structural studies of lipid systems · 6 biomembranes phosphatidylcholine (symmetric fatty acid)...
TRANSCRIPT
1
Structural studies of lipid systems
Hamburg, 22.10.2012
Regine Willumeit
Introduction
What is important to know about lipids?
What can be measured?
Example for structure determination
2
Lipids are important for:
Cells -> membranes
in sensors
Polymeric implants (MPC polymers)
Crystallisation
Joints
As co-factors or as HDL (lipoproteins)
Biomembranes
3
Lipids in Cells
@Lehninger Biochemistry
Composition of an Erythrocyte Membrane
@Lehninger Biochemistry
cell recognition
intra celluar signals
apoptosis signal
unknown
unknown
PEPC
PS
4
Lipid Names
Phospholipids = 4 letter code
first two letter: chains
last two letter: head group
POPC: Palmitoyl-Oleoyl Phosphatidyl Cholin
Structure of a Phospholipid
PE PC PS
5
Structure of a Phospholipid: Headgroups
neutral
negative negative chargecharge
ZetaZeta Potential:Potential:PC = 0PC = 0 PE = PE = --2525 PG = PG = --60 mV60 mV
neutralneutralneutralneutral
Biomembranes
958.48TetracosanoicLignoceroyl24:0
930.43TrocosanoicTrucisanoyl23:0
902.37DocosanoicBehenoyl22:0
874.32HeneicosanoicHeniecosanoyl21:0
846.27EicosanoicArachidoyl20:0
818.21NonadecanoicNonadecanoyl19:0
790.16OctadecanoicStearoyl18:0
762.10HeptadecanoicHeptadecanoyl17:0
846.273,7,11,15-tetra
methylhexadecanoicPhytanoyl16:0 [(CH3)4]
734.05HexadecanoicPalmitoyl16:0
706.00PentadecanoicPentadecanoyl15:0
677.94TetradecanoicMyristoyl14:0
649.89TridecanoicTridecanoyl13:0
621.84DodecanoicLauroyl12:0
593.78UndecanoicUndecanoyl11:0
565.73DecanoicCapryl10:0
537.67NonanoicNonanoyl9:0
509.62OctanoicCapryloyl8:0
481.57HeptanoicHeptanoyl7:0
453.51HexanoicCaproyl6:0
425.46PentanoicPentanoyl5:0
397.41TetranoicButanoyl4:0
369.35TrianoicPropionoyl3:0
M.W.IUPACTrivialCarbonNumber
Phosphatidylcholine (Symmetric Fatty Acid)
1,2-Diacyl-sn-Glycero-3-Phosphocholine (Saturated Series)
@Avanti Polar Lipids
DP
DM
6
Biomembranes@Avanti Polar LipidsPhosphatidylcholine (Symmetric Fatty Acid)
1,2-Diacyl-sn-Glycero-3-Phosphocholine (Unsaturated Series)
954.45 15-cis-
tetracosenoicNervonoyl24:1
878.184,7,10,13,16,19 (all
-cis) docosahexaenoic
DHA 22:6
898.3413-cis-docosenoicErucoyl22:1
830.145,8,11,14(all -cis) eicosatetraenoic
Arachidonoyl20:4
842.2311-cis-eicosenoicEicosenoyl20:1
778.069-cis-12-cis-15-
cisoctadecatrienoicLinolenoyl18:3
782.099-cis-12-cis-
octadecadienoicLinoleoyl18:2
786.139-trans-
octadecenoicElaidoyl18:1
786.159-cis-octadecenoicOleoyl18:1
786.136-cis-octadecenoicPetroselinoyl18:1
730.029-trans-
hexadecenoicPalmitelaidoyl16:1
730.029-cis-hexadecenoicPalmitoleoyl16:1
673.919-trans-
tetradecenoicMyristelaidoyl14:1
673.919-cis-tetradecenoicMyristoleoyl14:1
M.W.IUPACTrivialCarbon Number
Biomembranes@Avanti Polar Lipids
Phosphatidylcholine (Asymmetric Fatty Acid)
1-Acyl-2-Acyl-sn-Glycero-3-Phosphocholine
788.14StearoylOleoyl18:1-18:0
760.09PalmitoylOleoyl18:1-16:0
732.03MyristoylOleoyl18:1-14:0
834.17DocosahexaenoylStearoyl18:0-22:6
810.15ArachidonoylStearoyl18:0-20:4
786.13LinoleoylStearoyl18:0-18:2
788.14OleoylStearoyl18:0-18:1
762.10PalmitoylStearoyl18:0-16:0
734.05MyristoylStearoyl18:0-14:0
806.12DocosahexaenoylPalmitoyl16:0-22:6
782.09ArachidonoylPalmitoyl16:0-20:4
758.07LinoleoylPalmitoyl16:0-18:2
760.09OleoylPalmitoyl16:0-18:1
762.10StearoylPalmitoyl16:0-18:0
706.00MyristoylPalmitoyl16:0-14:0
734.05StearoylMyristoyl14:0-18:0
706.00PalmitoylMyristoyl14:0-16:0
M.W.2-Acyl1-AcylCarbonNumber
PO
PSPM
SO
7
Biomembranes
@Lehninger Biochemistry
Amphipatic Molecules in Solution
hydrophilic hydrophobic
highlyhighly diluteddiluted systemsystem increaseincrease of of concentrationconcentration
CMC = CMC = criticalcritical micellemicelle concentrationconcentration
@Lehninger Biochemistry
8
Amphipatic Molecules in Solution
POPGinverse POPE
POPC
DPPE, DPPC, POPC, POPE….
@Lehninger Biochemistry
Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31.
What can be measured?
monoolein/water
9
What can be measured?
PO=Palmitoyl-oleoyl (16:0-18:1); DP=Dipalmitoyl (16:0)
Data Base: LIPIDAT
Phase Behaviour of
Phospholipids
Lamellar Phase
Repeat Distance d
Bragg-equation:
nn = 2d= 2d··sinsin
4 Q = 2 k0 sin =
sin
Seddon Handbook of Biol Physics 1995
10
Small Angle Small Angle ScatteringScattering on on LipidLipid VesiclesVesicles also also isis DiffractionDiffraction
http://www.encapsula.com/products_01.html and Lehninger Biochemistry
Lipid unilamellarvesicle
multilamellarvesicle
total ensemble SAS
Diffraction
BiomembranesDiffraction of POPG Membranes (Neutrons)
Inte
nsi
ty
q-value
1. Order
11
BiomembranesDiffraction of POPG Membranes (Neutrons)In
ten
sity
q-value
1. Order
Peaks equidistant
Repeat distance = 2 / q
Peak Distance = Repeat distance/nn = 1, 2, 3, 4,….
Intensities & Phases ->
Scattering length density profile
12
Biomembranes(invers) hexagonal phase POPE Membrane (SAXS)
Biomembranes(invers) hexagonal phase POPE Membrane (SAXS)
Peaks NOT equidistant
Repeat distance = 2 / q
Peak distance = Repeat distance/nn = 1, 3, 2, 7, 3, 12, 13, …
13
Cubic Phase
Seddon Handbook of Biol Physics 1995
Cubic Phase
Peaks NOT equidistant
Repeat distance = 2 / q
Peak distance = Repeat distance/nn = 1, 2, 3, 2, 5, 6, 8, 3, …
Seddon Handbook of Biol Physics 1995
14
Sample Preparation
Solution of lipids in chloroform or methanol oder mixture
Drying of solution to obtain a lipid film
Hydratisation of the film with water / solvent
Drying of vesicles solution on support
LiposomesLiposomes
MultilamellarMultilamellar LayersLayers
Example for structure determination
Main problem: fast acquisition of antibiotic resistance by bacteria
Possible alternative to 'classic' antibiotics: discovery of natural and
synthetic antibiotic peptides
Belong to the innate immune system in most species
Peptide Antibiotics
'Infectious diseases are the leading cause of world-wide and third leading cause of death in the United States'
J.M. Hughes, Director of NCID & CDC, 1999
15
BiomembranesPeptide Antibiotics
Magainins
(Xenopus laevis,
Bombina variegata)
Melittin
Cecropins
(Hyalophora cecropia)
Thionine
Plant Defensines
(Heuchera sanguinea)
What is known about Peptide Antibiotics?
'Killing' mechanism:
Destruction of cytoplasmic membrane of bacteria
NO protein receptor! No resistance??
Hypothesis:
physical interaction with the lipids of the membrane
16
The peptide NK-2
NK-lysinIsolated from pig small intestine
ca. 8.9 kDa78 aminoacids
amphipathic5 -helices
33% identity to a gene product (NKG5) from activated T and NK cell
Function: antibacterial, cytotoxic
J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124
helix 3+4 = NK-2 (res. 39-65)
10 positive charges
hydrophilic
hydrophobic
The peptide NK-2
NK-lysinIsolated from pig small intestine
ca. 8.9 kDa78 aminoacids
amphipathic5 -helices
33% identity to a gene product (NKG5) from activated T and NK cell
Function: antibacterial, cytotoxic
J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124
helix 3+4 = NK-2 (res. 39-65)
10 positive charges
hydrophilic
hydrophobic
Common Common featuresfeatures of of peptidepeptide antibioticsantibiotics::smallsmall (15(15--30 AA)30 AA)
highlyhighly amphipathicamphipathic((positivelypositively) ) chargedcharged
NKNK--2:2:good good antibacterialantibacterial activityactivity (MIC < 1(MIC < 1M)M)
littlelittle hemolytichemolytic activityactivity (>> 10(>> 10M)M)littlelittle cytotoxicitycytotoxicity (>> 10(>> 10M)M)
CanCan wewe explainexplainSelectivitySelectivity
andandMode of ActionMode of Action??
17
Membrane Composition
PG = Phosphatidyl-glycerole PE = Phosphatidyl-ethanolamineCL = Cardiolipin PC = Phosphatidyl-cholineSM = Sphingomyeline PS = Phosphatidyl-serine
PG PE CL PC SM PSGram-negativeE.coli IM 6 82 12 0 0 0S. typhimurium 33 60 7 0 0 0P. cepacia 18 82 0 0 0 0Gram-positiveS.aureus 57 0 43 0 0 0B. subtilis 29 10 47 0 0 0
C.albicans 0 70 0 4 15 11Erythrocyte 0 30 0 33 24 13
negative
SAXS Results: POPC in 10 mM Na-Phosphate-Buffer, pH 7.4
18
SAXS Results: DPPC in 10 mM Na-Phosphate-Buffer, pH 5.2
pH 5.2
SAXS Results: POPG in Water
19
SAXS Results: POPG in Water
DMPG (negative) - FTIR
Willumeit et al. BBA 1669 (2005) 125 – 134
Stiffening
Decrease of =
Increase of ordering
DM=Dimyristoyl (14:0)
s(CH2) = symmetric stretching vibration
L
L
20
SAXS Results: POPE in 10 mM Na-Phosphate-Buffer, pH 7.4
POPE
POPE + NK-2 (1000:1)
Influence of NK-2 on POPENKNK--2 2 inducesinduces a negative a negative membranemembranecurvaturecurvature --> > breakingbreaking of of thethe membranemembrane!!
21
New derivatives of NK-2
Several derivatives, small (15-30 AA), highly amphipathic(positively) charged
NKCS-[FR]KKGTLIDKSIRRLRTRMIKKSVGRLIK
NKCS-[20K]KKSIRRLFTRMIKKSVGRLIK
NKCS-[15-27]KKGTLIDKSIRRL
NKCS-[14]FTRMIKKSVGRLIK
NKCS-[17]RRLFTRMIKKSVGRLIK
NK-CSKKGTLIDKSIRRLFTRMIKKSVGRLIK
NK-2-[CS]
New derivatives of NK-2
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
45 50 55 60 65 70 75 80 85temperature [°C]
rep
eat
dis
tan
ce [
nm
]
POPE
POPE+NKCS-[14] 1000:1
POPE+NKCS-[17] 1000:1
POPE+NKCS-[20K] 300:1
22
New derivatives of NK-2
Act
ivit
y
Sh
ift
of
HII
tran
siti
on
Shift for PE lipids (HII phase) is towards
higher temperatures!
Use of X-ray scattering to aid the design and delivery of membrane-active drugs
G. Pabst • D. Zweytick • R. Prassl • K. Lohner, Eur Biophys J (2012) 41:915–929
How could anaesthetics work?
2 mol% 2 mol% ketamineketamine = IC= IC5050 for a channel for a channel composed of bent helicescomposed of bent helices
23
Lipid Rafts
Homepage of Jarek Majewski of the Lujan Neutron Scattering Center at Los Alamos
Areas in membranes with specific lipid and protein composition
Combination of glycosphingolipids and protein receptors
Usually more densely packed and "floating" in the membrane
Regulating the Size and Stabilization of Lipid Raft-likeDomains and Using Calcium Ions as Their Probe
Or Szekely, Yaelle Schilt, Ariel Steiner, and Uri Raviv, Langmuir 2011, 27, 14767–14775
DOPC DPPCPOPC
ratio 1:1:1
24
Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid
Drug Delivery
A. ANGELOVA et al., ACCOUNTS OF CHEMICAL RESEARCH 44(2) (2011) 147–156
Molecules included in membranecan lead to channel swelling
Decrease in curvature
Better storage capabilities
Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid
Drug Delivery
A. ANGELOVA et al., ACCOUNTS OF CHEMICAL RESEARCH 44(2) (2011) 147–156
Change of structure
Release of drug
25
Crystallizing membrane proteins using lipidicmesophases
Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31.CholesterolProtein
-> precipitant solution
-> shift of equilibrium away
from stability in the cubic
membrane.
-> phase separation
-> protein molecules diffuse
from the continuous
bilayered reservoir into
the lattice of the
advancing crystal face
Lipidic cubic phase technologies formembrane protein structural studies
Vadim Cherezov, Current Opinion in Structural Biology 2011, 21:559–566
26
Stability of the lipid layers under shear
M. Kreuzer, M. Reinhardt, J. Stahn, M. Golub, R. Willumeit, R. Dahint, R. Steitz
Lubrication of synovial joints is most efficient
Friction coefficient in the range of 0.001-0.01
Constant under changing conditions
Combination of complex structure of cartilage and self-assembled structures formed by phospholipids and biomacromolecules
albumin proteoglycans such as lubricin and aggrecan polyglycosaminoglycan like hyaluronan surface active phospholipids (SAPLs)
Four main biological components of synovial fluid
M. Kreuzer, M. Reinhardt, J. Stahn, M. Golub, R. Willumeit, R. Dahint, R. Steitz
AMOR, PSI
Stability of the lipid layers under shear
27
Tresset PMC Biophysics 2009 2:3 doi:10.1186/1757-5036-2-3 / Technical Brief 2012 Volume 4
Thank you for your attention