Structural studies of lipid systems · 6 Biomembranes Phosphatidylcholine (Symmetric Fatty Acid) @Avanti Polar Lipids 1,2-Diacyl-sn-Glycero-3-Phosphocholine (Unsaturated Series) 954.45

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


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

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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

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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


Amphipatic Molecules in Solution

hydrophilic hydrophobic

highlyhighly diluteddiluted systemsystem increaseincrease of of concentrationconcentration

CMC = CMC = criticalcritical micellemicelle concentrationconcentration


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Amphipatic Molecules in Solution

POPGinverse POPE

POPC

DPPE, DPPC, POPC, POPE….


Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31.


monoolein/water

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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

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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

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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

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Biomembranes(invers) hexagonal phase POPE Membrane (SAXS)

Biomembranes(invers) hexagonal phase POPE Membrane (SAXS)

Peaks NOT equidistant


Peak distance = Repeat distance/nn = 1, 3, 2, 7, 3, 12, 13, …

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Cubic Phase


Cubic Phase

Peaks NOT equidistant


Peak distance = Repeat distance/nn = 1, 2, 3, 2, 5, 6, 8, 3, …


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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

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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

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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??

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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

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SAXS Results: DPPC in 10 mM Na-Phosphate-Buffer, pH 5.2

pH 5.2

SAXS Results: POPG in Water

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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

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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!!

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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]


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

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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

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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

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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

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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

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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

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Tresset PMC Biophysics 2009 2:3 doi:10.1186/1757-5036-2-3 / Technical Brief 2012 Volume 4

Thank you for your attention

Structural studies of lipid systems · 6 Biomembranes Phosphatidylcholine (Symmetric Fatty Acid) @Avanti Polar Lipids 1,2-Diacyl-sn-Glycero-3-Phosphocholine (Unsaturated Series) 954.45

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