Peter HöferProduct Manager EPRPittsburgh – April 2016

EPR in Structural Biology

Innovation with Integrity

EPR speciesnaturally occurring

• Metal Centers: Cu2+, Mn2+, Fe3+, Mo5+, …

• Radicals: tyrosine, tryptophan, quinol, carotenoid……

• Antioxidants: ascorbate, polyphenols, nitroaromatic drugs…

• Small Molecule: NO, H2O2, O2, OH…

• Defect Centers: O-vacancy, irradiation damage…

EPR speciesreporter molecules

• Spin Probes: TEMPOL, Trityl, DPPH…

• Spin Labels: PROXYL, MTSSL…

• Spin Traps: DMPO, DEPMPO, PBN, MNP; CMH…

N

O

SSO2CH3

CH3

CH3H3C

H3CHS Protein

N

O

S

CH3

CH3H3C

H3C

S Protein

+

CC

Cys

Cys

Not binding

Binding

Transforming

EPR samples

• Liquids and Solids

• Can be measured on the same instrument (same probehead)

• For low temperature VT accessories are required. T range 4-300K: He or cryogen-free VT, 100–500K: liq. and gaseous N2

• Sample concentration range and typical volume

• X-band: nM-M & 50 - 150 µlFor aqueous sample @ RT capillaries or flat cells are used instead of tubes

• Q-band: nM-mM & 5 – 15 µl

• No molecular size restriction!

• In-vitro and in-vivo

4/18/2016 5

(Most) proteins and nucleic acids don’t have unpaired electrons !    no EPR?! 

... but ...

we can introduce the wanted unpaired electron into almost any system under investigation 

and we can do this also (almost) wherever we want  

Spin Labelling Bio Molecules

Site‐directed spin labeling (SDSL)

4/18/2016 6

What Is a Spin Label?

…a stable chemical compound which possesses an unpaired electron (i.e. it is a stable radical) and a specific reactive group which binds to (bio)molecules.

The vast majority of spin labels are nitroxides, where the unpaired electron is located at an –NO group, which is usually part of a heterocyclic ring.

Functional groups contained within the spin label allow them to be attached to the molecule under investigation and determine their specificity. 

e.g.: Protein thiol groups (SH‐C) specifically react with the functional groups of the spin labelmethanethiosulfonate,maleimide, andiodoacetamide,

creating a covalent bond with cysteine. Any amino acid of the protein, one at the time, can be replaced with cysteine by site‐directed mutagenesis. If a natural Cys present –outside of the region of interest‐additional site‐directed mutagenesis step (Cys to Ser) is required, unless the native Cys is buried and hence not accessible to the spin label.

A little bit of history:Spin labels were first synthesized in the laboratory of H. M. McConnel in 1965.The first spin labelling studies have been performed using thiol‐specific functional groups to label natively occurring cysteins in proteins (e.g. in hemoglobin).Site‐directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W. L. Hubbell in the late 80s/early 90s.

4/18/2016 7

SDSL-EPR-Tools: Overview

• Spin Label Mobilityreflected in the EPR spectral lineshapes

=> provide a fingerprint of the protein foldand its dynamics

• Accessibilitytowards paramagnetic relaxation 

enhancement molecules (NiEDDA, CrOx, O2)=> Discrimination between lipid bilayer, aqueous phase and protein interior

• Polarityof the SL microenvironment

=> reflected in the Azz. Increasing Azz points to shifts in polar environment 

• Spin‐Spin Distance determination (~ 8 – 100 A)CW‐EPR:   8‐20 ADEER:        15‐100 A

Information about Structure and Dynamics

Spin Label Environment

Free tumbling

Strongly immobilized

Moderately immobilized

Effects from molecular motion N

Effects from relaxation

30 uM

15 mM

50 mM

N

Characterize the paramagnetic center environment

Spin Label MobilityEPR-SDSL & Intrinsically Disordered Proteins

EPR spectral shape is sensitive to the mobility of the label which is described by the rotational correlation time (c ) A spectral modification represents change in

the environment of the label affecting its mobility and thus reveals structural transitions such as folding events

Region of the intrinsically disordered NTAIL C-terminal domain that undergoes an α-helical-induced folding in the presence of the partner protein PXD .

EPR spectral shape broadening in the case of a disorder‐to order transition due to an induced folding mechanism

Before After folding

0.01 0.1 1 10 1000.1

1

P1/2

23.9 mW

11.1 mW

P / mW0.01 0.1 1 10 100

P1/2

3.9 mW

1.8 mW

P / mW

Solvent Accessibilityspectrum saturation

in air

in nitrogen

bound free

bound free

Lactate Dehydrogenase (LDH) – NAD+ crystal structure.

Calculated solvent accessible surface.

Spin Label attachment at

P1/2P1/2

In collaboration with W. Trommer24 mW

11 mW

4 mW

2 mW

April 18, 2016 11

Dependence of the isotropic hyperfine coupling, a0

N, on solvent polarity, for DOXYL (circles) and TOAC (squares) spin labels in proticsolvents

Polarity of the SL microenvironment

a0N

EMXnano, EMXmicro, EMXplusMulti purpose research instruments

EPR Product PortfolioCW-EPR

Innovation with Integrity

Distance measurementsElectron Spin as a Molecular Microscope

Pulse-EPR: ESEEM, HYSCORE, ENDOR

CW-ENDOR

S-I: < 8 Å

Pulse-EPR: DEER/PELDOR

S-S: 15 – 100 Å

CW-EPR

S-S: 5 - 20 ÅS-I: 0 - 4 Å

Electron-Nuclear Hyperfine Coupling

isotropic anisotropic

Pulse EPR: ESEEMLipid Membrane

/2

3p-ESEEM

2p-ESEEM

D2O solvent

#D2O seen by SL at the membrane surface

#D2O

Electron-Electron Dipolar Coupling

pump

D r - 3

EE

observe

0 g1 g2 e2

2 h r3= dd =dd

2 ( 3 cos2 –1 )

r / nm dd / MHz

1.5 15.42.0 6.52.5 3.33.0 1.9

Pulse EPR: Dipolar SpectroscopyDEER/PELDOR

observe

pump

3420 3450 3480 3510Field / G

3530

Dipolar oscillation

Background

/2

Pulse EPR: Dipolar EPR SpectroscopyDEER/PELDOR

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 00 .00 .10 .20 .30 .40 .50 .60 .70 .80 .91 .0

1 2 3 4 5 6D is ta n c e [n m ]

Nor

mal

ized

Ech

o A

mpl

itude

t [n s ]

NNOO

1

A BRAB

Pulsed Double Electron Electron Resonance (DEER) Spectroscopy:Measures the dipole–dipole interaction between two unpaired electron spins and is being used to determine long range distances (15 - 100 Å)

DEER vs other techniques

Innovation with Integrity

DEER covers the entire length scale

Pulsed EPR: Dipolar EPR SpectroscopyDEER/PELDOR

site-directedmutagenesis spin labeling

with MTSSLDEERExperiment&Analysis

Wild Type Cys

SH-OH-

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 00 .00 .10 .20 .30 .40 .50 .60 .70 .80 .91 .0

1 2 3 4 5 6D is ta n c e [n m ]

Nor

mal

ized

Ech

o A

mpl

itude

t [n s ]

Information content

Distance range: 15 - 100 Å

Distance distribution

Orientation information

Correlate structure and structural changes to

functionality

Pulsed EPR: Dipolar EPR SpectroscopyDEER/PELDOR

Samples: proteins, RNA, DNA,protein-protein, protein-RNA complexes

Types of paramagnetic centers Radicals endogenous: tyrosine tryptophan, quinone…

exogenous: nitroxide and trityl spin labels

Transition ion metals endogenous: Cu, Fe, Mo/W, Ni, Co, Mn…

exogenous: Mn, Cu, Gd, spin labels

Typical concentration 50 - 200 µM

Volume X-Band: 50 - 100 µl

Q-Band: 5 – 15 µl

Temperature: typically 50K-100K

Spin labels

Advantages:

No limitations on molecular size

Works in phospholipids

Works in-cell

Pulsed: Dipolar EPR SpectroscopyPELDOR / DEER: Proteins

The distance between a single pair of spin

labeled mutants is measured at a time

Distance determination between multiple spin

labels is possible however the analysis is

more complicated

Pulsed: Dipolar EPR SpectroscopyPELDOR / DEER: Membrane Proteins

Distance determination in various intermediate states direct observation of large conformational changes

Distance measurements in liposomes Explore structure and conformational dynamics in native-like

environment

Black=open, red=closed

Pulsed EPR: Dipolar EPR SpectroscopyPELDOR / DEER: RNA & DNA

In-Vivo

In-cell: RNA and DNA

In-vivo determination of intramolecular distances in nucleic acids understanding their conformational

flexibility

Strong change in vitro vs in cell

Pulsed EPR: Dipolar EPR SpectroscopyPELDOR / DEER: NMR Meets EPR

Combining NMR and EPR:

Powerful, novel approach for structure determination of large

protein–RNA complexes

Binding of RsmE protein to the RsmZ sRNA:

ELEXYS E580: DEER/PELDOR

Pulse EPR

ELEXYS E580

Q-Band ~ 25 min Acquisition time

X-Band ~ 22 h Acquisition time

Dedicated GUI for ease of use

Recent S/N improvements:

Going from X- to Q-Band (> 50)

Shaped pulse (> 3)

Gain in throughput!

References

• Spin Label Mobility (slide 9)– Martinho, M., et. al., Assessing induced folding within the intrinsically disordered C-terminal

domain of the Henipavirus necleoproteins by site-directed spin labeling EPR spectroscopy (2012) 13(5), p453. doi: 10.1080/07391102.2012.706068

• Polarity of Microenvironemnt (slide 11)– Marsh, D., Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies:

Transmembrane Profiles, Appl Magn Reson (2010) 37(1-4), p435. doi:10.1007/s00723-009-0078-3

• DEER (slide 22)– Lumme, C., et. al., Nucleoties and Substrates Trigger the Dynamics of the Toc34 GTPase

Homodimer Involved in Chloroplast Preprotein Translocation, Structure (2014), http://dx.doi.org/10.1016/j.str.2014.02.004

• DEER (slide 23)– Duerr, K. L., et. al., Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and

desensitized states. Cell (2014) 158(4), p 778. doi: 10.1016/j.cell.2014.07.023– Zou, P., et. al., Conformation Cycle of the ABC transporter MsbA in Liposomes. Detailed

Analysis using Double Electron-Electron Resonance Spectroscopy, J Mol. Biol. (2009) 393(3), p586. doi:10.1016/j.jmb.2009.08.050

– Mchaourab, H. S., et. al., Toward the Fourth Dimension of Membrand Protein Structure: Insight into Dynamics from Spin0labeling EPR Spectrscopy, Structure (2011) 19(11), p 1549. doi:10.1016/j.str.2011.10.009

© Copyright 2012 Bruker Corporation.

References

© Copyright 2012 Bruker Corporation.

• DEER (slide 24)– Krstic, I., et. al., Long-Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR

Spectroscopy. Angew. Chem. Int. Ed. (2011) 50(22), p 5070. doi: 10.1002/anie.201100886– Haensel, R., et. al., In-Cell NMR and EPR Spectroscopy of Biomacromolecules, Angew. Chem.

Int. Ed. (2014) 53(39), p10300. doi: 10.1002/anie.201311320• DEER (slide 25)

– Duss, O., et. al., EPR-aided approach for solution structure determination of large RNAs or protein-RNA complexes, Nature Comm. (2014) 5. doi: 10.1038/ncomms4669

– Duss, O., et. al., Structural basis of the non-coding RNA RsmZ acting as a protein sponge, Nature (2014) 509, p588. doi: 10.1038/nature13271

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