What is important to know about lipids? What can be measured? - - PDF document

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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

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Lipids are important for:

Cells -> membranes in sensors Polymeric implants (MPC polymers) Crystallisation Joints As co-factors or as HDL (lipoproteins)

Biomembranes

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Lipids in Cells

@Lehninger Biochemistry

Composition of an Erythrocyte Membrane

@Lehninger Biochemistry

cell recognition intra celluar signals apoptosis signal unknown unknown

PE PC PS

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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 charge charge Zeta Zeta Potential: Potential: PC = 0 PC = 0 PE = PE = -

• 25

25 PG = PG = -

• 60 mV

60 mV neutral neutral neutral neutral

Biomembranes

958.48 Tetracosanoic Lignoceroyl 24:0 930.43 Trocosanoic Trucisanoyl 23:0 902.37 Docosanoic Behenoyl 22:0 874.32 Heneicosanoic Heniecosanoyl 21:0 846.27 Eicosanoic Arachidoyl 20:0 818.21 Nonadecanoic Nonadecanoyl 19:0 790.16 Octadecanoic Stearoyl 18:0 762.10 Heptadecanoic Heptadecanoyl 17:0 846.27 3,7,11,15-tetra methylhexadecanoic Phytanoyl 16:0 [(CH3)4] 734.05 Hexadecanoic Palmitoyl 16:0 706.00 Pentadecanoic Pentadecanoyl 15:0 677.94 Tetradecanoic Myristoyl 14:0 649.89 Tridecanoic Tridecanoyl 13:0 621.84 Dodecanoic Lauroyl 12:0 593.78 Undecanoic Undecanoyl 11:0 565.73 Decanoic Capryl 10:0 537.67 Nonanoic Nonanoyl 9:0 509.62 Octanoic Capryloyl 8:0 481.57 Heptanoic Heptanoyl 7:0 453.51 Hexanoic Caproyl 6:0 425.46 Pentanoic Pentanoyl 5:0 397.41 Tetranoic Butanoyl 4:0 369.35 Trianoic Propionoyl 3:0 M.W. IUPAC Trivial Carbon Number

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 Lipids

Phosphatidylcholine (Symmetric Fatty Acid)

1,2-Diacyl-sn-Glycero-3-Phosphocholine (Unsaturated Series)

954.45 15-cis- tetracosenoic Nervonoyl 24:1 878.18 4,7,10,13,16,19 (all

• cis)

docosahexaenoic

DHA

22:6 898.34 13-cis-docosenoic Erucoyl 22:1 830.14 5,8,11,14(all -cis) eicosatetraenoic Arachidonoyl 20:4 842.23 11-cis-eicosenoic Eicosenoyl 20:1 778.06 9-cis-12-cis-15- cisoctadecatrienoic Linolenoyl 18:3 782.09 9-cis-12-cis-

• ctadecadienoic

Linoleoyl 18:2 786.13 9-trans-

• ctadecenoic

Elaidoyl 18:1 786.15 9-cis-octadecenoic Oleoyl 18:1 786.13 6-cis-octadecenoic Petroselinoyl 18:1 730.02 9-trans- hexadecenoic Palmitelaidoyl 16:1 730.02 9-cis-hexadecenoic Palmitoleoyl 16:1 673.91 9-trans- tetradecenoic Myristelaidoyl 14:1 673.91 9-cis-tetradecenoic Myristoleoyl 14:1 M.W. IUPAC Trivial Carbon Number

Biomembranes

@Avanti Polar Lipids

Phosphatidylcholine (Asymmetric Fatty Acid)

1-Acyl-2-Acyl-sn-Glycero-3-Phosphocholine

788.14 Stearoyl Oleoyl 18:1-18:0 760.09 Palmitoyl Oleoyl 18:1-16:0 732.03 Myristoyl Oleoyl 18:1-14:0 834.17 Docosahexaenoyl Stearoyl 18:0-22:6 810.15 Arachidonoyl Stearoyl 18:0-20:4 786.13 Linoleoyl Stearoyl 18:0-18:2 788.14 Oleoyl Stearoyl 18:0-18:1 762.10 Palmitoyl Stearoyl 18:0-16:0 734.05 Myristoyl Stearoyl 18:0-14:0 806.12 Docosahexaenoyl Palmitoyl 16:0-22:6 782.09 Arachidonoyl Palmitoyl 16:0-20:4 758.07 Linoleoyl Palmitoyl 16:0-18:2 760.09 Oleoyl Palmitoyl 16:0-18:1 762.10 Stearoyl Palmitoyl 16:0-18:0 706.00 Myristoyl Palmitoyl 16:0-14:0 734.05 Stearoyl Myristoyl 14:0-18:0 706.00 Palmitoyl Myristoyl 14:0-16:0 M.W. 2-Acyl 1-Acyl Carbon Number

PO PS PM SO

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Biomembranes

@Lehninger Biochemistry

Amphipatic Molecules in Solution

hydrophilic hydrophobic

highly highly diluted diluted system system increase increase of

• f concentration

concentration CMC = CMC = critical critical micelle micelle concentration concentration

@Lehninger Biochemistry

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Amphipatic Molecules in Solution

POPG inverse 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

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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:

n n  = 2d = 2d· ·sin sin 

4  Q = 2 k0 sin =  sin

Seddon Handbook of Biol Physics 1995

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Small Angle Small Angle Scattering Scattering on

• n

Lipid Lipid Vesicles Vesicles also also is is Diffraction Diffraction

http://www.encapsula.com/products_01.html and Lehninger Biochemistry

Lipid unilamellar vesicle multilamellar vesicle total ensemble SAS Diffraction

Biomembranes

Diffraction of POPG Membranes (Neutrons) Intensity q-value

• 1. Order

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Biomembranes

Diffraction of POPG Membranes (Neutrons) Intensity q-value

• 1. Order

Peaks equidistant Repeat distance = 2 / q Peak Distance = Repeat distance/n n = 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 Repeat distance = 2 / q Peak distance = Repeat distance/n n = 1, 3, 2, 7, 3, 12, 13, …

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Cubic Phase

Seddon Handbook of Biol Physics 1995

Cubic Phase

Peaks NOT equidistant Repeat distance = 2 / q Peak distance = Repeat distance/n n = 1, 2, 3, 2, 5, 6, 8, 3, …

Seddon Handbook of Biol Physics 1995

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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

Liposomes Liposomes Multilamellar Multilamellar Layers Layers

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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Biomembranes

Peptide 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-lysin

Isolated from pig small intestine

• ca. 8.9 kDa

78 aminoacids amphipathic 5 -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-lysin

Isolated from pig small intestine

• ca. 8.9 kDa

78 aminoacids amphipathic 5 -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 features features of

• f peptide

peptide antibiotics antibiotics: : small small (15 (15-

• 30 AA)

30 AA) highly highly amphipathic amphipathic ( (positively positively) ) charged charged NK NK-

• 2:

2: good good antibacterial antibacterial activity activity (MIC < 1 (MIC < 1 M) M) little little hemolytic hemolytic activity activity (>> 10 (>> 10 M) M) little little cytotoxicity cytotoxicity (>> 10 (>> 10 M) M) Can Can we we explain explain Selectivity Selectivity and and Mode of Action Mode of Action? ?

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Membrane Composition

PG = Phosphatidyl-glycerole PE = Phosphatidyl-ethanolamine CL = Cardiolipin PC = Phosphatidyl-choline SM = Sphingomyeline PS = Phosphatidyl-serine

PG PE CL PC SM PS Gram-negative E.coli IM 6 82 12

• S. typhimurium

33 60 7

• P. cepacia

18 82 Gram-positive S.aureus 57 43

• B. subtilis

29 10 47 C.albicans 70 4 15 11 Erythrocyte 30 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 POPE

NK NK-

• 2

2 induces induces a negative a negative membrane membrane curvature curvature -

• >

> breaking breaking of

• f the

the membrane membrane! !

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New derivatives of NK-2

Several derivatives, small (15-30 AA), highly amphipathic (positively) charged

NKCS-[FR] K K G T L I D K S I R R L R T R M I K K S V G R L I K NKCS-[20K] K K S I R R L F T R M I K K S V G R L I K

NKCS-[15-27]

K K G T L I D K S I R R L NKCS-[14] F T R M I K K S V G R L I K NKCS-[17] R R L F T R M I K K S V G R L I K NK-CS K K G T L I D K S I R R L F T R M I K K S V G R L I K

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 85 temperature [°C] repeat distance [nm] POPE POPE+NKCS-[14] 1000:1 POPE+NKCS-[17] 1000:1 POPE+NKCS-[20K] 300:1

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New derivatives of NK-2

Activity Shift of HII transition

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% ketamine ketamine = IC = IC50

50 for a channel

for a channel composed of bent helices composed 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-like Domains and Using Calcium Ions as Their Probe

Or Szekely, Yaelle Schilt, Ariel Steiner, and Uri Raviv, Langmuir 2011, 27, 14767–14775 DOPC DPPC POPC

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 membrane can 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 lipidic mesophases

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

• >

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 for membrane 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