.g. for recording three or higher dimensional spectra.
Chapter 4: Resonance assignment
101
Figure 32. Solid state NMR 1H detected 2D
1H-
15N correlation spectrum (2D hNH) of fully
protonated U-13
C,15
N-DGK in phospholipid bilayers. The spectrum was recorded on a Bruker
600 MHz spectrometer at ~278.15-283.15 K and a MAS rate of 111 kHz (Bruker 0.7 mm rotor,
~0.5 mg sample). It was conducted with 400 scans and a duty cycle of 0.8 s. The total
measurement time was ~12 h. Some, well-resolved residues from the extramembrane regions
(green) could be assigned based on the assignments from 13
C/15
N detected experiments
conducted at a MAS rate of 15.2 kHz (Table S5). The spectrum was recorded by Dr. Venita
Decker at Bruker BioSpin GmbH in Rheinstetten.
If the further optimization turns out to be successful, high quality data could be
collected, allowing 1H-based sequential assignments and the identification of 1H-1H
proximities, as shown for PR [251]. This would help to define a 3D structure of DGK by
MAS NMR. Additionally, well-resolved spectra would allow automated data analysis as
already used for solution NMR studies, resulting in a reliable and especially in a faster
assignment and structure determination.
Direct proton detection might be useful to selectively observe the mobile entities in a
fully protonated sample as well. With the application of INEPT-based spectroscopy,
many protein resonances with intrinsically narrow linewidths could be already
selectively observed (Figure 29). However, these resonances could only be tentatively
assigned. With the implementation of direct proton detection, the sensitivity can be
increased. Sensitivity enhancements of up to tenfold have been already reported for 2D
1H–13C INEPT HSQC experiments, when proton detection is compared to carbon
detection [248]. In addition, Ward et al. could show that proton-detected experiments
Chapter 4: Resonance assignment
102
can be easily extended to three dimensions through the incorporation of proton–proton
mixing. This step facilitates the detection of side chain protons, extending the spin
systems by the proton chemical shifts. Additionally, it provides the possibility of the
determination of inter-residue correlations, allowing a sequential assignment of the
mobile regions. Concerning DGK, especially the cytosolic loop, which is invisible in
dipolar coupling based experiments, would be of great interest, since it is reported to
be involved in nucleotide binding [6]. Changes in dynamics between apo and
nucleotide bound state might be observable.
In general, DGK could serve as a model system for 1H detection on α-helical
membrane proteins in phospholipids for further methodical developments of the
technique. Further tests would clearly benefit from DGK’s high stability.
Chapter 5: Functional studies
103
5 Functional studies based on chemical shift perturbations
5.1 Introduction
DGK catalyzes the ATP-dependent phosphorylation of diacylglycerol (DAG) to
phosphatic acid (PA) at the membrane/cytoplasm interface. Over the last decades, an
immense data set has been collected to find out, how this unique and complex enzyme
accomplishes this reaction:
Two 3D structures have been published for DGK: one obtained by solution NMR in
dodecylphosphocholine (DPC) micelles [7] and one by 3D crystallization in lipidic cubic
phases (LCP) composed of monoacylglycerols (MAGs) [5]. Additionally, several studies
were carried out using different techniques to map the active site. Functionally relevant
residues were identified by mutational studies [6, 7, 10], X-ray crystallization [6], MD
simulations [11] and solution NMR [7, 10]. Furthermore, based on observations from
the X-ray structures in combination with mutational studies [6, 7, 10] and MD
simulations [6], Caffrey and co-workers proposed a catalytic mechanism for DGK [6].
Despite this valuable data set, important long-standing questions regarding DGK’s
catalytic mechanism remain unsolved. It is unknown yet whether the three active sites
of DGK are in same or different states during catalysis and whether DGK undergoes a
substantial conformational change prior to the actual phosphoryl transfer.
In this chapter, these questions are addressed by multidimensional high field 13C,15N
MAS NMR. A considerable advantage of NMR as a technique for structural and
functional analysis is that it enables the investigation of membrane proteins without
attenuating its structural plasticity that is in most cases integral to the biological
function. This is contrary to X-ray studies on DGK, which stabilize a single molecular
conformation by the crystallization process itself and by the application of cryogenic
temperatures. Another advantage of solid state MAS NMR is the possibility of
performing experiments directly within the lipid bilayer [254], which brings it closer to
physiological conditions compared to other membrane mimicking environments such
as detergent micelles. The membrane environment is of key importance as it is a
strong structural factor [11, 19-21]. It is also directly linked to the catalytic activity of
DGK [11, 19]. Thus, MAS NMR can provide highly complementary data to X-ray
crystallization and solution NMR studies.
The apo state of DGK is compared with the substrate bound states on the basis of the
nearly complete assignment of DGK (84%), shown in chapter 4. Perturbations in peak
position and intensity of the substrate bound states were analysed for each of the
101 assigned residues in 3D and 2D heteronuclear correlation spectra. Substrate-
Chapter 5: Functional studies
104
induced chemical shift perturbations indicate structural changes while alterations in
peak intensities can be interpreted qualitatively in terms of altered dynamics: An
increase in mobility causes a reduction in peak intensities in experiments based on
cross polarisation. The nucleotide-bound state is based on
adenylylmethylenediphosphonate (AMP-PCP), a suitable non-hydrolysable ATP
analogue, which has been already used in solution NMR and X-ray crystallization
studies [6, 7]. The DAG-bound state is emulated by 1,2-dioctanoyl-sn-glycerol (DOG,
chain length n=8), which has been reported to act as a lipid substrate for DGK [107,
255].
5.2 Results
5.2.1 Establishing nucleotide- and DAG-bound states of DGK for NMR analysis
In order to find saturation conditions for DGK with Mg*AMP-PCP, a competitive
Mg*ATP inhibition assay was performed by monitoring the ATPase activity as a
function of Mg*AMP-PCP concentration (Figure 33a). It turned out that a concentration
of at least 10 mM of Mg*AMP-PCP (10-fold molar excess) is necessary to reduce
DGK’s activity below 10%, leading to saturation. The comparatively high Mg*AMP-
PCP-to-protein ratio, needed to gain saturation, is indicative of low-affinity nucleotide
binding. This finding is consistent with titration studies on DGK by solution NMR [7].
Additionally, 1H-31P cross polarization (CP) experiments were carried out, which show
that AMP-PCP binds to DGK under these conditions (Figure 33b). Furthermore, it could
be demonstrated that the fully saturated system is stable over at least 30 d at 4°C
without any significant evidence of degradation (Figure 33c). This ensures long MAS
NMR experiments such as 3D NCACX experiments with measurement times of up to
14 d.
Chapter 5: Functional studies
105
Figure 33. DGK in the AMP-PCP bound state. (a) Competitive inhibition assay verifies the
binding of Mg*AMP-PCP to the active sites of DGK. DGK proteoliposomes were incubated with
4 to 16 mM of Mg*AMP-PCP, equating 4 to 16-fold molar excess compared to DGK. Mg*ATP
(3 mM) was present in each sample. A concentration of at least 10 mM of Mg*AMP-PCP (10-
fold molar excess) is needed to decrease the activity of DGK below 10%, resulting in a fully
saturated system. 100% activity corresponds to the rate recorded with wtDGK in 90mol%
DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1
mg-1
. Experiments were repeated three times.
The activity was calculated as the mean value. Error bars correspond to standard deviations.
(b) The 31
P-CP MAS spectrum confirms the binding of AMP-PCP. For this purpose,
proteoliposomes were incubated with 14 mM Mg*AMP-PCP (pH 7.2). (c) The 2D NCA spectra
of U-13
C,15
N-DGK-I,L,V incubated with 14 mM Mg*AMP-PCP (pH 7.2), recorded immediately
after the incubation (black) and after 30 d (green), show that the fully saturated system is stable
over a long period of time without any significant evidence of degradation.
In order to generate a state of DGK with bound lipid substrate, the protein was
reconstituted into DMPC/DMPA liposomes, including 20 mol% DOG [107, 255].
Empirically, it turned out that 20 mol% of DOG is an applicable amount to keep the
liposomes still intact. In order to test, whether DOG can reach the active site, MgATP
was added to the sample. This way, DGK could transfer the phosphoryl group from
ATP to DOG, yielding DOG-PA. Phosphorylated DOG should then become visible in
31P spectra. Indeed, the DOG-PA signal could be monitored in 31P-cross polarization
(CP) and direct polarization (DP) spectra (Figure 34a). The DP spectrum shows all 31P-
species such as α- and β-ADP as well as DOG-PA. The CP spectrum on the other side
only displays membrane-bound 31P-species such as DOG-PA and phospholipids. Both
Chapter 5: Functional studies
106
the DP and CP spectra prove that DOG reaches the active sites of DGK in our
preparations. The same conditions were used to prepare a sample, in which DGK was
saturated with both Mg*AMP-PCP and DOG (80 mol% DMPC/DMPA and 20 mol%
DOG). In this case, DOG could not be phosphorylated, but bound Mg*AMP-PCP could
be observed as described above (Figure 34b).
Figure 34. (a) DGK in the DOG bound state. DGK was reconstituted into 80 mol%
DMPC/DMPA and 20 mol% DOG and incubated with 14 mM Mg*ATP (pH 7.2). DGK
phosphorylates DOG to DOG-PA, which can be observed by 31
P-MAS NMR, both by cross- and
direct polarization. The spectra prove that DOG can reach the active site of DGK under the here
applied experimental conditions. (b) DGK in the DOG+AMP-PCP bound state. 31
P-CP spectrum
of DGK reconstituted into 80 mol% DMPC/DMPA and 20 mol% DOG and incubated with 14 mM
Mg*AMP-PCP (pH 7.2). It illustrates 31
P species of the bound AMP-PCP, which demonstrates a
binding of the nucleotide to DGK.
5.2.2 DGK forms a symmetric trimer in its substrate bound states
Under the applied experimental conditions, no peak splitting could be detected for the
AMP-PCP and the DOG bound state during the assignment process. This is, as for the
apo state, already partly observable in the 2D NCA spectra, e.g. for well-resolved
peaks, such as Gly20, Asn27, Ala29, Glu88, Ala99 and Trp112 (Figure 35a, b). This
implies a symmetric conformation of DGK in its substrate bound states. Especially,
Gly83 and Gly91, framing the cytosolic loop, which is reported to be involved in
nucleotide binding [6], act as perfect probes for conformational asymmetries induced
by AMP-PCP. Clearly, both do not split in the AMP-PCP bound state (Figure 35a): Only
one peak for Gly83, indicating a random coil structure and one peak for Gly91,
suggesting a helical structure, could be detected. These findings support the
Chapter 5: Functional studies
107
assumption that the DGK trimer adopts a symmetric conformation in the AMP-PCP
bound state.
Figure 35. The DGK trimer adopts a symmetric conformation in its substrate bound states. (a)
Superposition of 2D NCA spectra of apo DGK (black) and AMP-PCP-bound DGK (green).
Regions for helical and random coil (rc) secondary structure are highlighted for glycines. (b)
Superposition of 2D NCA spectra of apo DGK (black) and DOG-bound DGK (yellow). For the
AMP-PCP and the DOG bound state no peak splitting can be observed.
5.2.3 Substrate-induced chemical shift and peak intensity perturbations
5.2.3.1 AMP-PCP bound state
In the AMP-PCP bound state, significant chemical shift perturbations (CSPs ≥ 0.2 ppm)
and alterations in peak intensities are visible for 57% of the assigned residues,
including backbone and side chains (Figure 36a-c, Table S6). Previous mutational
studies have identified the following residues of DGK to be catalytically relevant: T8,
R9, A13, S17, G20, E28, A30, F31, R32, E34, E69, N72, S73, E76, D80, R81, G83,
L89, S90, A93, K94, D95, G97, S98 and A100 [7, 256]. With the studies by MAS NMR
using heteronuclear 2D and 3D correlation experiments, 14 out of these 25 residues
could be confirmed to be affected by nucleotide binding, featuring significant CSPs
and/or changes in peak intensities (labelled bold). Additionally, further 43 residues
Chapter 5: Functional studies
108
could be identified: G15, Y16, K19, R22, A23, W25, I26, N27, A29, Q33, A37, L40,
A41, V43, A45, C46, W47, L48, D49, V50, D51, I53, R55, V56, L57, V62, V65, M66,
I67, I70, A74, A77, V79, I82, E88, G91, R92, A99, V101, L102, T111, I114 and L116.
Affected residues, showing significant CSPs and/or perturbations in peak intensity, are
highlighted in the DGK topology plot and mapped on the 3D structure in Figure 36d.
The picture illustrates that binding of the nucleotide has a higher impact on DGK as it
was assumed so far [7]. Largest CSPs ≥ 0.4 ppm are visible mainly for residues of the
cytosolic region: E28, F31, R32, Q33, E69, I70, V79, R81, I82, G91 and K94, but also
for the periplasmic loop: D49, I53 and for the transmembrane region: A45, V62. For
V62, D80 and K94. Representative sections from the 3D NCACX spectra are shown in
Figure 36c. Especially, glycines turned out to be sensitive to AMP-PCP binding.
Significant chemical shift perturbations could be detected for Gly15, Gly20, Gly91 and
Gly97 and an increased peak intensity was observed for Gly20 and Gly83 (Figure 36a,
b). Particularly, Gly83 located in the cytosolic loop (CL) features a notable increase in
signal intensity, suggesting a reduced mobility of CL. This is consistent with X-ray data,
implying a participation of the cytosolic loop in nucleotide binding via Glu85, Tyr86 and
His87 [6]. However, the most pronounced effect could be observed for Gly91, which is
present in the extramembranous part of helix 3. It shows a high shift of 1.7 ppm in the
N dimension to a lower ppm value. A reason could be the close proximity to the purine
ring of AMP-PCP [6], resulting in a higher shielding of 91GlyN and that in turn would
cause a shift to a lower ppm value in the N dimension. Overall, glycines act as perfect
sensors for nucleotide binding and accompanied conformational and dynamical
changes in the cytoplasmic region.
Chapter 5: Functional studies
109
Figure 36. Effect of nucleotide binding on DGK. (a) Superposition of 2D NCA spectra of DGK’s
apo (black) and AMP-PCP-bound (green) state. Representative pronounced shifts are
illustrated in subsections of 2D NCACX (b) and 3D NCACX (c) spectra. (d) The topology and
ribbon model of the DGK monomer are shown with residues highlighted that are affected by
AMP-PCP. In the topology maps, alterations in peak intensity and different levels of weighted
CSPs are distinguished. In the ribbon model of monomeric DGK, residues, which show a
response on AMP-PCP binding, are highlighted in green. The ribbon model is obtained from the
OPM database [116], using the PDB ID 4UXX from the X-ray structure [6].
5.2.3.2 DOG bound state
In the DOG bound state, overall CSPs and dynamical changes are considerably less
pronounced compared to the AMP-PCP bound state. Only 14% of the assigned
residues and mainly their side chains are affected (Figure 37a-c, Table S6). CSPs
Chapter 5: Functional studies
110
≥ 0.3 ppm are visible in side chain nuclei of K19, Q33, E69 and I70. Changes in peak
intensities appear more often, implying dynamical changes, which occur mainly in the
surface helix as shown in Figure 37d. These structural and dynamical changes might
take place due to a specific direct response on the lipid substrate in the active site, but
could be also caused by changes of the membrane due to 20 mol% of DOG, which is
inserted into the lipid bilayer. However, based on biochemical and structural data [6, 7,
11, 256], the perturbations concerning Arg9, Gln33 and Glu69 are likely related to
specific interactions with the lipid substrate bound to the active site. Glu69 is reported
to be conserved and relevant for the catalytic function of DGK. It is depicted to directly
interact with the lipid substrate [7, 256]. Its side chain peaks are raised during DOG
binding, which is especially observable for Cd. Furthermore, Cb shows a clear CSP
(Figure 37b). Both the signal increase and the CSP imply an interaction of the Glu69
side chain with the lipid substrate. Gln33, which features a significant CSP ≥ 0.3 ppm
for Cg (Figure 37b), is located in the active site [6] as well. Hence, it might be also a
possible interacting partner for DOG. Moreover, Arg9 and Lys12 that are located on the
surface helix are affected by DOG as well. Both, which are identified as highly mobile
residues in scalar coupling based experiments, are significantly reduced in the DOG
bound state compared to the apo state (Figure 37c), which is indicative for a decrease
of mobility. Arg9 has been already reported to be catalytically important, directly
interacting with the lipid substrate [6, 7, 11, 256].
Chapter 5: Functional studies
111
Figure 37. Effect of DOG binding on DGK. (a) Superposition of 2D NCA spectra of DGK’s apo
(black) and DOG-bound (yellow) state. (b) Representative extractions from the 3D NCACX
illustrate shifts for Glu69 and Gln33. (c) Superposition of 15
N and 13
C INEPT-based experiments
of the apo (black) and DOG-bound (yellow) state of DGK. In the DOG bound state, the INEPT
signals are decreased compared to the apo state, indicating a reduction in mobility. Arg9 and
Lys12, which could be assigned unambiguously, are highlighted. (d) The topology and ribbon
model of the DGK monomer highlight residues that are affected by DOG. In the topology maps,
alterations in peak intensity and different levels of weighted CSPs are distinguished. In the
ribbon model of monomeric DGK, residues, which show a respond on DOG, are highlighted in
green. The ribbon model is obtained from the OPM database [116], using the PDB ID 4UXX
from the X-ray structure [6].
Chapter 5: Functional studies
112
5.2.3.3 AMP-PCP + DOG bound state
The AMP-PCP + DOG bound state shows a similar fingerprint compared to the state
with only AMP-PCP bound (Figure 38).
Figure 38. Effect of AMP-PCP and DOG binding on DGK. (a) Superposition of 2D NCA spectra
of apo (black) and AMP-PCP-bound (green) DGK. (b) Superposition of 2D NCA spectra of
AMP-PCP-bound (green) and AMP-PCP+DOG-bound (pink) DGK. Both the AMP-PCP bound
and AMP-PCP+DOG bound states feature a similar fingerprint with significant alterations
compared to the apo state.
5.3 Discussion
5.3.1 DGK forms a symmetric trimer in its substrate-bound states
Under the applied experimental conditions, the DGK trimer adopts a symmetric
conformation in the AMP-PCP bound and the DOG bound state. This suggests that all
three active sites are occupied by the respective substrate at the same time. This in
turn indicates that under high substrate concentration, the three active sites are
possibly in the same state during catalysis, which is contrary to the hypothesis by
Caffrey and co-workers. The crystal structure of Δ4DGK co-crystallized with AMP-PCP
shows that only one active site is occupied by the nucleotide substrate, even though a
high concentration of AMP-PCP (10 mM) was used like in this study [6]. Differences in
Chapter 5: Functional studies
113
the number of occupied active sites between NMR and X-ray data might occur due to
different experimental conditions. The investigations by MAS NMR were carried out on
wild-type DGK in DMPC/DMPA-liposomes under a physiological pH of 7.2 and at
~275 K. Crystallization of the thermostable Δ4-mutant took place under acidic
conditions (pH 5.6) in lipidic cubic phases formed by monoacylglycerols (MAGs), which
also act as lipid substrates. Its structure could be affected by crystallization contacts.
5.3.2 AMP-PCP bound state
This study demonstrates for the first time that not only the cytosolic part but also long
ranges of the transmembrane domains are affected by nucleotide binding Figure 36d.
The nucleotide most likely governs DGK into its catalytic active form. This is consistent
with the fact that DGK’s high nucleotide specificity is mainly observed in form of
reductions in kcat for ATP analogues [257]. It is also supported by the evidence that
the tetraphosphate-linked bisubstrate analogue served as a good inhibitor [257].
Furthermore, it is not surprising that nucleotide binding is noticed on a large scale in
this small kinase. With regard to the size of DGK, the nucleotide is comparatively bulky.
Additionally, the impact of AMP-PCP on DGK is likely even enhanced by the
circumstances that under the here applied experimental conditions three nucleotide
molecules are bound to one kinase molecule. These findings are accompanied by data
of Jia and co-workers, who demonstrated that the membrane is essential for stabilizing
the small structure of DGK, enabling the orientation of the substrates [11]. A similar
substantial conformational change was found for other kinases catalyzing direct
phosphoryl transfer as well [258-260].
Accordingly, binding of nucleotide induces an information transfer through the whole
enzyme towards the opposite site, which might occur in preparation of DGK for binding
the lipid substrate. The respective residues are possibly oriented for a proper binding of
the lipid. This implies positive heteroallostery, emanating from the nucleotide substrate,
which is in-line with kinetic studies of DGK, indicating that binding of the nucleotide
substrate does result in an enhanced affinity of the lipid substrate [4, 257].
For the bound states of DGK with either AMP-PCP + DOG or only AMP-PCP, a similar
fingerprint was detected (Figure 38). This suggests that the nucleotide substrate
induces a substantial conformational change, which is possibly required to trigger the
actual phosphoryl transfer reaction. Convincing kinetic and structural data support a
direct, in-line phosphoryl transfer based on a close proximity of the γ-phosphate of the
nucleotide and the 1-OH of the lipid substrate [6, 107, 257]. Respectively, our data
Chapter 5: Functional studies
114
indicate that the specific conformation induced by the nucleotide is most likely the
catalytic active conformation of DGK, which is needed to bring both substrates in close
proximity.
5.3.2.1 Comparison of the AMP-PCP bound state with solution NMR data
Next to the investigation of the AMP-PCP bound state of DGK by solid state NMR,
titration studies of DGK with up to 16 mM AMP-PCP were carried out by solution NMR
[7]. Both studies provide significant chemical shift changes (CSPs) concerning the
backbone of several residues. For 4 residues, namely E69, N72, G91 and K94, both
studies are in agreement, showing significant CSPs, whereas they deviate for a large
number of other residues. For instance, the solution NMR study reveals for G83 the
highest CSP. This is in contrast to our data, which feature for G83 only changes in
peak intensity, but no significant CSP (Figure 36a, b). Furthermore, e.g. for G20, A29,
D49, I70, I82, E88 and G97 clearly significant CSPs could be observed by solid state
NMR (Figure 36a, b), which are not visible by solution NMR [7]. The observed
differences occur presumably due to the manner, in which the proteins are present at
the time of substrate binding. In one case, the protein is embedded in a DPC micelle at
45°C [7]. In the other case, it is incorporated in DMPC/DMPC liposomes at 4°C. The
applied pH and AMP-PCP concentration were in a similar range. The shorter chain
length, the very high curvature and the small hydrophobic thickness of DPC micelles
(30–40 Å diameters) [119] could interfere with the approximate diameter of the DGK
trimer (~100 Å) [7], which is in contrast to almost planar lipid bilayers. This is in
agreement with notable differences, which are already visible in the secondary
structure obtained by solution and solid state NMR: In contrast to the ssNMR data, the
solution NMR structure features two small distortions in Y16 within the surface helix
(SH) and I70/L70 of helix 2 (H2). In addition, there are small deviations around the
interhelical turn (T) between H1 and SH and around the periplasmic loop (PL) between
H1 and H2 as well as the cytoplasmic loop (CL) between H2 and H3, which are 3, 1
and 6 residues longer in DPC micelles compared to lipid bilayers. It should be noted
that the variations in the secondary structures in different environments all arise in
catalytically critical regions, such as the SH, cytosolic regions of H2 and H3 and the
CL. These differences most likely reflect the impact of the environment, in which the
membrane protein is embedded.
Chapter 5: Functional studies
115
5.3.3 DOG bound state
In the DOG bound state, overall changes are significantly less pronounced. Especially,
the transmembrane domains do not show any reaction on the acyl chains of DOG
(Figure 37d). However, few significant perturbations could be determined at the
cytoplasm/membrane interface for Arg9, Lys12, Gln33 and Glu69 (Figure 37a-c).
Based on the crystal structure, all four of them are located in the active site in close
proximity to the headgroup of the lipid substrate [6]. Their side chains possibly interact
with the proximal OH group or the carbonyl oxygen at the ester linkages of DOG,
keeping the headgroup of the lipid substrate in position (Figure 39). Especially, Arg9
and Glu69 have been already reported to directly interact with the headgroup of the
lipid substrate via H-bonds [6, 11]. Hence, these data imply that the lipid substrate is
primarily recognized by its headgroup and not by its acyl chains, which is in agreement
with observations by Walsh et al. [255].
Figure 39. Enlarged view from the membrane plane, illustrating the crystal structure of Δ4 DGK
(PDB 3ZE5) [5], accommodating the lipid substrate (orange) in the hydrophobic pocket.
Possible interactions between the side chain of Arg9, Lys12, Gln33 and Glu69 with the proximal
OH group of the lipid substrate are depicted.
Changes in DGK induced by DOG might take place, as just mentioned, due to a
specific direct response on the lipid substrate in the active site, but could be also
provoked by the altered order parameter of proximate phospholipids due to inserted
DOG (20 mol%) into the lipid bilayer. It is known that DAGs with a short chain length
(DOG, chain length n = 8) induce an ordering effect close to the headgroup of the
phospholipid side chains [261]. The DOG molecules intercalate between the bulky
headgroups of the phospholipids and promote a tighter contact between their side
chains in the region close to the headgroups, thereby increasing their order
Chapter 5: Functional studies
116
parameters. The lower segments of the phospholipid side chains, which cannot be
reached by DOG, are slightly more disordered in the presence of DOG.
5.4 Summary and Outlook
This study provides an almost complete resonance assignment of wtDGK within the
lipid bilayer in its apo-, nucleotide- and lipid substrate-bound states. This way, the
overall response of DGK towards substrate binding could be mapped. It could be
shown that all three active sites can be occupied concurrently by both AMP-PCP and
DOG. Under high substrate concentration, the three active sites are most likely in the
same state during catalysis. Additionally, it could be demonstrated for the first time that
not only the cytosolic region but also large parts of the transmembrane domains are
affected by nucleotide binding. This possibly supports the enzyme in binding of the lipid
substrate, implying positive heteroallostery. Furthermore, the substantial
conformational change induced by the nucleotide seems to set the enzyme into a
catalytic active state, triggering the actual phosphoryl transfer reaction.
In order to obtain further insights into DGK’s catalytic mechanism, the protonation state
of several residues could be investigated. Unfortunately, an unambiguous
determination at this stage was not feasible due to signal overlap and low signal
intensity in the 3D NMR spectra. For this purpose, a strategic labelling scheme to
follow these specific residues by 2D experiments has to be developed. This way, they
could be specifically monitored in the apo, AMP-PCP-, DOG- and AMP-PCP+DOG-
bound state.
Furthermore, nucleotide-protein and DAG-protein interactions could be investigated in
the AMP-PCP and DOG bound state using DNP-enhanced MAS NMR. Labelling
schemes could be decided with respect to the X-ray structure (PDB 4UXX) [6].
Additionally, the role of the surface helix in sensing osmolality and altered lateral
pressure in the lipid bilayer [5] could be probed by multidimensional high field MAS
NMR based on the almost complete resonance assignment shown in chapter 4.
Different concentrations of DAG could be inserted into the lipid bilayer, modulating the
membrane order parameter. In order to probe how DGK, especially its SH, responds to
changes of the membrane lateral pressure, chemical shift perturbations could be
examined and site-resolved order parameters and relaxation rates could be
determined.
Chapter 6: Long-range contacts
117
6 Long-range contacts
6.1 Introduction
Inter-residue contacts are the cement of a protein structure, stabilizing its fold. Thus,
they have been of great interest for the investigation of the mechanisms of protein
folding and stability [262, 263]. Contacts also provide a platform for crosstalk between
single residues and thus, in some cases between different domains or subunits. Hence,
they take up functional roles as well [264, 265]. Contacts in proteins can be of different
nature. Hydrogen bonds are formed between two electronegative atoms, such as
nitrogen and oxygen, by sharing a hydrogen atom [266]. It has been demonstrated that
even weak hydrogen bonds could provide inter-residue contacts [267, 268]. Ionic
bonds are based on interactions between oppositely charged groups of a molecule,
e.g. between the positively charged basic side chains of lysine and arginine, and the
negatively charged carboxyl groups of glutamate and aspartate [269]. In contrast, van
der Waals interactions are weak forces [270]. They act not only between polar
molecules but also between electrically neutral atoms and molecules. This is due to the
shifting of electrons in the outer shell of the atoms, which temporarily cause charge
movements and so-called polarization. Charged areas with different signs are then
attracted to one another and hence provide an attraction between two atoms, even if
they are electrically neutral overall.
DGK appears as a homotrimer and features a remarkable stability in native
membranes [8, 9]. Its oligomeric arrangement is of direct functional relevance, since its
complex forming monomers alone are not functional. Each active site is built by
components of two monomers, based on the composite shared site model [5-7, 10].
This implies that the substrate-bound state of one site is relayed to the other sites,
suggesting cooperativity in substrate binding [5, 6], which is not uncommon for many
oligomeric proteins [271]. The assumption of cooperativity is supported by the
presence of the amphiphilic surface helix, which suggests itself as a mediator
transmitting inter-subunit information [5, 6]. However, understanding DGK’s remarkable
stability and the cross-talk between its subunits demands the identification of key intra-
and interprotomer contacts, which are of structural or functional importance.
Chapter 6: Long-range contacts
118
6.2 Intraprotomer contacts visualized by high field MAS NMR
6.2.1 Results and Discussion
In order to identify long-range contacts in the apo state of DGK, 13C-13C DARR and 2D
NCOCX spectra with long mixing times were recorded using high field MAS NMR. For
this purpose, the extensive side chain assignment obtained in chapter 4 could be used.
The spectra were analyzed with focus on long-range, non-sequential side chain-side
chain cross peaks. This way, some intraprotomer contacts could be determined:
Crosspeaks between Ca of Ser61 located in helix 2 and side chain carbons of Trp112
present in helix 3 (Figure 40a) reveal close proximity of both residues. This is
consistent with findings of Caffrey and co-workers, who suggested a hydrogen bond
between Ne of Trp112 and the OH-group of Ser61 (Figure 40b) [6]. The contact
between Trp112 and Ser61 has most likely a stabilizing effect on the transmembrane
region of each monomer (Figure 40b). This is in agreement with mutational studies by
Lau and Bowie, who demonstrated that the mutant of DGK, in which Trp112 is replaced
by Phe, was susceptible to aggregation and showed a low specific activity [9].
Figure 40. Intraprotomer interactions in the transmembrane region of DGK between Trp112 and
Ser61. (a) 2D 13
C-13
C DARR spectrum of U-13
C,15
N-DGK-ILV with 800 ms mixing time.
Crosspeaks appear between Ca of Ser61 and the side chain carbons of Trp112 revealing an
intraprotomer contact between helices 2 (Ser61) and 3 (Trp112). (b) Visualization of the
intraprotomer contact between Trp112 and Ser61 in the crystal structure of Δ4 DGK (PDB
Chapter 6: Long-range contacts
119
4UXX) [6]. Enlarged view from the membrane plane, accommodating the lipid substrate (yellow)
in the hydrophobic pocket (left). View from the periplasm, depicting the three monomers in
different shades of grey (right). Trp112 (H3) is secured by a hydrogen bond to Ser61 (H2) in
the lower region of the hydrophobic pocket.
Furthermore, crosspeaks between side chain nitrogens, Ne and Nh1/2, of Arg32 with
25TrpCO, 28GluCb and 29AlaCa,Cb,CO were detected (Figure 41a). Arg32 is located
at the membrane/cytoplasm interface of helix 1, while Trp25, Glu28 and Ala29 are
present in the SH, interhelical turn and the cytoplasmic part of H1 (Figure 41b). These
contacts are described for the first time. Additionally, a R32A mutant was prepared,
which features a strongly reduced activity (Figure 49, grey bar). This is in agreement
with previous mutational studies [6, 7]. The functional relevance of Arg32 could be
explained by its role in forming the contacts with Trp25, Glu28 and Ala29, which have
most likely a strengthening effect on the joint between H1 and SH, stabilizing the SH
and thus the active site as well (Figure 41b).
Figure 41. Intraprotomer interactions in the cytoplasmic region of DGK between Arg32 and
Trp25/Glu28/Ala29. (a) 2D NCOCX spectrum of U-13
C,15
N-DGK with a 400ms DARR mixing
step. Crosspeaks between 32ArgNh1/2 and 32ArgNe with 25TrpCO, Glu28Cb and
29AlaCa/Cb/CO are determined, caused by an intraprotomer contact between these residues in
helix 1 and the surface helix. (b) Depiction of the intraprotomer contact involving Arg32 in the
crystal structure of Δ4 DGK (PDB 3ZE5) [5]. Enlarged view from the membrane plane,
illustrating the intraprotomer contacts for 32ArgNe/Nh1,2 with 25TrpCO, Glu28Cb and
29AlaCa/b/O.
6.3 Interprotomer contacts visualized by DNP-enhanced MAS NMR
6.3.1 Introduction
Unfortunately, no cross-protomer contacts could be detected by conventional high field
MAS NMR. The identification of interactions at protomer interfaces is in general a more
complex and challenging task. Most data available so far are from crystal structures of
Chapter 6: Long-range contacts
120
membrane proteins. Crystal structures have been reported for DGK as well [5, 6],
suggesting several interprotomer contacts as illustrated in Figure 42.
Figure 42. Representative possible interprotomer contacts in DGK suggested by the crystal
structure of Δ4 DGK (PDB 3ZE5) [5]. Enlarged view from the cytoplasm (a) and membrane
plane (b, c), illustrating a possible interprotomer contact for Arg81 and Glu88 (a), Arg92 and
Asn27 (b) as well as Lys19 and Asp95 (c).
However, the predicted contacts are in general debatable due to incompleteness of the
electron density concerning relevant side chains (Arg81, Glu88, Arg92, Asn27 and
Lys19: PDB 3ZE5 electron density map). Additionally, contacts in crystal structures can
be assailable due to the difficulty to distinguish between biologically relevant
interactions from those induced by crystal contacts [272, 273]. This highlights the need
for complementary spectroscopic data in the membrane environment. Solid state NMR
is able to provide such interaction data. It could be shown that it is possible to
investigate protein−protein contacts by directly observing interpeptide dipole couplings
[274] or indirectly through paramagnetic relaxation enhancement [275]. Concerning
amyloid fibrils, mixed labelled samples were utilized to determine interprotein distance
constraints [276, 277].
By dis- and reassembling of trimeric DGK, mixed labelled 13C−15N complexes can be
gained, which feature a unique isotope labelling pattern at their protomer interfaces.
15N−13C transferred echo double resonance (TEDOR) spectroscopy [135, 136] enables
the detection of specific interprotomer contacts. Conventional ssNMR would be barely
suitable for the identification of interprotomer contacts [129], since reassembling of
different labelled protomers to the oligomeric state is based on a statistical distribution,
leading to a comparably low number of mixed labelled interfaces. Hence, sensitivity
enhancement is needed. Dynamic nuclear polarization (DNP) has been developed to
increase the sensitivity of MAS NMR by orders of magnitude [278]. There are several
studies under cryogenic conditions, which benefited from DNP, as presented for
Chapter 6: Long-range contacts
121
various membrane proteins [134, 279-281]. Maciejko et al. could verify for the first time
that DNP-enhanced ssNMR can be used to detect interprotomer contacts within a
homo-oligomeric membrane protein embedded into lipid bilayers [129, 134]. This work
was done in this lab using pentameric green proteorhodopsin. In this chapter, it is
demonstrated that the basic principles of the work from Maciejko et al. can be applied
to trimeric DGK as well. An extensive study was carried out to establish a procedure for
dis- and reassembling of homo-trimeric DGK to produce active mixed labelled
complexes, which is verified by LILBID-MS [282], BN-PAGE [283] and the coupled
activity assay [18]. A labelling scheme is utilized, which enables the detection of
possible cross-protomer interactions (Arg/Lys−Asp/Glu/Asn), as predicted from the
crystal structures of DGK (Figure 42) [5, 6]. DNP-enhanced 13C−15N TEDOR
experiments are conducted to monitor these contacts, while single-site mutations are
inserted in order to assign them. Additionally, the mixed labelled trimers were saturated
with the ATP analogue, AMP-PCP, to determine, if these contacts are involved in
nucleotide binding.
The theoretical background of DNP and the TEDOR experiment is explained in detail in
section 1.3.3.
6.3.2 Results
6.3.2.1 Creating mixed labelled trimers of DGK
In order to monitor 13C−15N side chain contacts using DNP-enhanced TEDOR
experiments, mixed labelled complexes, consisting of neighbouring 13C and 15N
protomers, can be gained by dis- and reassembling of homo-trimeric DGK. As shown in
chapter 3, the BN-PAGE (Figure 17b) shows one main trimer population for DGK
surrounded by DDM micelles, which is in-line with literature [7, 18]. It is also confirmed
by LILBID-MS (Figure 17c), a well-tested and unambiguous method for determining the
mass of macromolecular complexes [284]. It verifies that BN-PAGE analysis offers a
reliable assessment of the oligomeric state. DGK in its trimeric form has been shown to
be remarkable stable [9, 84, 89, 93, 108]. Data by Lau and Bowie indicate an
impressive unfolding free energy of 16 kcal/mol for the transmembrane domain and
even 6 kcal/mol for the cytoplasmic domain [9]. Thus, harsh conditions are necessary
to disrupt the oligomeric state. Thermal denaturation of membrane proteins is usually
irreversible as shown for DGK [141] as well as many other membrane proteins, such as
bacteriorhodopsin [285-287], erythrocyte band 3 [288, 289], cytochrome c oxidase
[290, 291] and photosystem II [292-294]. In contrast, it is known from a number of
Chapter 6: Long-range contacts
122
studies that numerous membrane proteins can be refolded after denaturation with
chemical denaturants, such as guanidine hydrochloride (GuHCl), urea or SDS [295-
299]. Unlike GuHCl and urea, SDS is able to build mixed micelles with other
detergents, providing an environment for the protein, in which the α-helical content of
membrane proteins is often preserved [300, 301]. Lau and Bowie could show that even
after complete denaturation of DGK by the anionic SDS, the helical content is retained.
Additionally, 90% of the enzymatic activity could be recovered by dilution of the SDS-
denatured DGK into a DM solution [9]. Consequently, the detergent of choice for
disassembling trimeric DGK was SDS, which should enable the creation of active
mixed labelled trimers. For this purpose, a complete separation of the trimers into
monomers is beneficial, since it results in the largest number of interprotomer 13C−15N
interfaces after reassembling. Thus, the optimal ratio of protein-to-SDS concentration
had to be determined. Since DGK is more stable at higher protein concentrations [141],
a low concentration of 0.2 mg/ml was used for disruption. The BN-PAGE (Figure 43b)
illustrates the effect of different detergent concentrations on disassembling of trimeric
DGK. The higher the SDS concentration, the higher is the degree of disassembly into
monomers. The disruption into monomers is incomplete for SDS concentrations from
0.5%-1.5%, since clearly a population of dimers is visible. Starting from 2% SDS, the
separation into monomers seems to be total, which is therefore, used for disassembly
of trimeric DGK in the subsequent studies, allowing sufficient mixing of differently
labelled protomers. LILBID-MS was applied as well, confirming the predominantly
monomeric state of DGK in SDS micelles (Figure 43c). The LILBID mass spectrum was
recorded by Oliver Peetz of the research group of Prof. Dr. Nina Morgner (Institute of
Physical and Theoretical Chemistry, Goethe University Frankfurt am Main).
Furthermore, it was tested, if the trimeric form of DGK can be regained, when SDS is
removed. The BN-PAGE (Figure 43d) shows that a transition from monomeric to
trimeric DGK can be obtained, when SDS is completely displaced by DDM through
extensive washing. A scheme for the creation of mixed labelled trimers is presented in
Figure 43a. In order to examine, whether the disrupting detergent affects the activity of
DGK, the coupled activity assay was carried out. For this purpose, DGK trimers in DDM
micelles (-SDS), DGK monomers in SDS micelles (+SDS) and DGK trimers in DDM
micelles after SDS treatment (±SDS) were reconstituted into lipid bilayers. As
reconstitution method BioBeads were applied, reported to be suitable not only for DDM,
but also for SDS removal [302]. Figure 43e illustrates that trimers reconstituted from
DDM micelles before and after SDS treatment feature a similar activity. Monomers from
SDS micelles lead to a reduced activity below 20%, indicating that monomers, known
Chapter 6: Long-range contacts
123
to be separately not functional [5, 7, 10], do not reassemble to trimers upon
reconstitution.
Figure 43. Creation of active mixed labelled trimers of DGK. (a) Differently labelled trimers of
DGK are separately expressed, solubilized, purified and eluted in DDM. Subsequently, they are
disassembled into monomers or dimers by SDS and mixed in a 1:1 ratio. Then, SDS is removed
and replaced by DDM, resulting in mixed labelled DGK trimers, which can then be reconstituted
into liposomes. (b) BN-PAGE of DGK (0.2 mg/ml) in different SDS concentrations: (1) 0.5%, (2)
1%, (3) 1.5%, (4) 2%, (5) 3%. The higher the SDS concentration, the higher is the degree of
disassembly into monomers. (c) LILBID-MS confirms the predominantly monomeric state of
DGK in SDS micelles: The signals for the monomeric, dimeric and trimeric form of DGK are
labelled by “1”, “2” and “3”, respectively. They occur at a charged state of −1. The LILBID mass
spectrum was recorded by Oliver Peetz of the research group of Prof. Dr. Nina Morgner
(Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt am Main). (d) The
BN-PAGE shows DGK as trimer in DDM micelles before (1) and after (3) SDS treatment and
mostly in its monomeric state in SDS micelles (2). (e) Activity of DGK reconstituted from
different detergent environments: DGK trimers from DDM micelles before SDS treatment (-SDS,
dark grey) and after SDS treatment (±SDS, green) as well as DGK monomers from SDS
micelles (+SDS, yellow) were reconstituted. 100% activity corresponds to the rate recorded with
wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1
mg-1
. Experiments were
repeated three times. The activity was calculated as the mean value. Error bars correspond to
standard deviations.
Chapter 6: Long-range contacts
124
Using the procedure above, samples were prepared, in which the trimer is composed
of (i) 13C- and 15N-DGK protomers ([CN]-DGK), (ii) 13C-DGK and 15N-Arg-Lys-DGK
protomers ([CN(Arg,Lys)]-DGK) and (iii) 13C-DGK ([CC]-DGK). The last one serves as
control sample for the differentiation of 13C−15N contacts from naturally occurring 13C-
or 15N-isotopes. In order to decrease natural abundance signals, all 15N-labelled
protomers were 13C-depleted by applying [12C6]glucose (99.5%) as a carbon source,
leading to a 50% lowering of the 13C background.
6.3.2.2 Validation of the application of AMUPol as biradical
DNP-enhancement was obtained by doping the sample with the biradical polarizing
agent AMUPol, serving as a source for unpaired electrons [133]. The enhancement (ε)
is determined by the ratio of NMR signal intensities with and without microwave
irradiation [131]. 45-fold signal heightening could be achieved, when the
proteoliposome pellet (~40 µl) was split into two small ones (~20 µl) for ~20 h of
incubation each with ~20 µl AMUPol solution (Figure 44a). In contrast, if just the unsplit
pellet of ~40 µl was incubated with ~40 µl AMUPol, a signal enhancement of only 23-
fold was determined after ≥ 24 h of incubation. Figure 44b proves that the presence of
AMUPol around the protein has no influence on DGK’s activity, since a similar activity
with and without the biradical was reached.
Figure 44. Validation of the application of AMUPol. (a) DNP enhancement shown for a 13
C−CP
spectrum of DGK incubated with 20 mM AMUPol. Upon microwave irradiation, a 45-fold
sensitivity enhancement is reached. (b) Activity of DGK with (+) and without (-) AMUPol,
Chapter 6: Long-range contacts
125
indicating that the presence of the biradical has no influence on the activity. 100% activity
corresponds to the rate recorded with wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9)
µmol min-1
mg-1
. Experiments were repeated three times. The activity was calculated as the
mean value. Error bars correspond to standard deviations.
6.3.2.3 DNP-enhanced 15N−13C TEDOR experiments
6.3.2.3.1 Finding the best mixing time using 1D TEDOR spectra
The achieved sensitivity enhancement facilitated 15N−13C TEDOR experiments on the
mixed labelled trimers of DGK. By using TEDOR spectra, through-space dipole−dipole
contacts between 13C and 15N spins can be identified. Firstly, several 1D-TEDOR
spectra were recorded with different rotor periods (L0 = 4 – 40) to find the best mixing
time for our experiments. L0 describes the number of 180° pulses during tmix/4. Figure
45 illustrates TEDOR spectra of the control sample ([CC]-DGK).
Figure 45. DNP-enhanced 1D-TEDOR spectra of the control sample ([CC]-DGK) at different
mixing times. The spectra were recorded with 4096 scans at a 400 MHz spectrometer, ~105 K,
pH 7.2 and a spinning speed of 8 kHz. The 263 GHz gyrotron was operated at a collector
current of 70 mA. Six spectra were recorded with L0 (rotor periods) = 4, 8, 16, 24, 32, and 40.
The spectrum with short mixing time (L0 = 4) features TEDOR signals in the CO (170-
185 ppm) and the Ca (50 - 70 ppm) range. The signal at ~96 ppm represents the
Chapter 6: Long-range contacts
126
spinning sideband of the carbonyl peak. Concerning higher mixing times (L0 = 16 - 40),
both the CO and the Ca signals decrease. However, small signals arise with increasing
mixing times in the aliphatic side chain range (10 - 40 ppm). All these signals originate
from the natural abundance of 15N in 13C-protomers. The CO signals arise from the
backbone peptide bonds, linking covalently two consecutive amino acids, whereas the
Ca signals derive from covalently bonded neighbouring backbone atoms within the
same amino acid. With 121 residues, DGK has 120 peptide bonds. Thus, a natural 15N
abundance of ~0.4% leads to statistically ~0.5 15N per DGK monomer, which is
obviously enough to be detected through DNP enhancement. The TEDOR signals for
covalently bonded nuclei are dominant at shorter mixing times due to their short
distances (CO-N: 1.32 Å, N-Ca: 1.45 Å [303]). In contrast, with longer mixing times, i.e.
with more 180° pulses in the 15N channel, longer 15N-13C distances can be determined,
such as the amide 15N and the side chain carbons. These signals peak at L0 = 24 and
decrease slightly with higher L0s.
Mixed labelled [CN]-DGK shows a similar behaviour concerning the mixing time as the
control sample ([CC]-DGK) (Figure 46). It features signals in the CO and Ca range from
naturally appearing 15N-isotopes (~0.4%) in the 13C and 13C-isotopes (~0.5%) in the 15N
labelled protomers, which reduce with higher mixing times. Also the behaviour of the
TEDOR signals in the aliphatic side chain range is comparable to the control sample.
They peak with L0 = 24, with the difference that they appear clearly stronger, indicating
further 15N contacts to aliphatic carbons. They most likely arise from protein-protein
contacts between backbone 15N amide and aliphatic 13C side chains of neighbouring
protomers. These signals are additional to natural abundance signals, suggesting that
the preparation of mixed labelled trimers ([CN]-DGK) was successful, providing
interprotomer 13C-15N contacts. Since signals in the aliphatic side chain range peak
with L0=24 (6.25 ms), it turned out that 6.25 ms is the perfect total mixing time to
optimize the signal intensity of anticipated long-range interprotomer couplings in the
DGK sample.
Chapter 6: Long-range contacts
127
Figure 46. DNP-enhanced 1D-TEDOR spectra of [CN]-DGK) at different mixing times. The
spectra were recorded with 3520 scans at a 400 MHz spectrometer, ~105 K, pH 7.2 and a
spinning speed of 8 kHz. The 263 GHz gyrotron was operated at a collector current of 70 mA.
Six spectra were recorded with L0 (rotor periods) = 4, 8, 16, 24, 32, and 40.
6.3.2.3.2 Visualizing interprotomer contacts using 2D TEDOR spectra
In order to monitor specific 13C- 15N contacts between the protomers, 2D-TEDOR
spectra were recorded (Figure 47). All three spectra exhibit certain natural abundance
signals originating from N−CO and N−Ca single bond contacts as well as from long-
range N−Cx couplings. In addition, arginine intraresidue 13C−15N contacts between
ArgNe,n and ArgCz are visible. These natural abundance signals appear, as already
implied for the 1D TEDOR spectra, from naturally arising 13C-isotopes (~0.5%) in the
15N labelled protomers that are still included in spite of depletion by 12C. 12C6-glucose is
unfortunately not available with purity higher than 99.5%. In addition, the 15N natural
abundance in 13C-labelled protomers amounts to ∼0.4%.
The comparison of the spectrum of mixed labelled [CN]-DGK with the [CC]-DGK
control spectrum offers one additional cross-peak, which implies at least one cross-
protomer 13C−15N contact. The peak appears in the 15N chemical shift range of
arginine, suggesting that this amino acid type participates in cross-protomer
interactions. This is supported by the selectively labelled [CN(Arg,Lys)]-DGK sample,
which illustrates the same cross-protomer contact as [CN]-DGK. The cross-peak
Chapter 6: Long-range contacts
128
reveals a correlation between the 15N resonance of Arg-Ne,n with 13C resonances of
carboxyl groups from Asp-Cg and/or Glu-Cd and/or the carbonyl group from Asn-Cg,
indicating possible cross-protomer salt bridge or H-bond contacts between these
residues. The selectively labelled sample was not only arginine, but also lysine
labelled, since the crystal structures of DGK predict one interprotomer contact with a
distance of ~3.7 Å between 19Lys-Nz (34 ppm) and 95Asp-Cg (179 ppm) as well
(Figure 42c) [5, 6]. However, both spectra for [CN]-DGK and [CN(Arg,Lys)]-DGK do not
feature a significant cross peak involving lysine, implying that the distance between
these two residues is too large in the wild type to be detectable. Nevertheless, the data
concerning the cross peak involving arginine reveal that DNP-enhanced TEDOR
spectra enable investigating 13C−15N contacts across the protomer interface of trimeric
and not only of higher-oligomeric membrane proteins. Further data are required to
assign the cross-peak to distinct residues and to clarify its impact on structural stability
and functionality.
Chapter 6: Long-range contacts
129
Figure 47. DNP-enhanced 15
N−13
C-TEDOR spectra (tmix = 6.25 ms) of [CN]-DGK,
[CN(Arg,Lys)]-DGK and the control sample, [CC]-DGK. All spectra reveal cross-peaks
originating from natural abundance intramolecular backbone 13
C−15
N-contacts (highlighted in
grey). Further cross-peaks (highlighted green) are detected in [CN]-DGK and [CN(Arg,Lys)]-
DGK. They can be assigned to cross-protomer contacts, reflecting a through-space correlation
between Arg and Asn/Asp/Glu. These cross-peaks demonstrate that salt bridges or H-bonds
between Asn/Asp/Glu and Arg must be present at the protomer interfaces.
Chapter 6: Long-range contacts
130
6.3.2.3.3 Attemps to assign the cross-peak by RxA-mutants
Single-site mutations were chosen to investigate the impact of the respective arginine
on DGK’s oligomerization, structural stability and functionality. For creating single-site
mutants, alanine was used for substitution. It is usually the first choice for mutational
scanning [304-306]. It is non-bulky and chemically inert due to its methyl group. A
mutation to alanine is generally equivalent to simply truncating a side chain back to Cb,
the first side chain atom. It has the propensity to form α-helices, but can also occur in
β-sheets, since the position of Cb depends upon the backbone dihedral angles of the
polypeptide and is part of the main chain structure of the protein. Glycine, for instance,
which removes Cb, is unusually flexible and can adopt polypeptide backbone
conformations that are generally not allowed by other amino acids. Consequently, a
mutation to glycine would cause flexibility and possible conformational changes,
making interpretation more complex than for alanine. Replacing side chains with larger,
more constrained amino acids with a branched pattern, more polar, differently charged,
or more hydrophobic atoms might cause changes in structure and conformation along
with the side chain chemistry. Thus, they would complicate the analyses of results
more than alanine as well.
Since DGK contains in total six arginines, the single-site mutations R9A, R22A, R32A,
R55A, R81A and R92A were introduced into the protein. These RxA-mutations not only
enable to investigate, if the side chain of the respective arginine has an impact on
DGK’s oligomerization, stability and function, but also can be used for cross-peak
identification. The mixed labelled single mutants were prepared as the wild type and
analysed by BN-PAGE. Figure 48 shows for all mutants a similar oligomerization
behaviour compared to wtDGK: All mutants form trimers in DDM that can be disrupted
to mainly monomers by SDS and reassemble mostly to trimers, when SDS is replaced
by DDM. There are also small deviations visible. Some mutants feature a weak dimer
population in SDS and/or in DDM after SDS treatment, which cannot be exclusively
explained by the inserted mutation, since also small deviations in handling during the
preparation process might be possible. In order to clarify, if solely the mutation is the
reason for this slight difference, all mutants would have to be prepared for at least three
times.
Chapter 6: Long-range contacts
131
Figure 48. Characterization of the oligomeric state of the single-site RxA mutants in comparison
to wtDGK by BN-PAGE. The BN-PAGES show DGK as trimer in DDM micelles before (1) and
after (3) SDS treatment and mainly as monomers in SDS micelles (2). All RxA mutants show a
similar oligomerization behavior as the wt, indicating that the respective arginines located in
extramembranous regions of DGK are not necessary for the trimer formation.
The next characterization step should verify whether the respective introduced
mutation and/or the SDS treatment affects the activity of DGK. Thus, the coupled
activity assay was performed. For this purpose, RxA-DGK trimers in DDM micelles
before and after SDS treatment were reconstituted into lipid bilayers. Figure 49
indicates that all six arginines in DGK play a functional role, since the activity is clearly
reduced for all RxA-mutants compared to the wild type. Especially, the mutation of
Arg9, Arg32, Arg81 and Arg92 caused a reduction of activity below 25%. The SDS
treatment of the RxA mutants led to a loss of activity of only up to 20%, which is
comparable to the wt. Thus, they are only slightly prone to SDS.
Chapter 6: Long-range contacts
132
Figure 49. Kinase activity of DGK affected by single site RxA mutations, expressed as a
percentage of wt activity. DGK trimers from DDM micelles (dark grey) and DGK trimers from
DDM micelles after SDS treatment (green) were reconstituted into lipid bilayers and then
measured. The activity is clearly reduced for all RxA-mutants compared to the wild type. The
SDS treatment of the RxA mutants led to a loss of activity of only up to 20%, which is
comparable to the wt. 100% activity corresponds to the rate recorded with wtDGK in 90mol%
DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1
mg-1
. Experiments were repeated three times.
The activity was calculated as the mean value. Error bars correspond to standard deviations.
Overall, the data suggest that the six arginines are not essential for the trimer formation
of DGK, but they are all more or less functionally relevant. Mixed labelled complexes of
all mutants ([CN]-DGK-RxA) were analysed by DNP-enhanced TEDOR experiments
and compared with the wild type spectrum as well as among each other in order to spot
any differences (Figure 50). Since only one cross-peak in the arginine range (Arg-
Asp/Glu/Asn) had been observed, the spectral width was reduced and the offset
adjusted to focus the peak of interest, which allows to use more scans, leading to a
better signal-to-noise ratio. Unfortunately, the cross-peak does not disappear for each
of the six possible arginine mutants ([CN]-DGK-RxA), indicating that multiple arginines
contribute to these interactions. However, a reduction of the cross peak intensities for
R81A- and R92A-DGK could be observed compared to the wild type, suggesting that
both arginines are involved in the cross-protomer interactions. The activity of both RxA-
mutants is also clearly decreased (Figure 49).
Chapter 6: Long-range contacts
133
6.3.2.3.4 AMP-PCP bound state of mixed labelled DGK
Additionally, it was tried to determine, if bound nucleotide substrate affects the cross-
protomer contacts significantly. For this purpose, DGK was saturated with the ATP
analogue adenylylmethylenediphosphonate (AMP-PCP) as described in chapter 2 and
5. Its characterization revealed a fully saturated system, which is in addition highly
stable, enabling long MAS NMR experiments. However, an obvious chemical shift
perturbation or a (dis)appearance of cross-peaks is not observable (Figure 50).
Figure 50. DNP-enhanced 15
N−13
C-TEDOR spectra of mixed labelled trimers (U-13
C/U15
N12
C-
DGK). The interprotomer crosspeak does not disappear for each single-site RxA-mutant,
demonstrating that more than one Arg contributes to these interactions. Based on the 3D crystal
structure [6], only Arg81 and Arg92 have an appropriate location at the interface to be involved
in these interactions. This would be in-line with the observed reduction of the cross peak
intensities for R81A- and R92A-DGK. For the AMP-PCP bound state of DGK, no significant
changes of the cross-peak are observable.
6.3.3 Discussion
6.3.3.1 Creating mixed labelled trimers of DGK
The overall procedure to monitor interprotomer contacts is technically challenging. The
first obstacle is to find a proper method for reassembling differently labelled protomers
to active mixed labelled complexes, which includes firstly the separation of oligomers to
protomers. Here, BN-PAGE and LILBID-MS analysis confirmed that DGK is present in
its trimeric form in DDM micelles (Figure 17b, c). For disassembling of DGK, suitable
conditions could be carved out, which enabled to disrupt DGK mainly into monomers,
Chapter 6: Long-range contacts
134
despite the high stability of its trimeric state. Therefore, harsh conditions including the
application of the anionic detergent SDS are necessary to disrupt the oligomeric state,
which however implicates a risk for irreversible denaturation. The conditions, such as
the ratio of protein-to-SDS concentration, have to be fine-tuned. Since DGK is more
stable at higher protein concentrations [141], a low concentration of 0.2 mg/ml had to
be used for disruption by 2% SDS. The concentration dependence can be explained by
the evidence that dissociation and unfolding occurs prior inactivation, which is provided
by the correlation between thermodynamic stability and kinetic stability [141]. If the
protein concentration is too high, the disruption would have been incomplete or
extreme high SDS concentrations would have been necessary. Most important, it could
be verified that the effects of 2% SDS on DGK are reversible. If SDS is displaced by
DDM, the trimeric form of DGK can be retrieved, shown by BN-PAGE analysis (Figure
43d). Additionally, 90% of the enzymatic activity could be recovered this way, illustrated
by the coupled activity assay (Figure 43e). These data are in-line with the findings of
Lau and Bowie, who reported that DGK can be reversibly unfolded by SDS and retains
much of its helical content [9]. Besides, this study shows for the first time that it is
necessary to eliminate SDS completely prior to the reconstitution step. DGK monomers
seem not to reassemble in correctly refolded and active trimers within the lipid bilayer.
Although BioBeads have been reported to be suitable as reconstitution method for SDS
removal [302], they might absorb SDS too fast, leading to aggregation of the unfolded
DGK before a proper refolding and trimer formation can occur. SDS removal via
dialysis instead of BioBeads might be a suitable alternative, since dialysis enables a
slower elimination of detergent. But this can take up to two weeks. The quickest and
more reliable procedure is replacing SDS completely by DDM to generate refolded
trimeric DGK before the reconstitution into lipids occurs, which leads to active
complexes. Taken all, this study demonstrates that mixed labelled oligomers with
preserved activity can be generated for comparably small oligomers like DGK (43 kDa).
6.3.3.2 Statistical analysis of unique interfaces in mixed labelled DGK
The second obstacle results from the intrinsically low sensitivity of MAS NMR. The low
number of cross-protomer spin pairs would be beneath the detection limit of
conventional ssNMR, even if large amounts of membrane protein were present.
Additionally, the number of contacts cannot be controlled during the mixing process,
since the assembly of mixed complexes is statistically defined. The association of 13C-
Chapter 6: Long-range contacts
135
and 15N-labelled monomers to [CN]-DGK causes a certain number of configurations
that are unequally populated.
The population P for a specific configuration is calculated by:
𝑃(𝑁, 𝑘) =𝑁!
𝑘! (𝑁 − 𝑘)! (23)
where N represents the number of monomers (3) and k the number of 15N-labelled
protomers within the mixed labelled complex of [CN]-DGK (0, 1, 2, 3). Each
configuration leads to a certain number of 13C−15N interfaces, I(N,k) (0, 2, 2, 0). The
average number of interfaces per DGK trimer is determined by:
𝐼𝑎𝑣𝑔 = ∑ 𝑃(𝑁, 𝑘) 𝐼(𝑁, 𝑘)𝑁
𝑘=1
∑ 𝑃(𝑁, 𝑘)𝑁𝑘=1
= ∑ 𝑃(3, 𝑘) × 𝐼(3, 𝑘)3
𝑘=0
∑ 𝑃(3, 𝑘)3𝑘=0
= (1 × 0) + (3 × 2) + (3 × 2) + (1 × 0)
8
(24)
= 12
8= 1.5
For a trimer, four distinct configurations are possible, leading to an average number of
1.5 N−C interfaces per complex, of which only 50% are unique interfaces (N→C vs.
C→N) that are conducive to the 15N−13C TEDOR spectra:
𝐼𝑢𝑛𝑖𝑞𝑢𝑒𝑎𝑣𝑔
= ∑ 𝑃(3, 𝑘) × 𝐼𝑢𝑛𝑖𝑞𝑢𝑒(3, 𝑘)3
𝑘=0
∑ 𝑃(3, 𝑘)3𝑘=0
= (1 × 0) + (3 × 1) + (3 × 1) + (1 × 0)
8=
6
8= 0.75 (25)
Thus, just 0.75 specifically labelled interfaces are calculated per DGK trimer, leading to
only 0.75 TEDOR-active Arg-Asp/Glu/Asn interactions. Consequently, this work would
simply not be feasible based on conventional NMR. Fortunately, this hurdle can be
overcome by dynamic nuclear polarization (DNP).
N 3
k 0 1 2 3
P 1 3 3 1
I 0 2 2 0
Iunique 0 1 1 0
Chapter 6: Long-range contacts
136
6.3.3.3 DNP-enhanced 15N-13C TEDOR experiments
Dynamic nuclear polarization using the biradical AMUPol offered a maximal signal
enhancement for DGK of 45-fold (Figure 44a). This level of enhancement could be
reached by an increase of the accessibility and a reduction of the diffusion time of
AMUPol via reducing the volume of the pelleted proteoliposome sample for the
incubation with the biradical. The improvement of the diffusion efficiency might be
necessary due to the comparably low molar protein-lipid-ratio of 1:50. Taken all, these
data confirm that AMUPol is suitable for DNP-enhanced ssNMR on membrane
proteins.
A drawback of DNP are signals from naturally appearing 15N- (~0.4%) as well as 13C-
isotopes (~0.5%), contributing to the spectra pattern of [CN]DGK. They can be
identified by using control experiments ([CC]DGK), which enable to distinguish specific
cross-protomer from natural abundance signals.
This study demonstrates that DNP-enhanced TEDOR experiments allow the detection
of 13C-15N contacts across the protomer interface of trimeric DGK. A cross-peak, which
exposes in the range of Arg-Ne,n, could be visualized (Figure 47).
6.3.3.4 Attemps to assign the cross-peak by RxA-mutants
In order to assign this cross-peak to specific residues, suitably chosen single-site
mutations were introduced into DGK. Therefore, six RxA-mutants were prepared,
covering all arginines (R9, R22, R32, R55, R81 and R92) in DGK. Mixed labelled
complexes of these mutants ([CN]-DGK-RxA) could be produced in the same way as
the wild type, since they feature a similar oligomerization behaviour (Figure 48): All
mutants form trimers in DDM that can be disrupted to mainly monomers by SDS and
reassemble mostly to trimers, when SDS is replaced by DDM. This suggests that the
respective arginines, which are all located in extramembranous parts of DGK, are not
essential for the trimer formation. The driving force for the oligomerization is most likely
the transmembrane domain, which is reported to be more stable than the
extramembranous regions. Lau and Bowie could demonstrate that the denaturation by
SDS occurs in two steps. The first one includes the extramembranous parts, leading to
a partially unfolded intermediate, whereas the second one affects the transmembrane
domain, resulting in a completely unfolded or denatured protein [9].
However, the observed cross-peak did not disappear for each of the six RxA-mutants
(Figure 50), which suggests more than just one interprotomer contact, involving several
arginines. Whereupon, R81A- and R92A-DGK show a reduced cross peak compared
Chapter 6: Long-range contacts
137
to the wild type. Both, Arg81 and Arg92, could be shown by mutational studies to be
functionally relevant (Figure 49) [6, 7], though they are not reported to interact directly
with the nucleotide or lipid substrate [6]. A reason could be a participation in forming
interprotomer contacts, allowing a cross-talk between the protomers or simply by
stabilizing the active site. Due to the crystal structure, a possible interacting partner for
Arg81 is Glu88, located in the CL of the adjacent subunit (Figure 42a) [6]. This contact
might stabilize the loop, which is reported to participate in binding of the nucleotide [6].
It is suggested that the Tyr86 side chain of CL acts as a cover of the nucleotide binding
site in the nucleotide bound state [5]. The Arg81-Glu88 contact possibly keeps the
cover open in the apo state to facilitate binding of the nucleotide. For Arg92, a possible
interacting residue is Asn27, located in the interhelical turn of the adjacent subunit,
linking SH and H1 (Figure 42b) [6]. The possible contact most likely holds both the SH
and the H1 in close proximity towards H2 and H3, stabilizing the active site.
Unfortunately, the contacts cannot be distinguished from one another in the DNP-
enhanced TEDOR spectra (Figure 50). They overlap due to bad spectral resolution,
caused by DNP conditions, involving low field (1H frequency of 400 MHz) and low
temperature (~105 K).
6.3.3.4.1 Drawback of mutations
Not only the bad resolution of DNP spectra poses an obstacle, but also the negative
side-effects of mutations. All RxA-mutants of DGK, used to assign the interprotomer
contacts to specific residues and to elucidate their importance for oligomerization,
feature a significant reduction in activity. This suggests that these mutations have
conformational impact on the protein. Furthermore, it is likely that, if one contact is
omitted, the loss is compensated by another contact. Evidence for that is found in the
TEDOR spectra of R81A- and R92A-DGK with a higher spectral width, allowing the
detection of a possible cross-peak in the lysine range as well (Figure 51). R81A- and
R92A-DGK feature both a significant cross peak in the 15N chemical shift range of
lysine, indicating a participation of this amino acid type in forming an interprotomer
contact, which is not observable for the wild type. This suggests that if either Arg81 or
Arg92 is eliminated, an interprotomer contact involving lysine is formed or strengthened
to compensate the loss of the respective arginine. The arising cross-peak exposes
most likely a salt bridge between 19Lys-Nz and 95Asp-Cg at the protomer interface, as
predicted by the crystal structures (Figure 42c) [5, 6]. Because of the negative side-
Chapter 6: Long-range contacts
138
effects of mutations, double mutants for the identification of cross peaks should not be
taken into account.
Figure 51. DNP-enhanced 2D-TEDOR spectra (tmix = 6.25 ms) of the RxA mutants [CN]-DGK-
R81A and [CN]-DGK-R92A compared to wild-type [CN]-DGK and [CN(Arg,Lys)]-DGK. [CN]-
DGK-R81A and [CN]-DGK-R92A feature both a significant cross peak in the 15
N chemical shift
range of lysine (*), indicating a participation of this amino acid type in forming an interprotomer
contact, which is not observable for the wild type.
6.3.3.5 Assessing the interprotomer contacts during nucleotide binding
Additionally, it was tried to determine, if bound nucleotide substrate affects the cross-
protomer contacts significantly. For this purpose, DGK was saturated with the ATP
analogue AMP-PCP, as described in chapter 5. However, an obvious chemical shift
perturbation or a (dis)appearance of cross-peaks is not observable (Figure 50),
indicating at the first sight no involvement of these Arg-contacts in nucleotide binding.
However, a more detailed look at higher field and temperature with better spectral
resolution would be necessary to judge, if the contacts are really excluded from
nucleotide binding, since a proper detection of conformational changes, identified upon
significant chemical shift changes, is not possible due to the worse line broadening,
Chapter 6: Long-range contacts
139
caused by DNP conditions. Only drastic changes, such as appearance or
disappearance of signals, would lead to unambiguous information about the system
during substrate binding.
6.4 Summary and Outlook
Overall, functionally relevant intra (Arg32-Trp25/Glu28/Ala29; Trp112-Ser61)- and
interprotomer (Arg-contacts) long-range interactions could be detected, which possibly
stabilize the active sites and/or transmit information about substrate binding or changes
of the surrounding lipid bilayer within and between protomers.
Additionally, it could be proven that the highly stable trimeric form of DGK can be
disrupted into monomers by SDS and that, most notably, the effects of SDS on the
protein are reversible, enabling the preparation of fully active mixed labelled trimers.
Furthermore, this study shows that DNP-enhanced TEDOR experiments allow the
detection of 13C-15N contacts across the protomer interface of trimeric DGK. Here,
overlapping cross-peaks, which expose a correlation between Arg-Nn,e and Asp-Cg/
Glu-Cd/ Asn-Cg, could be visualized.
Moreover, this study includes an overall picture with valuable information about all
arginines in DGK, which illustrates that all of them are functionally relevant, but not
essential for the trimer formation.
Next to it, this work demonstrates that mutational studies might not always be the best
choice for the assignment of certain signals, since a mutation holds the risk of
unwanted conformational changes within the protein, leading to ambiguous results.
Unfortunately, working under DNP conditions, involving low field and low temperature,
goes along with bad spectral resolution. Thus, the cross peaks, involving most likely
more than one arginine, could not be assigned to specific residues, since they strongly
overlap. Additionally, a proper detection of conformational changes induced by
nucleotide binding is not possible due to worse line broadening. However, drastic
changes of the cross-peaks during nucleotide binding are not observable.
In order to overcome the issue of bad resolution caused by severe line broadening
associated with the necessity to perform experiments at cryogenic temperatures, DNP
NMR at high magnetic field (800 MHz) in combination with fast MAS (40 kHz) could be
applied. Jaudzems et al. could demonstrate that these conditions yield enhanced
resolution and long coherence lifetimes enabling the acquisition of resolved 2D
correlation spectra. This in turn would allow the detection of better resolved longrange
contacts that can not be observed at room temperature [307].
Chapter 6: Long-range contacts
140
Another option to still assign the detected cross-peaks to specific residues by low field
DNP (400 MHz) and slow MAS (8 kHz), might be the application of more complex
labelling schemes. Here, specific labelling, in detail unique pair labelling, in
combination with a 2D NCOCX experiment could lead to the identification.
Appendix
141
Appendix
Supplementary tables
Table S1. List of chemicals. All were obtained with pro analysis (p.a.) quality.
Chemical Molecular formula Molar mass (g mol-1) Manufacturer
Adenine C5H5N5 135.13 AppliChem
Agar-agar -- -- Roth
Amino acids -- -- AppliChem
Ammonium chloride NH4Cl 53.49 AppliChem
15N-ammonium chloride
15NH4Cl 54.48 Cambridge Isotope Lab.
Ampicilin Natriumsalz C16H18N3NaO4S 371.39 Roth
AMUPol C36H62N4O11 726.90 Bruker
ATP C10H14N5Na2O13P3 551.10 AppliChem
BisTris C8H19NO5 209.24 AppliChem
Calcium chloride CaCl2 * 2H2O 147.01 Sigma-Aldrich
Chloroform CHCl3 119.38 Roth
Cholesterol C27H46O 386.67 Avanti
Coomassie Brilliant Blue G250 C47H48N3NaO7S2 840.01 AppliChem
Cytosine C4H5N3O 111.1 AppliChem
DDM C24H46O11 510.63 AppliChem
Disodium hydrogen phosphat Na2HPO4 141.96 AppliChem
DMPA C31H60O8PNa 614.76 Avanti
DMPC C36H72NO8P 677.93 Avanti
DNase I -- ~31000 AppliChem
DPC C17H38NO4P 351.5 Anatrace
EDTA C10H16N2O8 292.25 AppliChem
Ethanol (96%) C2H6O 46.07 Roth
Glukose C6H12O6 180.16 AppliChem
12C-glucose
12C6H12O6 180.09 Cambridge Isotope Lab.
13C-glucose
13C6H12O6 186.11 Cambridge Isotope Lab.
HEPES C8H18N2O4S 238.31 Roth
Imidazole C3H4N2 68.08 AppliChem
IPTG C9H18O5S 238.30 AppliChem
Isotope labelled amino acids -- -- Cambridge Isotope Lab.
LB-Medium -- -- Roth
LDS Sample Buffer 4X -- -- Thermofisher
Lithiumchlorid LiCl 42.39 abcr
Magnesium chloride MgCl2 * 6H2O 203.30 AppliChem
Magnesium sulfate MgSO4 * 7H2O 246.48 AppliChem
Methanol (99%) CH4O 32.04 Roth
Monopotassium phosphate KH2PO4 136.09 AppliChem
NADH C21H27N7Na2O14P2 709.41 AppliChem
Ni-NTA agarose -- -- Macherey-Nagel
OG C14H28O6 292.38 AppliChem
Phosphoenolpyruvate monopotassium salt C3H4KO6P 206.13 AppliChem
PIPES C8H18N2O6S2 302.37 Roth
Protease Inhibitor cOmplete -- -- Sigma Aldrich
Sodium chloride NaCl 58.44 AppliChem
Thymine C5H6N2O2 126.11 AppliChem
Appendix
142
Uracil C4H4N2O2 112.09 AppliChem
Centrum vitamin tablets -- -- Pfizer Consumer Healthcare
Table S2. List of consumable materials.
Material
Type Manufacturer
Amicon® Ultra-15 Ultracel
® - 10K Merck Millipore Ltd.
Bio-Beads™ SM-2 Adsorbent Media Bio-Rad
Blue Native PAGE Gel Native PAGE Novex BisTris Gel 4-10%, 10 wells Invitrogen
Falcon tube 50, 15 ml Sigma
Glycerol stock beads Roti-Store cryo vials Roth
Microfuge tube Polypropylene 1.5 ml Beckman Coulter®
PD10 column GE17-0851-01-ColumnPD10 GE Healthcare
Petri dishes 90 mm Ø Roth
Plasmid DNA extraction kit NucleoSpin Plasmid Macherey-Nagel
Pipette tips 5000 µl, 1000 µl, 200 µl, 20 µl, 2.5 µl Eppendorf
Plastic tubes 1.5 ml Eppendorf
SDS-PAGE gel RunBlue SDS Gel 4-20% Expedeon
Sterile filter 0.2 µm pore size Sartourius Stedim Biotech
Table S3. List of equipment.
Equipment Type Manufacturer
Autoclave V-75 (75 l volume) Systec
Blue Native PAGE equipment XCell SureLock Mini-Cell System Life Techonologies
Centrifuges
Allegra 21R
Beckman Coulter
Avanti J-E Beckman Coulter
Biofuge Pico Heraeus
GS-15R Beckman Coulter
Gyrotron 263 GHz Bruker
HPLC systems
Äkta Prime GE Healthcare
Äkta Purifier GE Healthcare
Incubators
Innova 44 New Brunswick Scientific
Thermomixer Compact Eppendorf
NMR spectrometers
850 MHz WB Avance III Bruker
400 MHz WB Avance II Bruker
pH meter SevenEasy Mettler Toledo
Rotary evaporator Rotavapor R-200 Büchi
SDS-PAGE equipment Mini-PROTEAN Tetra Biorad
Spectrophotometers V-550 Jasco
NanpDrop 1000 Thermo Scientific
Infinite M200 Tecan Reader
Ultracentrifuge Optima LE-80K Beckman Coulter
Appendix
143
Table S4. Experimental parameters for all multidimensional and dipolar coupling based spectra of DGK in its apo state (white), saturated with AMP-
PCP (light grey), DOG (middle grey) and with AMP-PCP + DOG (dark grey).
dimensionality
2D
experiment PDSD DARR NCA
sample U-13
C,15
N-DGK U-13
C,15
N-DGK-I,L,V U-13
C,15
N-DGK-I,L,V U-13
C,15
N-DGK U-13
C,15
N-DGK + AMP-PCP U-13
C,15
N-DGK + DOG U-13
C,15
N-DGK + AMP-PCP + DOG
figure 21, 22 40a 24 24, 27, 35a/b, 36a, 37a, 38a 33, 35a, 36a, 38a/b 35b, 37a 38b
probehead HCN E-free E-free HCN E-free E-free HCN
recycle delay [s] 2.5 0.8 0.8 3.5 1.0 0.8 2.5
transfer 1 HC-CP HC-CP HC-CP HN-CP HN-CP HN-CP HN-CP
field [kHz] 84(H) 55.6(C) 75.7(H) 55.6(C) 70.9(H) 55.6(C) 72.8(H) 41.7(N) 74.3(H) 41.7(N) 72.1(H) 41.7(N) 76.7(H) 41.7(N)
shape (ramp) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H)
contact time [ms] 1.25 1.20 1.30 1.40 1.40 1.50 1.20
carrier [ppm] 113 113 118.2 118.2 118.2 118.2 118.2
transfer 2 PDSD DARR NCA-DCP NCA-DCP NCA-DCP NCA-DCP NCA-DCP
field [kHz] - 11.8(H) 38(N) 22.8(C) 83.3(H) 38(N) 22.8(C) 100(H) 38(N) 22.8(C) 83.3(H) 38(N) 22.8(C) 83.3(H) 38(N) 22.8(C) 83.3(H)
shape (ramp) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C)
contact/mixing time [ms] 20 800 4.8 3.5 5.0 4.4 3.8
carrier [ppm] 60.9 60.9 60.9 60.9 60.9
T1 increments 1344 1344 176 160 165 176 160
spectral width [kHz] 55.6 55.6 3.8 3.8 3.8 3.8 3.8
aqu. time [ms] 12.0 12.0 23.2 21.0 21.7 23.2 21.1
T2 increments 3390 3390 3380 3390 3380 3380 3380
spectral width [kHz] 100 100 100 100 100 100 100
aqu. time [ms] 17 17 17 17 17 17 17
1H SPINAL decoupling [kHz] 100 83.3 83.3 100 83.3 83.3 83.3
number of scans 72 192 496 208 336 336 128
total measurement time 2d21h 4d21h 20h 1d9h 16h 14h 14h
Appendix
144
dimensionality 2D
experiment NCACX NCOCX
sample U-13
C,15
N-DGK U-13
C,15
N-DGK + AMP-PCP U-13
C,15
N-DGK
figure 36b 36b 41a
probehead HCN E-free E-free
recycle delay [s] 3.0 1.0 1.2
transfer 1 HN-CP HN-CP HN-CP
field [kHz] 88(H) 41.7(N) 74.3(H) 41.7(N) 73.3(H) 41.7(N)
shape (ramp) 80.100 (H) 80.100 (H) 80.100 (H)
contact time [ms] 1.4 1.4 1.4
carrier [ppm] 118.2 118.2 99
transfer 2 NCA-DCP NCA-DCP NCO-DCP
field [kHz] 38(N) 22.8(C) 100(H) 38(N) 22.8(C) 83.3(H) 38(N) 53.2(C)
83.3(H)
shape (ramp) 90.100 (C) 90.100 (C) 90.100 (C)
contact/mixing time [ms] 4.6 4.2 4.6
carrier [ppm] 57.6 57.6 165
transfer 3 DARR DARR DARR
field [kHz] 14.1 (H) 11.9 (H) 11.8 (H)
contact/ mixing time [ms] 50 50 400
carrier [ppm] 57.6 57.6 165
t1 increments 128 120 104
spectral width [kHz] 3.04 3.04 7.6
aqu. time [ms] 21 19.7 6.8
t2 increments 3390 3390 3390
spectral width [kHz] 100 100 100
aqu. time [ms] 17 17 17
1H SPINAL decoupling [kHz] 100 83.3 83.3
number of scans 1464 1600 2960
total measurement time 6d17h 2d21h 7d6h
Appendix
145
dimensionality 3D
experiment NCACX NCOCX CONCA
sample U-13
C,15
N-DGK U-13
C,15
N-DGK-I,LV U-13
C,15
N-DGK + AMP-PCP U-13
C,15
N-DGK + DOG U-13
C,15
N-DGK U-13
C,15
N-DGK-I,LV U-13
C,15
N-DGK U-13
C,15
N-DGK-I,LV
figure 26, 36c, 37b not shown 36c 37b 26 not shown 26 not shown
probehead HCN E-free E-free HCN HCN E-free HCN E-free
recycle delay [s] 2.5 0.8 1.0 2.5 2.5 1.0 3.0 1.0
transfer 1 HN-CP HN-CP HN-CP HN-CP HN-CP HN-CP HC-CP HC-CP
field [kHz] 74.1(H) 41.7(N) 60.4(H) 41.7(N) 74.3(H) 41.7(N) 74.7(H) 41.7(N) 72.8(H) 41.7(N) 60.4(H) 41.7(N) 74.1(H) 55.6© 77.1(H) 55.6©
shape (ramp) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H) 80.100 (H)
contact time [ms] 1.80 0.95 1.40 1.40 1.25 0.95 1.50 1.70
carrier [ppm] 118.2 118.2 118.2 118.2 118.2 118.2 176.6 176.6
transfer 2 NCA-DCP NCA-DCP NCA-DCP NCA-DCP NCO-DCP NCO-DCP CON-DCP CON-DCP
field [kHz] 38(N) 22.8© 100(H) 38(N) 22.8© 83.3(H) 38(N) 22.8© 83.3(H) 38(N) 22.8© 100(H) 38(N) 53.2© 100(H) 38(N) 53.2© 83.3(H) 38(N) 53.2© 100(H) 38(N) 53.2© 83.3(H)
shape (ramp) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C) 90.100 (C)
contact/mixing time [ms] 4.0 4.7 4.6 4.5 4.0 4.8 8.0 7.1
carrier [ppm] 57.6 57.6 57.6 57.6 176.6 176.6 118.2 118.2
transfer 3 DARR DARR DARR DARR DARR DARR NCA-DCP NCA-DCP
field [kHz] 12.5 (H) 11.8 (H) 11.9 (H) 13.6 (H) 12.5 (H) 11.8 (H) 38(N) 22.8© 100(H) 38(N) 22.8© 83.3(H)
shape (ramp) 90.100 (C) 90.100 (C)
contact/ mixing time [ms] 50 50 50 50 100 100 5.0 4.8
carrier [ppm] 57.6 57.6 57.6 57.6 176.6 176.6 57.6 57.6
t1 increments 80 74 80 80 64 64 40 46
spectral width [kHz] 3.04 3.04 3.04 3.04 3.04 3.04 2.5 2.5
aqu. time [ms] 13.2 12.2 13.2 13.2 10.5 10.5 7.9 9.1
t2 increments 64 94 72 64 50 50 48 50
spectral width [kHz] 5.1 6.6 5,07 5,07 2.5 2.5 2.5 2.5
aqu. time [ms] 6.3 7.1 7.1 6.3 9.9 9.9 9.5 9.9
t3 increments 3390 3390 3390 3390 3390 3390 2988 2988
spectral width [kHz] 100 100 100 100 100 100 100 100
aqu. time [ms] 17 17 17 17 17 17 15 15
1H SPINAL decoupling [kHz] 100 83.3 83.3 100 100 83.3 100 83.3
number of scans 64 136 88 88 72 152 96 240
total measurement time 9d19h 9d16h 6d9h 13d12h 7d1h 6d3h 6d12h 6d16h
Appendix
146
Table S5. Resonance assignments of wild-type DGK in DMPC/DMPA liposomes by MAS NMR experiments. Chemical shifts are given in ppm.
ResID N CO Ca Cb Cd Cd1 Cd2 Hd Ce Ce2 Ce3 Ne He Cg Cg1 Cg2 Ch2 Nh1/2 Cz Cz2 Cz3
1 Ala
2 Asn
3 Asn
4 Thr
5 Thr
6 Gly
7 Phe
8 Thr
9 Arg
43.30
3.16
84.50 7.35 27.24
71.69
10 Ile
11 Ile
12 Lys
29.21
42.05
2.98
13 Ala
14 Ala
174.70
15 Gly 113.27 177.97 47.07
16 Tyr 119.52 176.61 59.46
133.85
117.47
17 Ser
18 Trp
19 Lys 116.77 180.05 59.25 32.57
25.75
20 Gly 109.11 174.01 46.56
21 Leu 121.55 177.73 57.50 41.67
23.45
26.02
22 Arg 120.40 177.03 59.16 29.78 44.30
29.82
23 Ala 118.36 180.19 54.41 18.39
24 Ala 121.80 176.86 54.81 15.75
Appendix
147
25 Trp 116.31 176.51 60.75 29.28
126.59
110.67
26 Ile 113.33 177.81 63.94 38.49
13.28
28.84 17.01
27 Asn 111.21 174.96 55.36 41.22
176.90
28 Glu 117.46 174.69 53.42 28.63 180.87
32.91
29 Ala 130.72 178.95 55.33 18.24
30 Ala 116.25 179.25 54.83 18.01
31 Phe 114.63 176.77 61.36 39.31
131.20
139.33
32 Arg 116.47 178.04 58.93 31.50 43.94
83.32
27.90
69.98 158.75
33 Gln 115.09 179.52 59.36 27.86
33.84
34 Glu 115.68 177.02 59.05 26.95
34.39
35 Gly 107.72 174.59 46.99
36 Val 119.17 176.50 67.07 32.15
21.55 21.55
37 Ala 118.85 178.14 54.92 19.35
38 Val 117.04 178.16 67.30 31.08
39 Leu 117.44 178.32
40 Leu 117.35 177.74 58.25 41.14
26.94
41 Ala 117.24 179.25 55.11 18.01
42 Val 118.13 177.96 67.26 31.02
22.94 22.94
43 Val 119.61 177.79 67.72 30.75
22.26 22.26
44 Ile 118.74 177.77 65.96 37.56
13.33
29.75 17.01
45 Ala 120.51 179.15 54.95 18.83
46 Cys 113.21 172.57 63.16 27.62
47 Trp 121.36 176.97 58.17 30.72
48 Leu 116.03 176.56 54.88 44.48
23.35
26.37
49 Asp 123.00 176.03 52.59 39.28
50 Val 109.85 174.87 58.02 33.38
20.29 17.35
Appendix
148
51 Asp 117.78 174.53 52.71 41.67
180.04
52 Ala 121.13 178.54 55.39 19.18
53 Ile 116.01 177.28 65.31 37.14
13.35
29.67 18.89
54 Thr 115.95 175.45 67.45
21.12
55 Arg 119.97 177.62 60.87 29.37 42.70
27.97
56 Val 116.13 178.36 67.23 31.01
24.37 23.39
57 Leu 122.24 179.87 57.67 42.60
26.62
58 Leu 120.34 179.05 57.67 39.96
25.74 24.39
27.02
59 Ile 116.14 178.89 65.29 38.99
16.12
29.00 17.93
60 Ser 116.17 177.13 62.56
61 Ser 115.07 177.34 61.49 63.01
62 Val 114.40 177.57 64.24 30.41
23.31 19.38
63 Met 119.86 179.25 57.15 32.89
30.61
64 Leu 120.25 178.18 57.71 39.98
22.43
25.13
65 Val 115.30 176.57 66.77 31.18
21.99 20.26
66 Met 112.89 177.98 56.64 31.47
67 Ile 119.09 176.63 65.77 38.01
14.24
28.93 17.25
68 Val 116.36 177.23 66.70 31.02
23.23 22.87
69 Glu 120.91 179.11 58.01 29.52 181.23
35.33
70 Ile 122.38 177.26 66.32 37.18
14.83
26.56 19.48
71 Leu 121.20 178.28 57.95 41.68
22.05
26.56
72 Asn 118.20 176.83 56.50 38.45
178.26
73 Ser 115.97 176.06 62.25 63.04
74 Ala 126.91 178.33 55.37 17.54
75 Ile 118.39 177.28 65.09 36.97
12.81
29.34 16.61
76 Glu 119.75 176.88 59.99 28.77 182.67
35.65
Appendix
149
77 Ala 119.70 178.92 55.01 17.00
78 Val 116.34 177.02 66.27 30.86
23.53 22.96
79 Val 119.21 179.05 67.17 30.95
24.33 22.95
80 Asp 120.95 178.21 56.46 39.41
81 Arg 122.54 178.18 57.45
42.24
84.30
27.50
71.70 158.13
82 Ile 121.11 176.84 64.49 37.96
15.16
28.86 16.32
83 Gly 105.00 172.69 44.96
84 Ser 121.37 179.12 58.34
85 Glu
86 Tyr
87 His
88 Glu 129.84 179.21 59.55 29.52
35.36
89 Leu 121.29 179.02 57.31 41.66
27.08
90 Ser 116.49 175.47 62.30 62.56
91 Gly 106.74 174.75 47.04
92 Arg 120.47 177.89 59.02 31.36 44.27
80.77
25.16
76.04 159.39
93 Ala 119.23 179.12 55.82 18.80
94 Lys 114.08 179.99 59.41 32.43 29.86
41.71
26.55
95 Asp 122.43 178.72 57.58 39.11
96 Met 119.97 177.09 60.26 33.92
16.91
32.09
97 Gly 106.66 175.57 47.55
98 Ser 113.65 177.28 61.82 62.57
99 Ala 125.37 178.14 54.34 17.85
100 Ala 120.41 178.62 55.58 17.94
101 Val 116.25 177.43 66.17 31.30
21.17
102 Leu 119.23 178.20 58.16 40.74
23.80 22.02
27.04
Appendix
150
103 Ile 115.82 177.00 64.63 36.09
11.07
27.45 18.79
104 Ala 121.52 179.59 55.96 17.02
105 Ile 120.16 149.30 66.03 37.44
12.98
30.73 16.95
106 Ile 120.13 177.49 66.12 37.14
12.92
29.40 16.71
107 Val 118.35 178.58 67.08 31.06
22.88 22.88
108 Ala 126.54 178.22 56.07 16.67
109 Val 117.62 178.17 67.19 30.99
22.00 22.91
110 Ile 118.96 176.72 66.29 38.40
13.27
29.78 17.04
111 Thr 116.61 175.14 67.90 68.50
20.77
112 Trp 120.50 177.88 62.90 27.39
125.68 130.41
137.55 118.98 130.66
123.76
114.76 120.84
113 Cys 115.39 176.33 64.95 27.45
114 Ile 116.27 178.76 65.74 37.70
13.75
27.38
115 Leu 116.25 180.15 57.44 40.71
22.52
26.63
116 Leu 118.76 178.24 57.69 40.39
21.84
27.09
117 Trp 115.61 175.24 64.57 26.34
118 Ser 115.44 175.38
119 His
120 Phe
121 Gly
Appendix
151
Table S6. Summary of all significant perturbations in peak position and intensity
during the interaction of DGK with its substrates.
*Arg9 and Lys12 are detected in scalar coupling based experiments. The other
residues are observed in dipolar coupling based experiments.
AMP-PCP DOG
weighted CSP peak intensity weighted CSP peak intensity
9 Arg Cg x reduced*
Cd x reduced*
Ne x reduced*
Nh1/h2 x reduced*
12 Lys Cd x reduced*
Ce x reduced*
15 Gly Ca x disappeared x reduced
CO x disappeared x reduced
16 Tyr Ca x disappeared
Cb x disappeared x disappeared
Cd1 x disappeared
Ce2 x disappeared x disappeared
CO x disappeared x reduced
19 Lys Cb x disappeared
Cg 0.39 increased
CO x reduced
20 Gly Ca 0.22 increased
CO 0.22 increased
22 Arg Cd x disappeared
23 Ala Cb 0.35 x
CO 0.26 x
25 Trp Cg 0.38 x
26 Ile Cb 0.22 reduced
Cg1 0.22 x
Cg2 x increased
Appendix
152
Cd x increased x disappeared
27 Asn Cb 0.20 x
Cg 0.21 x
28 Glu Cg 0.40 reduced x reduced
Cd x disappeared
CO 0.21 x
29 Ala Ca 0.22 x
Cb 0.23 x
CO 0.31 x
31 Phe Cb 0.27 x
Cg 0.58 reduced x reduced
Cd 0.57 x x reduced
Ce x appears
32 Arg Cb 0.24 reduced
Cg 0.41 x
Cd 0.25 reduced
33 Gln Cb x reduced x reduced
Cg 0.54 reduced 0.31 reduced
CO 0.23 reduced
37 Ala Ca x reduced
Cb 0.21 reduced
CO x reduced
40 Leu Cd x appears
41 Ala Cb 0.30 x
43 Val Cb 0.35 x
Cgb 0.36 x
45 Ala Cb 0.22 x
CO 0.47 x
46 Cys Ca 0.23 reduced
Cb 0.27 reduced
Appendix
153
CO 0.22 reduced
47 Trp Cb x disappeared
48 Leu Cd 0.24 x
49 Asp Ca 0.34 x
Cb 0.38 increased x increased
CO 1.29 increased x increased
50 Val Ca x disappeared x increased
Cb x disappeared x increased
Cgb x disappeared x increased
Cga x disappeared x increased
CO x disappeared x increased
51 Asp Ca x reduced
Cb x reduced
Cg 0.33 reduced
CO x reduced
53 Ile Cg2 0.40 x
CO 0.28 x
55 Arg Cb 0.27 reduced
Cg x reduced
Cd 0.28 reduced
56 Val Cg1 0.32 x
57 Leu CO 0.22 reduced
62 Val Cb 0.38 x
Cg2 0.23 x
Cg1 0.43 x
65 Val CO 0.29 x
66 Met Ca 0.21 reduced
Cb x reduced
CO 0.27 x
Appendix
154
67 Ile Cg1 x reduced
Cd x reduced
69 Glu Cb 1.20 reduced 0.35 increased
Cg x disappeared
Cd x disappeared x increased
70 Ile Ca 0.28 x
Cb 0.38 x
Cg1 0.31 x 0.35 reduced
Cg2 0.39 x 0.24 x
Cd 0.47 increased 0.29 increased
CO 0.27 x
72 Asn Ca 0.20 x
74 Ala Cb 0.32 x
76 Glu Cb x reduced
Cg 0.26 reduced
Cd x disappeared x increased
77 Ala CO 0.22 x
79 Val Cb 0.28 x
Cg1 0.45 x
Cg2 0.28 x
CO 0.44 x
80 Asp Ca 0.36 x
Cb 0.25 x
CO 0.28 x
81 Arg Cg 0.39 x
Cd 0.62 x
Cz x reduced
82 Ile Ca 0.62 reduced
Cb 0.44 reduced
Cg1 0.43 reduced
Cg2 0.54 reduced
Cd2 0.48 reduced
Appendix
155
CO 0.54 reduced
83 Gly Ca x increased
CO x increased
88 Glu Ca 0.39 reduced
Cb 0.29 reduced
Cg x disappeared
CO 0.21 reduced
91 Gly Ca 0.70 x
CO 0.80 x
92 Arg Ca x reduced
Cb 0.24 reduced
Cg x reduced
Cd x reduced
CO x reduced
94 Lys Cb 0.27 x
Cg 0.28 x
Cd 0.76 x
Ce 0.20 x
95 Asp Cb x reduced
97 Gly Ca 0.23 x
CO 0.31 x
98 Ser Ca x reduced
Cb x reduced
CO x reduced
99 Ala Ca x reduced
Cb x reduced
CO x reduced
101 Val Cg1 0.30 x
102 Leu Ca 0.29 x
Cd1 0.21 x
Appendix
156
111 Thr CO 0.28 x
114 Ile Cg1 x increased
Cd x increased
116 Leu Ca x disappeared
Cb x reduced
Cg x reduced
Cd x disappeared
CO x disappeared
Appendix
157
List of abbreviations
ADP adenosine 5‘-diphosphate
AMP-PCP adenylylmethylenediphosphonate
ATP adenosine 5‘-triphosphate
a.u. arbitrary units
BMRB biological magnetic resonance bank
BN-PAGE blue native-polyacrylamid gel electrophoresis
BSA albumin from bovine serum
CE cross effect
CHAPSO 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate
CL cytosolic loop
CMC critical micelle concentration
CP cross polarization
Cryo-EM cryo electron microscopy
CSA chemical shift anisotropy
CW continuous wave
DAG diacylglycerol
DCP double cross polarization
ddH2O double-distilled water
DGK diacylglycerol kinase
DARR dipolar assisted rotational resonance
DBG 1,2-dibutyryl-sn-glycerol
DM n-decyl-β-glucopyranoside
DDM n-dodecyl-β-glucopyranoside
DMPA 1,2-dimyristoyl-sn-glycero-3-phosphate
DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
DNA deoxyribonucleic acid
DNP dynamic nuclear polarization
DOG 1,2-dioctanoyl-sn-glycerol
DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol
DPC n-dodecylphosphocholine
DPPC 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine
DSS 4,4-dimethyl-4-silapentane-1-sulfonic acid
EDTA ethylenediaminetetraacetic acid
FID free induction decay
Gd3+
-DOTA gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
GPCR G-protein coupled receptor
H1-3 helix 1-3
HEPES 4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid
HETCOR heternuclear correlation experiment
HPLC high-performance liquid chromatography
IMAC immobilized metal affinity chromatography
IPTG isopropyl β-D-1-thiogalactopyranoside
KD dissociation constant
KM Michaelis-Menten constant
Appendix
158
LB lysogeny broth
LCP lipidic cubic phase
LDH lactate dehydrogenase
LDS lithium dodecyl sulfate
LILBID-MS liquid beam ionization/desorption mass spectrometry
M molecular weight
MAG monoacylglycerol
MAS magic angle spinning
MDO membrane derived oligosaccharides
MDS molecular dynamics simulation
NAD nicotinamide adenine dinucleotide
Ni-NTA Nickel-nitrilotriacetic acid
NMR nuclear magnetic resonance
ns number of scans
OD optical density
OE Overhauser effect
OG n-octyl-β-D-glucopyranoside
PA phosphatidic acid
PC phosphocholine
PCR polymerase chain reaction
PDB protein data bank
PDSD proton driven spin diffusion
PE phosphoethanolamine
PG phosphatidyl glycerol
Pi inorganic phosphorus
PK pyruvate kinase
PKC protein kinase C
PL periplasmic loop
ppm parts per million
PRE paramagnetic relaxation enhancement
REDOR rotational echo double resonance
RF radiofrequency
RT room temperature
S/N signal-to-noise ratio
SDS sodium dodecyl sulfate
SDS-PAGE SDS polyacrylamid gel electrophoresis
SE solid effect
SEC size exclusion chromatography
SH surface helix
SPINAL small phase incremental alternation
ssNMR solid state nuclear magnetic resonance
T interhelical turn
T1 spin lattice relaxation constant
T1ρ spin lattice relaxation constant in the rotating frame
T2 spin-spin relaxation constant
TEDOR Transferred echo Double Resonance
TEPS triethylphosphine
Appendix
159
TM transmembrane (helix)
TPPM two phase pulse modulation
Tris tris(hydroxymethyl)aminomethane
UV/VI ultraviolet/visible
v/v volume per volume
wt wild type
w/v weigth per volume
1D one dimensional
2D two dimensional
Appendix
160
List of figures
Figure 1. Physiological role of E.coli diacylglycerol kinase (DGK) in recycling during the
biosynthesis of membrane-derived oligosaccharides (MDOs) [1, 2] that are largely
generated in response to environmental stress, such as low osmolarity [15, 16]. DGK is
located within the inner membrane, where it catalyzes the ATP-dependent
phosphorylation of potentially membrane-disruptive diacylglycerol (DAG) to non-toxic
phosphatic acid (PA), providing the basis for restoring phosphatidylglycerol (PG), which
is consumed in the MDO cycle. The cartoon is based on the X-ray structure using the
PDB ID 4UXX [6]. The figure is adapted from Van Horn et al. [84]. ............................... 9
Figure 2. Sequence alignment of wild-type DGK and the two thermostable mutants,
Δ4- and Δ7-DGK [5]. The mutations in Δ4- and Δ7-DGK are labelled green. The N-
terminal tag is highlighted orange. .............................................................................. 14
Figure 3. Topology plot of wild-type DGK. The plot was created based on the DGK X-
ray structure [5] and refined by the CSI values obtained from chemical shifts in this
study (Table S5). The membrane is indicated by two solid black lines as calculated in
the PPM server [116]. The secondary structure elements of DGK are denoted as: CL,
cytoplasmic loop; H1-3, helices 1-3; PL, periplasmic loop and SH, surface helix. ....... 15
Figure 4. Comparison of the solution NMR (PDB 2KDC, wild-type DGK) and crystal
structure of DGK (PDB 3ZE5, Δ4-DGK). A view of the crystal (right) and solution NMR
(left) structure from the cytoplasm (a) and the membrane (b) plane. .......................... 17
Figure 5. Comparison of the DGK secondary structures obtained from solution NMR
(PDB 2KDC, wild-type DGK, chain A), solid state NMR [14], and X-ray crystallography
(PDB 3ZE5, Δ4-DGK, chain A): Rectangles symbolize α-helical regions, whereas solid
lines reflect deviations from helicity. Residues that were not resolved are illustrated by
dashed lines. The differences between the secondary structures are highlighted in
green. Both the ssNMR and the X-ray studies used a thermostabilized mutant, whereas
the wild type was used for solution NMR structure determination. ............................... 19
Figure 6. Substrate-binding sites determined in the X-ray structure (Δ4-DGK, PDB
4UXX) [6]. (a) Structure-based and possible interactions with the non-hydrolysable ATP
analogue adenylylmethylenediphosphonate (AMP-PCP, blue) and its two counterions
(Zn, orange). The figure is adapted and modified from Li et al. [6]. (b) Possible
interactions of Ser17, Glu69 and Ser98 with the lipid substrate monoacylglycerol (MAG,
yellow). ....................................................................................................................... 22
Appendix
161
Figure 7. Impact of magic angle spinning (MAS) at 54.74° on solid state NMR spectra.
(a) Depiction of a MAS rotor that is tilted in the magic angle β = 54.74° with respect to
the magnetic field B0. 15N (b)- and 1H (c)-NMR spectra of the microcrystalline tri-peptide
N-formyl-Met-Leu-Phe-OH under static (blue) and MAS (red) conditions. In the static
15N-NMR spectrum, the isotropic (δiso) and the anisotropic (δaniso) chemical shift as well
as the CSA parameters δxx, δyy and δzz are labelled accordingly. Comparing the 15N-
and 1H-NMR spectra under MAS of 25 kHz, it becomes obvious that higher spinning
speeds are needed to obtain well-resolved 1H-NMR spectra (see chapter 4, outlook),
whereas the 15N-NMR spectrum features already a good resolution at 25 kHz. The
figures are adapted from the lecture script “Solid state NMR”, prepared by Prof.
Clemens Glaubitz, Goethe University Frankfurt am Main, summer semester 2015. .... 32
Figure 8. Pulse sequence of a cross polarization (CP) experiment according the
Hartmann-Hahn condition, in which magnetization is transferred from highly abundant I
spins to dilute S spins. ................................................................................................ 34
Figure 9. Pulse sequence of a 2D 13C-13C PDSD experiment. During the preparation
time, the magnetization is transferred from 1H to 13C via CP step. This is followed by
the t1 period, when the 13C chemical shift evolves, while 1H nuclei are decoupled.
Thereafter, the magnetization is transferred back on the z-axis through a 90° pulse on
the 13C spins and the mixing step takes place, in which the proton driven spin diffusion
between 13C nuclei occur through space by flip-flop interactions. For detection, the 13C
spins are transferred back in the x,y-plane and the FID is recorded under 1H
decoupling. ................................................................................................................. 36
Figure 10. (a) Pulse sequence of the NCACX and NCOCX experiment. Both are 15N-
13C correlation transfer experiments with a subsequent 13C-13C mixing step. During the
preparation period, a broad-band 1H15N-CP step is used to generate 15N polarization
that evolves during t1 under proton decoupling. For the 15N-13C transfer, optimized spin
lock fields on the 15N and 13C channel are applied under proton decoupling. The 13C off-
set is centered in the Cα region for NCA and in the CO region for NCO. After the
double cross polarization (DCP), evolution on 13Cα/13CO takes place under proton
decoupling during t2. Subsequently, a DARR step follows, which transfers the
magnetization to any other proximate 13C nuclei. Therefore, two 90° pulses at an off-set
of 100 ppm were applied for excitation and reconversion of longitudinal magnetization.
The detection of 13C magnetization (t3) represents the final step, during which the
protons are decoupled. (b) Polarization transfer pathway for the NCACX (left) and
NCOCX (right) pulse sequence, schematically illustrated for a di-peptide. The selected
Appendix
162
off-set frequencies on Cα or CO enable a magnetization transfer within the same amino
acid [i] along the side chain, resulting in cross peaks in the NCACX spectrum, or along
the side chain of the previous amino acid [i-1], leading to cross peaks in the NCOCX
spectrum, respectively (black arrows). Additional through-space dipolar-assisted
pathways are possible as well (grey arrows). .............................................................. 38
Figure 11. (a) Pulse sequence of the CONCA experiment. It is a 15N-13C correlation
transfer experiment accomplished by three CP steps. During the preparation period, a
selective 1H13C-CP step is used to generate 13C polarization that evolves during t1
under proton decoupling. This is followed by a second selective CP step, which
transfers magnetization from 13CO[i-1] to 15N[i]. The 13C off-set is centered in the CO
region for CON. The magnetization then evolves on 15N[i] during t2 under proton
decoupling. Subsequently, the third CP step takes place, transferring magnetization
from 15N[i] to 13Cα[i]. The 13C off-set is centered in the Cα region for NCA. The
detection of 13C magnetization (t3) represents the final step, during which the protons
are decoupled. (b) Polarization transfer pathway for the CONCA pulse sequence,
schematically illustrated for a di-peptide. The selected off-set frequencies on CO and
later Cα enable a magnetization transfer from CO of the previous amino acid [i-1] via N
towards Cα (black arrow) and sometimes Cβ (grey arrow) of the following amino acid
[i], leading to cross peaks in the CONCA spectrum (black arrows). ............................. 39
Figure 12. Pulse sequence of the 13C-13C TOBSY experiment. The magnetization is
transferred from 1H to attached 13C nuclei through a refocused INEPT step based on J-
couplings: After the initial 90º 1H pulse, 1H chemical shift evolution during the variable
t1 period takes place. The evolution delay is fixed to achieve antiphase 1H
magnetization with respect to 13C via JHC (JHC ~200 Hz). The magnetization is
transferred to 13C by applying simultaneous 90º 1H and 13C pulses. The 13C chemical
shift evolution during the variable t2 period takes place. This is followed by isotropic 13C
mixing using TOBSY. The 13C-13C correlation is established through carbon-carbon J-
couplings (JCC ~35-53 Hz). The detection of 13C magnetization represents the final step
(t3), during which the protons are decoupled. .............................................................. 40
Figure 13. The expected low number of interacting spin pairs make dynamic nuclear
polarization for signal enhancement essential, which is obtained by the three spin cross
effect using the biradical AMUPol as a source for unpaired electrons. (a) Reconstituted
mixed labelled DGK doped with AMUPol [133] is depicted. (b) It is subjected to
continuous wave microwave irradiation, resulting in polarization transfer from electrons
via protons to the sites of interest................................................................................ 42
Appendix
163
Figure 14. TEDOR pulse sequence [136]. The sequence starts with a CP-step,
transferring magnetization from 1H to 13C nuclei. This is followed by two REDOR-steps
(tmix/2) to reintroduce heteronuclear dipolar couplings, which are enclosed by 90°
pulses. L0 describes the number of 180° pulses during tmix/4. The evolution time (t1)
takes place in-between the two REDOR steps. The detection of 13C magnetization (t2)
represents the final step, during which the protons are decoupled. ............................. 44
Figure 15. Sequence alignment of wild-type DGK and the thermostable mutant, Δ4-
DGK [89]. The mutations in Δ4-DGK are labelled green. The N-terminal tag is
highlighted orange. ..................................................................................................... 45
Figure 16. (a) Comparison of the activity of wt- (dark grey) and Δ4-DGK (red)
reconstituted into DMPC/DMPA. Both samples are prepared the same way. (b)
Comparison of the activity of reconstituted wtDGK, prepared in DDM (dark grey) and
DPC (red). 100% activity corresponds to the rate recorded with wtDGK in 90mol%
DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1 mg-1. All activity measurements were
repeated three times. The activity was calculated as the mean value. Error bars
correspond to standard deviations. (c) Size exclusion chromatography (SEC) of wtDGK
in 0.05% DDM (c1) and 0.5% DPC (c2), performed after several weeks at 4°C or
immediately after the IMAC step, respectively............................................................. 68
Figure 17. Characterization of the purity and oligomeric state of wtDGK in DDM
micelles. (a) The SDS-PAGE verifies the purity of the protein solution after IMAC
purification. (b) The BN-PAGE offers a reliable assessment of the oligomeric state,
clearly showing wtDGK as trimer in DDM micelles. (c) The trimeric state of wtDGK in
DDM micelles is confirmed by LILBID-MS: The signals for the monomeric, dimeric and
trimeric form of wtDGK are labelled by “1”, “2” and “3”, respectively. They occur at
charged states of −1 and −2. The LILBID mass spectrum was recorded by Oliver Peetz
of the research group of Prof. Dr. Nina Morgner (Institute of Physical and Theoretical
Chemistry, Goethe University Frankfurt am Main). ...................................................... 69
Figure 18. Superimposed 2D 13C-13C PDSD spectra of U-13C,15N-DGK embedded into
90mol% DMPC/ 10mol% DMPA (black) or 67.3mol% DMPC/ 32.7mol% cholesterol
(red), showing a similar fingerprint. The enlargement of a representative region in the
2D 13C-13C PDSD spectra displays clearly a reduced resolution for 67.3mol% DMPC/
32.7mol% cholesterol (red). For instance, the selected peak, P, features a line width in
F1 dimension of 138 Hz and 406 Hz and in F2 dimension of 574 Hz and 1006 Hz for
Appendix
164
90mol% DMPC/ 10mol% DMPA (black) and 67.3mol% DMPC/ 32.7mol% cholesterol
(red), respectively. The line widths were obtained from CCPN analysis 2.4.1 [175]. ... 73
Figure 19. Comparison of the activity of wtDGK reconstituted into DMPC/DMPA with
different molar protein-to-lipid ratios increasing from 1:80 to 1:20. The ratio of 1:50
(grey) is used for all subsequent studies. 100% activity corresponds to the rate
recorded with wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1 mg-1.
Experiments were repeated three times. The activity was calculated as the mean value.
Error bars correspond to standard deviations.............................................................. 74
Figure 20. Sucrose density gradient (40%-10%) centrifugation of empty liposomes
(left) and wtDGK reconstituted in DMPC/DMPA in a molar protein-to-lipid ratio of 1:50
(right), revealing homogeneous size distribution of proteoliposomes without any empty
liposomes observable. ................................................................................................ 74
Figure 21. Evaluation of the proteoliposom sample by MAS NMR. 1D 13C and 15N
cross polarization (CP) spectra of U-13C,15N-wtDGK reconstituted into DMPC/DMPA,
exhibit a good spectral resolution (a). 2D 13C-13C PDSD spectrum of U-13C,15N-wtDGK
reconstituted into DMPC/DMPA. A mixing time of 20 ms was used to yield one bond
correlations between aliphatic atoms. The spectrum evaluates structural homogeneity,
resolution and secondary structure. The high number of well resolved peaks
demonstrates a homogeneous sample preparation (b). .............................................. 75
Figure 22. Comparison of MAS ssNMR data of the wild type with its thermostable
mutant. 2D 13C-13C PDSD spectrum of U-13C,15N-wtDGK, recorded with a mixing time
of 20 ms (left). Enlargement of the selected region in the 2D 13C-13C PDSD spectrum
(right). The spectrum is compared with the assignment of the thermostable mutant of
DGK gained by MAS NMR [14]. The comparison shows that a transfer of these
assignments to the wild type sample is not possible. The red cross peaks were
obtained with CCPN analysis 2.4.1 [175]. ................................................................... 77
Figure 23. Examples of membrane protein structures determined in phospholipids by
solid state NMR. (a) Anabaena sensory rhodopsin, ASR: PDB 2m3g, with bound retinal
(yellow) [174]. (b) human chemokine receptor, CXCR1: PDB 2lnl [179]. (c) M2 1H
channel from influenza virus: PDB 2l0j [180, 181]. (d) Bacterial inner membrane protein
DsbB: PDB 2leg [182, 183]. (e) Mycobacterium cell division protein, CrgA: PDB 2mmu
[184]. (f) Membrane-inserted form of the fd bacteriophage coat protein: PDB 1mzt
[185]. The figure is adapted from Marassi and Opella [186]. ....................................... 78
Appendix
165
Figure 24. Comparison of the 2D NCA spectra of uniform labelled U-13C,15N-wtDGK
(black) and reverse labelled U-13C,15N-wtDGK-I,L,V (green). The 15N and 13Ca signals
of the uniform labelled U-13C,15N-wtDGK exhibit already comparable good average
linewidths of approximately 105 and 185 Hz, respectively. To resolve residual
ambiguities, Ile, Leu and Val are specifically unlabelled in the reverse labelled sample.
The inscriptions for Ile, Leu and Val are labelled in red and for the other amino acids in
black. In the 2D NCA of the reverse labelled sample, the peaks for Ile, Leu and Val are
clearly missing as expected. Apart from that, the two NCA spectra do not remarkably
differ from each other. ................................................................................................. 86
Figure 25. 15N CP spectra of U-13C,15N-DGK. The spectra were recorded with an E-
free (green) and with a standard (black) 3.2 mm triple-resonance HCN MAS probehead
(Bruker). In both cases 128 scans were applied. The E-free probehead enables to use
a recycle delay of 0.8 s, saving ~3x of the measurement time compared to the standard
probehead with a recycle delay of 2.5 s. The E-free probehead was custom-built and is
still under development. .............................................................................................. 87
Figure 26. Resonance assignment of U-13C,15N-wtDGK based on a set of 3D NCACX,
NCOCX and CONCA spectra. A representative sequential walk from I26 to A29 is
shown. Each set of three spectra represents a Cx[i−1]–N[i]–Cx[i] spin system. For
example, the N27 NCACX peaks are connected to the I26-N27 CONCA peak via the
same N and Ca. The I26 NCOCX peaks are linked to the I26–N27 CONCA peak
through the same N and CO, resulting in a Cx[i−1]–N[i]–Cx[i] system, which is linked
with the preceding system through all carbon shifts of I26 that are visible in both
NCACX and NCOCX spectra. The assignments are depicted by lines. ....................... 89
Figure 27. 2D NCA spectrum of U-13C,15N-DGK with assigned peaks labelled. .......... 90
Figure 28. Automated resonance assignment by ssFLYA confirms 91.5% of the
backbone and 89.1% of all (backbone + side chains) assignments obtained manually.
Assignments are classified as strong, if ≥ 80% of the individual chemical shift values
from 20 independent runs of the algorithm differ by less than 0.55 ppm from the
consensus value (strong colors). Other assignments by ssFLYA are graded as weak
(light colors). From other studies by ssFLYA, they are known to be erroneous for 39 –
72% [13]. Each assignment for an atom is symbolized by a colored rectangle: green -
assignment by ssFLYA agrees with the manual reference assignment within a
tolerance of 0.55 ppm; red - assignment does not match with the reference; blue -
assigned by ssFLYA, but not manually; black – assigned manually, but not by ssFLYA.
Appendix
166
The second row illustrates backbone assignments for N, Ca, and CO. The third to
eighth row represent the side chain assignments. For branched side chains, the
relevant row is subdivided into an upper part for one branch and a lower part for the
other branch. ssFLYA was performed by Dr. Sina Kazemi of the research group of
Prof. Dr. Peter Güntert (Institute for Biophysical Chemistry, Goethe University Frankfurt
am Main). He also kindly provided this figure. ............................................................. 91
Figure 29. 2D scalar coupling based 1H-15N HETCOR (a), 1H-13C HETCOR (b) and
13C-13C TOBSY (c) of U-13C,15N-DGK with tentative assignments. All residues, which
could not be detected and assigned by dipolar coupling based experiments are
considered as possible candidates for detection by experiments based on scalar
coupling. INEPT and TOBSY were applied for 1H-15N or 1H-13C heteronuclear
polarization and 13C-13C homonuclear mixing, respectively. Peaks for Arg9 and Lys12
are labelled green, as they could be assigned unambiguously. Peaks for the aromatic
rings were folded in the indirect dimension to save measurement time. Amino acids
that refer to the His-tag are labelled by ‘tag’. ............................................................... 93
Figure 30. Resonance assignment of DGK. (a) Each assignment for an atom is
symbolized by a blue rectangle: The second row illustrates backbone assignments for
N, Ca, and CO. The third to eighth row represent the side chain assignments. For
branched side chains, the relevant row is subdivided into an upper part for one branch
and a lower part for the other branch. This figure was kindly provided by Dr. Sina
Kazemi of the research group of Prof. Dr. Peter Güntert (Institute for Biophysical
Chemistry, Goethe University Frankfurt am Main). (b) The assigned residues are
mapped on the topology plot of DGK. The plot was created with respect to the X-ray
structure of DGK (PDB 3ZE5) [5] and refined by CSI values obtained from chemical
shifts (Table S5). The membrane is depicted by two solid black lines. 84% residues of
DGK were assigned by dipolar and scalar coupling based experiments...................... 94
Figure 31. Secondary structure analysis based on the chemical shifts. The chemical
shift index (CSI) Δδ is derived from the difference between the experimentally
determined MAS NMR chemical shifts (exp) for Ca and Cb and their random coil
standard chemical shifts (rc) according to Δδ =[δCa(exp)-δCa(rc)] - [δCb(exp)-δCb(rc)]
[206]. For Gly residues and residues without any assignment of Cb, only Ca secondary
shifts were used. Strongly positive (≥ 1.5 ppm) values of the CSI imply an α-helical
structure, whereas negative or near-zero values indicate deviations from helicity. (a)
The secondary structure of wild-type DGK determined by MAS NMR is compared with
the MAS NMR structure of the thermostable mutant [14] and the X-ray structure of
Appendix
167
wtDGK (PDB 3ZE4, chain A) [6]. Rectangles represent α-helical regions involving the
surface helix (SH) and the three transmembrane helices (H1-3), whereas solid lines
symbolize deviations from helicity including the interhelical turn (T), the periplasmic
(PL) as well as the cytoplasmic loop (CL). Residues that were not resolved by ssNMR
or by X-ray crystallography are depicted by dashed lines. Disparities between the three
secondary structures are highlighted in green. (b) The 2D NCACX spectrum of U-
13C,15N-DGK shows all assigned glycines. The regions for helical and random coil (rc)
secondary structure are coloured. Gly83 and Gly91 are labelled bold, since the DGK X-
ray structure exhibits asymmetries for both residues: Both were observed within a
helical and a random coil structure [5]. These asymmetries are not detectable by MAS
NMR. .......................................................................................................................... 96
Figure 32. Solid state NMR 1H detected 2D 1H-15N correlation spectrum (2D hNH) of
fully protonated U-13C,15N-DGK in phospholipid bilayers. The spectrum was recorded
on a Bruker 600 MHz spectrometer at ~278.15-283.15 K and a MAS rate of 111 kHz
(Bruker 0.7 mm rotor, ~0.5 mg sample). It was conducted with 400 scans and a duty
cycle of 0.8 s. The total measurement time was ~12 h. Some, well-resolved residues
from the extramembrane regions (green) could be assigned based on the assignments
from 13C/15N detected experiments conducted at a MAS rate of 15.2 kHz (Table S5).
The spectrum was recorded by Dr. Venita Decker at Bruker BioSpin GmbH in
Rheinstetten. ............................................................................................................ 101
Figure 33. DGK in the AMP-PCP bound state. (a) Competitive inhibition assay verifies
the binding of Mg*AMP-PCP to the active sites of DGK. DGK proteoliposomes were
incubated with 4 to 16 mM of Mg*AMP-PCP, equating 4 to 16-fold molar excess
compared to DGK. Mg*ATP (3 mM) was present in each sample. A concentration of at
least 10 mM of Mg*AMP-PCP (10-fold molar excess) is needed to decrease the activity
of DGK below 10%, resulting in a fully saturated system. 100% activity corresponds to
the rate recorded with wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9) µmol
min-1 mg-1. Experiments were repeated three times. The activity was calculated as the
mean value. Error bars correspond to standard deviations. (b) The 31P-CP MAS
spectrum confirms the binding of AMP-PCP. For this purpose, proteoliposomes were
incubated with 14 mM Mg*AMP-PCP (pH 7.2). (c) The 2D NCA spectra of U-13C,15N-
DGK-I,L,V incubated with 14 mM Mg*AMP-PCP (pH 7.2), recorded immediately after
the incubation (black) and after 30 d (green), show that the fully saturated system is
stable over a long period of time without any significant evidence of degradation. .... 105
Appendix
168
Figure 34. (a) DGK in the DOG bound state. DGK was reconstituted into 80 mol%
DMPC/DMPA and 20 mol% DOG and incubated with 14 mM Mg*ATP (pH 7.2). DGK
phosphorylates DOG to DOG-PA, which can be observed by 31P-MAS NMR, both by
cross- and direct polarization. The spectra prove that DOG can reach the active site of
DGK under the here applied experimental conditions. (b) DGK in the DOG+AMP-PCP
bound state. 31P-CP spectrum of DGK reconstituted into 80 mol% DMPC/DMPA and
20 mol% DOG and incubated with 14 mM Mg*AMP-PCP (pH 7.2). It illustrates 31P
species of the bound AMP-PCP, which demonstrates a binding of the nucleotide to
DGK. ......................................................................................................................... 106
Figure 35. The DGK trimer adopts a symmetric conformation in its substrate bound
states. (a) Superposition of 2D NCA spectra of apo DGK (black) and AMP-PCP-bound
DGK (green). Regions for helical and random coil (rc) secondary structure are
highlighted for glycines. (b) Superposition of 2D NCA spectra of apo DGK (black) and
DOG-bound DGK (yellow). For the AMP-PCP and the DOG bound state no peak
splitting can be observed. ......................................................................................... 107
Figure 36. Effect of nucleotide binding on DGK. (a) Superposition of 2D NCA spectra
of DGK’s apo (black) and AMP-PCP-bound (green) state. Representative pronounced
shifts are illustrated in subsections of 2D NCACX (b) and 3D NCACX (c) spectra. (d)
The topology and ribbon model of the DGK monomer are shown with residues
highlighted that are affected by AMP-PCP. In the topology maps, alterations in peak
intensity and different levels of weighted CSPs are distinguished. In the ribbon model of
monomeric DGK, residues, which show a response on AMP-PCP binding, are
highlighted in green. The ribbon model is obtained from the OPM database [116], using
the PDB ID 4UXX from the X-ray structure [6]. .......................................................... 109
Figure 37. Effect of DOG binding on DGK. (a) Superposition of 2D NCA spectra of
DGK’s apo (black) and DOG-bound (yellow) state. (b) Representative extractions from
the 3D NCACX illustrate shifts for Glu69 and Gln33. (c) Superposition of 15N and 13C
INEPT-based experiments of the apo (black) and DOG-bound (yellow) state of DGK. In
the DOG bound state, the INEPT signals are decreased compared to the apo state,
indicating a reduction in mobility. Arg9 and Lys12, which could be assigned
unambiguously, are highlighted. (d) The topology and ribbon model of the DGK
monomer highlight residues that are affected by DOG. In the topology maps,
alterations in peak intensity and different levels of weighted CSPs are distinguished. In
the ribbon model of monomeric DGK, residues, which show a respond on DOG, are
Appendix
169
highlighted in green. The ribbon model is obtained from the OPM database [116], using
the PDB ID 4UXX from the X-ray structure [6]. .......................................................... 111
Figure 38. Effect of AMP-PCP and DOG binding on DGK. (a) Superposition of 2D NCA
spectra of apo (black) and AMP-PCP-bound (green) DGK. (b) Superposition of 2D
NCA spectra of AMP-PCP-bound (green) and AMP-PCP+DOG-bound (pink) DGK.
Both the AMP-PCP bound and AMP-PCP+DOG bound states feature a similar
fingerprint with significant alterations compared to the apo state. ............................. 112
Figure 39. Enlarged view from the membrane plane, illustrating the crystal structure of
Δ4 DGK (PDB 3ZE5) [5], accommodating the lipid substrate (orange) in the
hydrophobic pocket. Possible interactions between the side chain of Arg9, Lys12,
Gln33 and Glu69 with the proximal OH group of the lipid substrate are depicted. ..... 115
Figure 40. Intraprotomer interactions in the transmembrane region of DGK between
Trp112 and Ser61. (a) 2D 13C-13C DARR spectrum of U-13C,15N-DGK-ILV with 800 ms
mixing time. Crosspeaks appear between Ca of Ser61 and the side chain carbons of
Trp112 revealing an intraprotomer contact between helices 2 (Ser61) and 3 (Trp112).
(b) Visualization of the intraprotomer contact between Trp112 and Ser61 in the crystal
structure of Δ4 DGK (PDB 4UXX) [6]. Enlarged view from the membrane plane,
accommodating the lipid substrate (yellow) in the hydrophobic pocket (left). View from
the periplasm, depicting the three monomers in different shades of grey (right). Trp112
(H3) is secured by a hydrogen bond to Ser61 (H2) in the lower region of the
hydrophobic pocket. .................................................................................................. 118
Figure 41. Intraprotomer interactions in the cytoplasmic region of DGK between Arg32
and Trp25/Glu28/Ala29. (a) 2D NCOCX spectrum of U-13C,15N-DGK with a 400ms
DARR mixing step. Crosspeaks between 32ArgNh1/2 and 32ArgNe with 25TrpCO,
Glu28Cb and 29AlaCa/Cb/CO are determined, caused by an intraprotomer contact
between these residues in helix 1 and the surface helix. (b) Depiction of the
intraprotomer contact involving Arg32 in the crystal structure of Δ4 DGK (PDB 3ZE5)
[5]. Enlarged view from the membrane plane, illustrating the intraprotomer contacts for
32ArgNe/Nh1,2 with 25TrpCO, Glu28Cb and 29AlaCa/b/O. ..................................... 119
Figure 42. Representative possible interprotomer contacts in DGK suggested by the
crystal structure of Δ4 DGK (PDB 3ZE5) [5]. Enlarged view from the cytoplasm (a) and
membrane plane (b, c), illustrating a possible interprotomer contact for Arg81 and
Glu88 (a), Arg92 and Asn27 (b) as well as Lys19 and Asp95 (c). ............................. 120
Appendix
170
Figure 43. Creation of active mixed labelled trimers of DGK. (a) Differently labelled
trimers of DGK are separately expressed, solubilized, purified and eluted in DDM.
Subsequently, they are disassembled into monomers or dimers by SDS and mixed in a
1:1 ratio. Then, SDS is removed and replaced by DDM, resulting in mixed labelled
DGK trimers, which can then be reconstituted into liposomes. (b) BN-PAGE of DGK
(0.2 mg/ml) in different SDS concentrations: (1) 0.5%, (2) 1%, (3) 1.5%, (4) 2%, (5) 3%.
The higher the SDS concentration, the higher is the degree of disassembly into
monomers. (c) LILBID-MS confirms the predominantly monomeric state of DGK in SDS
micelles: The signals for the monomeric, dimeric and trimeric form of DGK are labelled
by “1”, “2” and “3”, respectively. They occur at a charged state of −1. The LILBID mass
spectrum was recorded by Oliver Peetz of the research group of Prof. Dr. Nina
Morgner (Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt
am Main). (d) The BN-PAGE shows DGK as trimer in DDM micelles before (1) and
after (3) SDS treatment and mostly in its monomeric state in SDS micelles (2). (e)
Activity of DGK reconstituted from different detergent environments: DGK trimers from
DDM micelles before SDS treatment (-SDS, dark grey) and after SDS treatment
(±SDS, green) as well as DGK monomers from SDS micelles (+SDS, yellow) were
reconstituted. 100% activity corresponds to the rate recorded with wtDGK in 90mol%
DMPC/ 10mol% DMPA of 90 (± 9.9) µmol min-1 mg-1. Experiments were repeated three
times. The activity was calculated as the mean value. Error bars correspond to
standard deviations. .................................................................................................. 123
Figure 44. Validation of the application of AMUPol. (a) DNP enhancement shown for a
13C−CP spectrum of DGK incubated with 20 mM AMUPol. Upon microwave irradiation,
a 45-fold sensitivity enhancement is reached. (b) Activity of DGK with (+) and without (-
) AMUPol, indicating that the presence of the biradical has no influence on the activity.
100% activity corresponds to the rate recorded with wtDGK in 90mol% DMPC/ 10mol%
DMPA of 90 (± 9.9) µmol min-1 mg-1. Experiments were repeated three times. The
activity was calculated as the mean value. Error bars correspond to standard
deviations. ................................................................................................................ 124
Figure 45. DNP-enhanced 1D-TEDOR spectra of the control sample ([CC]-DGK) at
different mixing times. The spectra were recorded with 4096 scans at a 400 MHz
spectrometer, ~105 K, pH 7.2 and a spinning speed of 8 kHz. The 263 GHz gyrotron
was operated at a collector current of 70 mA. Six spectra were recorded with L0 (rotor
periods) = 4, 8, 16, 24, 32, and 40. ........................................................................... 125
Appendix
171
Figure 46. DNP-enhanced 1D-TEDOR spectra of [CN]-DGK) at different mixing times.
The spectra were recorded with 3520 scans at a 400 MHz spectrometer, ~105 K,
pH 7.2 and a spinning speed of 8 kHz. The 263 GHz gyrotron was operated at a
collector current of 70 mA. Six spectra were recorded with L0 (rotor periods) = 4, 8, 16,
24, 32, and 40. .......................................................................................................... 127
Figure 47. DNP-enhanced 15N−13C-TEDOR spectra (tmix = 6.25 ms) of [CN]-DGK,
[CN(Arg,Lys)]-DGK and the control sample, [CC]-DGK. All spectra reveal cross-peaks
originating from natural abundance intramolecular backbone 13C−15N-contacts
(highlighted in grey). Further cross-peaks (highlighted green) are detected in [CN]-DGK
and [CN(Arg,Lys)]-DGK. They can be assigned to cross-protomer contacts, reflecting a
through-space correlation between Arg and Asn/Asp/Glu. These cross-peaks
demonstrate that salt bridges or H-bonds between Asn/Asp/Glu and Arg must be
present at the protomer interfaces. ........................................................................... 129
Figure 48. Characterization of the oligomeric state of the single-site RxA mutants in
comparison to wtDGK by BN-PAGE. The BN-PAGES show DGK as trimer in DDM
micelles before (1) and after (3) SDS treatment and mainly as monomers in SDS
micelles (2). All RxA mutants show a similar oligomerization behavior as the wt,
indicating that the respective arginines located in extramembranous regions of DGK
are not necessary for the trimer formation. ................................................................ 131
Figure 49. Kinase activity of DGK affected by single site RxA mutations, expressed as
a percentage of wt activity. DGK trimers from DDM micelles (dark grey) and DGK
trimers from DDM micelles after SDS treatment (green) were reconstituted into lipid
bilayers and then measured. The activity is clearly reduced for all RxA-mutants
compared to the wild type. The SDS treatment of the RxA mutants led to a loss of
activity of only up to 20%, which is comparable to the wt. 100% activity corresponds to
the rate recorded with wtDGK in 90mol% DMPC/ 10mol% DMPA of 90 (± 9.9) µmol
min-1 mg-1. Experiments were repeated three times. The activity was calculated as the
mean value. Error bars correspond to standard deviations. ...................................... 132
Figure 50. DNP-enhanced 15N−13C-TEDOR spectra of mixed labelled trimers (U-
13C/U15N12C-DGK). The interprotomer crosspeak does not disappear for each single-
site RxA-mutant, demonstrating that more than one Arg contributes to these
interactions. Based on the 3D crystal structure [6], only Arg81 and Arg92 have an
appropriate location at the interface to be involved in these interactions. This would be
in-line with the observed reduction of the cross peak intensities for R81A- and R92A-
Appendix
172
DGK. For the AMP-PCP bound state of DGK, no significant changes of the cross-peak
are observable. ......................................................................................................... 133
Figure 51. DNP-enhanced 2D-TEDOR spectra (tmix = 6.25 ms) of the RxA mutants
[CN]-DGK-R81A and [CN]-DGK-R92A compared to wild-type [CN]-DGK and
[CN(Arg,Lys)]-DGK. [CN]-DGK-R81A and [CN]-DGK-R92A feature both a significant
cross peak in the 15N chemical shift range of lysine (*), indicating a participation of this
amino acid type in forming an interprotomer contact, which is not observable for the
wild type. ................................................................................................................... 138
List of tables
Table 1. Mapping of the active site through the identification of functionally relevant
residues by mutational studies, X-ray crystallization, MD simulations and solution NMR
................................................................................................................................... 23
Table 2. Primer sequences for single-site mutations .................................................. 46
Table 3. Components of PCR reaction mixture ........................................................... 47
Table 4. Standard PCR program used for mutagenesis of DGK ................................. 47
Table 5. Composition of M9 minimal medium for the expression of unlabelled DGK .. 49
Table 6. Composition of M9 minimal medium for the expression of U-13C,15N-DGK .... 49
Table 7. Composition of M9 minimal medium for the expression of U-13C,15N-DGK-I,L,V
................................................................................................................................... 50
Table 8. Composition of M9 minimal medium for the expression of U-13C-DGK .......... 52
Table 9. Composition of M9 minimal medium for the expression of U-12C,15N-DGK .... 52
Table 10. Composition of M9 minimal medium for the expression of U-12C,15NArg,Lys-
DGK ............................................................................................................................ 52
Table 11. Sample preparation for BN-PAGE .............................................................. 55
Table 12. Detergents used and compared in this dissertation. The classification, CMC
and concentration range are shown. ........................................................................... 70
Table 13. Amino acid composition of wild-type DGK. The numbers in brackets belong
to the His6-tag and linker. The most frequent hydrophobic amino acids: Ile, Leu and
Val, which were chosen for reverse labelling, are highlighted orange. ........................ 80
Table S1. List of chemicals. All were obtained with pro analysis (p.a.) quality. ......... 141
Table S2. List of consumable materials. ................................................................... 142
Appendix
173
Table S3. List of equipment. ..................................................................................... 142
Table S4. Experimental parameters for all multidimensional and dipolar coupling based
spectra of DGK in its apo state (white), saturated with AMP-PCP (light grey), DOG
(middle grey) and with AMP-PCP + DOG (dark grey). .............................................. 143
Table S5. Resonance assignments of wild-type DGK in DMPC/DMPA liposomes by
MAS NMR experiments. Chemical shifts are given in ppm........................................ 146
Table S6. Summary of all significant perturbations in peak position and intensity ..... 151
References
174
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Declaration of contributions
192
Declaration of contributions
Except where stated otherwise by reference or acknowledgment, the work presented
was generated by myself under the supervision of my advisors during my doctoral
studies. All contributions from colleagues are explicitly referenced in the thesis. The
material listed below was obtained in the context of collaborative research:
Figure 17c
o Title
Characterization of the purity and oligomeric state of wtDGK in DDM micelles.
(c) The trimeric state of wtDGK in DDM micelles is confirmed by LILBID-MS.
o Collaboration partner
The LILBID mass spectrum was recorded by Oliver Peetz of the research group
of Prof. Dr. Nina Morgner, Institute of Physical and Theoretical Chemistry,
Goethe University Frankfurt am Main.
o My contribution
I provided the sample for the LILBID MS measurements and modified the
figure.
Figure 28
o Title
Automated resonance assignment by ssFLYA confirms 91.5% of the backbone
and 89.1% of all (backbone + side chains) assignments obtained manually.
o Collaboration partner
ssFLYA was performed by Dr. Sina Kazemi of the research group of Prof. Dr.
Peter Güntert, Institute for Biophysical Chemistry, Goethe University Frankfurt
am Main. He also created this figure.
o My contribution
I provided the peak lists of the 3D NCACX, NCOCX and CONCA spectra of
uniform and reverse labelled DGK.
Figure 32
o Title
Solid state NMR 1H detected 2D 1H-15N correlation spectrum (2D hNH) of U-
13C,15N-DGK in phospholipid bilayers.
Declaration of contributions
193
o Collaboration partner
The 2D 1H-15N correlation (2D hNH) spectrum was recorded by Dr. Venita
Decker at Bruker BioSpin GmbH in Rheinstetten.
o My contribution
I provided the sample for the 1H detected 2D 1H-15N correlation experiment and
transferred the assignments from 13C/15N detected experiments on well-
resolved residues in this spectrum.
Figure 43c
o Title
Creation of active mixed labelled trimers of DGK. (c) LILBID-MS confirms the
predominantly monomeric state of DGK in SDS micelles.
o Collaboration partner
The LILBID mass spectrum was recorded by Oliver Peetz of the research group
of Prof. Dr. Nina Morgner, Institute of Physical and Theoretical Chemistry,
Goethe University Frankfurt am Main.
o My contribution
I provided the sample for the LILBID MS measurements and modified the
figure.
1,2-Dibutyrylglycerol (DBG)
o Collaboration partner
The synthesis of 1,2-dibutyrylglycerol (DBG) was carried out by Andreas Jakob
of the research group of Prof. Dr. Alexander Heckel, Institute of Organic
Chemistry and Chemical Biology, Goethe University Frankfurt am Main.
Publications
The sections 3.2.3, 3.2.5, 3.2.6, 4.1, 4.2.1, 4.2.2, 4.2.3, 4.2.3.1, 4.2.4, 4.2.5, 4.2.6,
4.2.7, 5.2, 5.3, 6.2, 6.3.2.1, 6.3.2.2, 6.3.2.3.2, 6.3.2.3.3 correspond (in part) to the
manuscript Möbius et al.: Global response of wild-type E. coli diacylglycerol kinase
towards nucleotide and lipid substrate binding observed by 3D and 2D MAS NMR.
Figure 1; Figure 17a,b; Figure 20; Figure 21a,b; Figure 22; Figure 24; Figure 26; Figure
27; Figure 28; Figure 29a,b,c; Figure 30b; Figure 31a; Figure 33a,b,c; Figure 34a,b;
Figure 35a,b; Figure 36a,b,c,d; Figure 37a,b,c,d; Figure 38a,b; Figure 39; Figure 40a,b;
Figure 41a,b; Figure 42a,b; Figure 43a,d; Figure 44a,b; Figure 47; Figure 48; Figure 49
and Figure 50 as well as Table S4; Table S5 and Table S6 are adapted from this
manuscript.
194