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

Structural studies of lipid systems Regine Willumeit Hamburg, 22.10.2012 Introduction What is important to know about lipids? What can be measured? Example for structure determination 1

Lipids are important for: Cells -> membranes As co-factors or as HDL (lipoproteins) Joints Crystallisation in sensors Polymeric implants (MPC polymers) Biomembranes 2

Lipids in Cells @Lehninger Biochemistry Composition of an Erythrocyte Membrane PE unknown PC unknown cell recognition PS apoptosis signal intra celluar signals @Lehninger Biochemistry 3

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 4

Structure of a Phospholipid: Headgroups neutral neutral neutral neutral neutral negative charge negative charge Zeta Potential: Potential: Zeta PC = 0 PC = 0 PE = PE = - -25 25 PG = - PG = -60 mV 60 mV @Avanti Polar Lipids Phosphatidylcholine (Symmetric Fatty Acid) Biomembranes 1,2-Diacyl- sn -Glycero-3-Phosphocholine (Saturated Series) Carbon Trivial IUPAC M.W. Number 3:0 Propionoyl Trianoic 369.35 4:0 Butanoyl Tetranoic 397.41 5:0 Pentanoyl Pentanoic 425.46 6:0 Caproyl Hexanoic 453.51 7:0 Heptanoyl Heptanoic 481.57 8:0 Capryloyl Octanoic 509.62 9:0 Nonanoyl Nonanoic 537.67 10:0 Capryl Decanoic 565.73 11:0 Undecanoyl Undecanoic 593.78 12:0 Lauroyl Dodecanoic 621.84 13:0 Tridecanoyl Tridecanoic 649.89 14:0 Myristoyl Tetradecanoic 677.94 DM 15:0 Pentadecanoyl Pentadecanoic 706.00 DP 16:0 Palmitoyl Hexadecanoic 734.05 3,7,11,15-tetra 16:0 [(CH 3 ) 4 ] Phytanoyl 846.27 methylhexadecanoic 17:0 Heptadecanoyl Heptadecanoic 762.10 18:0 Stearoyl Octadecanoic 790.16 19:0 Nonadecanoyl Nonadecanoic 818.21 20:0 Arachidoyl Eicosanoic 846.27 21:0 Heniecosanoyl Heneicosanoic 874.32 22:0 Behenoyl Docosanoic 902.37 23:0 Trucisanoyl Trocosanoic 930.43 24:0 Lignoceroyl Tetracosanoic 958.48 5

Phosphatidylcholine (Symmetric Fatty Acid) @Avanti Polar Lipids 1,2-Diacyl- sn -Glycero-3-Phosphocholine (Unsaturated Series) Biomembranes Carbon Number Trivial IUPAC M.W. 14:1 Myristoleoyl 9-cis-tetradecenoic 673.91 9-trans- 14:1 Myristelaidoyl 673.91 tetradecenoic 16:1 Palmitoleoyl 9-cis-hexadecenoic 730.02 9-trans- 16:1 Palmitelaidoyl 730.02 hexadecenoic 18:1 Petroselinoyl 6-cis-octadecenoic 786.13 18:1 Oleoyl 9-cis-octadecenoic 786.15 9-trans- 18:1 Elaidoyl 786.13 octadecenoic 9-cis-12-cis- 18:2 Linoleoyl 782.09 octadecadienoic 9-cis-12-cis-15- 18:3 Linolenoyl 778.06 cisoctadecatrienoic 20:1 Eicosenoyl 11-cis-eicosenoic 842.23 5,8,11,14(all -cis) 20:4 Arachidonoyl 830.14 eicosatetraenoic 22:1 Erucoyl 13-cis-docosenoic 898.34 4,7,10,13,16,19 (all DHA 22:6 -cis) 878.18 docosahexaenoic 15-cis- 24:1 Nervonoyl 954.45 tetracosenoic @Avanti Polar Lipids Phosphatidylcholine (Asymmetric Fatty Acid) Biomembranes 1-Acyl-2-Acyl- sn -Glycero-3-Phosphocholine Carbon 1-Acyl 2-Acyl M.W. Number 14:0-16:0 Myristoyl Palmitoyl 706.00 14:0-18:0 Myristoyl Stearoyl 734.05 PM 16:0-14:0 Palmitoyl Myristoyl 706.00 PS 16:0-18:0 Palmitoyl Stearoyl 762.10 PO 16:0-18:1 Palmitoyl Oleoyl 760.09 16:0-18:2 Palmitoyl Linoleoyl 758.07 16:0-20:4 Palmitoyl Arachidonoyl 782.09 16:0-22:6 Palmitoyl Docosahexaenoyl 806.12 18:0-14:0 Stearoyl Myristoyl 734.05 18:0-16:0 Stearoyl Palmitoyl 762.10 SO 18:0-18:1 Stearoyl Oleoyl 788.14 18:0-18:2 Stearoyl Linoleoyl 786.13 18:0-20:4 Stearoyl Arachidonoyl 810.15 18:0-22:6 Stearoyl Docosahexaenoyl 834.17 18:1-14:0 Oleoyl Myristoyl 732.03 18:1-16:0 Oleoyl Palmitoyl 760.09 18:1-18:0 Oleoyl Stearoyl 788.14 6

Biomembranes @Lehninger Biochemistry Amphipatic Molecules in Solution hydrophilic hydrophobic CMC = CMC = critical micelle critical micelle concentration concentration increase of of concentration concentration increase highly diluted diluted system system highly @Lehninger Biochemistry 7

Amphipatic Molecules in Solution POPC POPG inverse POPE DPPE, DPPC, POPC, POPE…. @Lehninger Biochemistry What can be measured? monoolein/water Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31. 8

What can be measured? Phase Behaviour of Phospholipids Data Base: LIPIDAT PO=Palmitoyl-oleoyl (16:0-18:1); DP=Dipalmitoyl (16:0) Lamellar Phase Repeat Distance d Bragg-equation: n   = 2d sin   = 2d· ·sin n 4  Q = 2 k 0 sin  = sin   Seddon Handbook of Biol Physics 1995 9

Small Angle Scattering Scattering on on Small Angle Lipid Vesicles Vesicles also also is is Diffraction Diffraction Lipid Lipid unilamellar vesicle multilamellar vesicle SAS total ensemble Diffraction http://www.encapsula.com/products_01.html and Lehninger Biochemistry 1. Order Diffraction of POPG Membranes (Neutrons) Biomembranes Intensity q-value 10

1. Order Diffraction of POPG Membranes (Neutrons) Biomembranes Peaks equidistant Repeat distance = 2  / q Intensity Peak Distance = Repeat distance/n n = 1, 2, 3, 4,…. Intensities & Phases -> q-value Scattering length density profile 11

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, … 12

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 13

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 Liposomes Liposomes Drying of vesicles solution on support Multilamellar Layers Layers Multilamellar Example for structure determination 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 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 14

Peptide Antibiotics Biomembranes Melittin Magainins ( Xenopus laevis, Bombina variegata ) Thionine Cecropins Plant Defensines ( Hyalophora cecropia ) ( 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 15

The peptide NK-2 NK-lysin Isolated from pig small intestine 78 aminoacids ca. 8.9 kDa 33% identity to a gene product (NKG5) from activated T and NK cell helix 3+4 = NK-2 (res. 39-65) Function: antibacterial, cytotoxic hydrophilic 5  -helices amphipathic hydrophobic 10 positive charges J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124 Common features features of of peptide peptide antibiotics antibiotics: : Common The peptide NK-2 small (15 small (15- -30 AA) 30 AA) highly amphipathic highly amphipathic NK-lysin Isolated from pig small intestine (positively positively) ) charged charged ( 78 aminoacids ca. 8.9 kDa NK- NK -2: 2: 33% identity to a gene product (NKG5) from activated T and NK cell  M) (MIC < 1  good antibacterial antibacterial activity activity (MIC < 1 M) good helix 3+4 = NK-2 (res. 39-65) Function: antibacterial, cytotoxic  M) (>> 10  little hemolytic hemolytic activity activity (>> 10 hydrophilic M) little 5  -helices amphipathic (>> 10   M) little cytotoxicity cytotoxicity (>> 10 M) little Can we Can we explain explain Selectivity Selectivity and and hydrophobic Mode of Action? ? Mode of Action 10 positive charges J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124 16

Membrane Composition PG PE CL PC SM PS negative Gram-negative E.coli IM 6 82 12 0 0 0 S. typhimurium 33 60 7 0 0 0 P. cepacia 18 82 0 0 0 0 Gram-positive S.aureus 57 0 43 0 0 0 B. subtilis 29 10 47 0 0 0 C.albicans 0 70 0 4 15 11 Erythrocyte 0 30 0 33 24 13 PG = Phosphatidyl-glycerole PE = Phosphatidyl-ethanolamine CL = Cardiolipin PC = Phosphatidyl-choline SM = Sphingomyeline PS = Phosphatidyl-serine SAXS Results: POPC in 10 mM Na- Phosphate-Buffer, pH 7.4 17

SAXS Results: DPPC in 10 mM Na- Phosphate-Buffer, pH 5.2 pH 5.2 SAXS Results: POPG in Water 18

SAXS Results: POPG in Water DMPG (negative) - FTIR Stiffening Decrease of  = L  Increase of ordering L  DM=Dimyristoyl (14:0)  s (CH 2 ) = symmetric stretching vibration Willumeit et al. BBA 1669 (2005) 125 – 134 19

SAXS Results: POPE in 10 mM Na-Phosphate-Buffer, pH 7.4 POPE + NK-2 (1000:1) POPE NK- -2 2 induces induces a negative a negative membrane membrane NK Influence of NK-2 on POPE curvature - curvature -> > breaking breaking of of the the membrane membrane! ! 20