Protein-Lipid Interactions: Structural and Functional Effects Anthony Lee (Southampton)

Transcription

1 Saulieu ctober 2004 Protein-Lipid Interactions: Structural and Functional Effects Anthony Lee (Southampton) The membrane as a system Co-evolution of lipids and membrane proteins

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3 R P - R Phosphatidylcholine + Me3N + N H3 Phosphatidylserine H Phosphatidic acid

4 Lipid as solvent Lipid as cofactor Bacteriorhodopsin K+ channel KcsA hydrophobic core Anionic lipid Boundary or Annular Lipid Cofactor or non-annular lipid

5 Reconstitution of Membrane Proteins Solubilization Detergent Reconstitution Detergent Removal Dilution Excess lipid Dialysis Biobeads Membrane Fragments Sealed vesicles

6 The importance of Annular Lipid The Lipid Headgroup Region The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

7 Ca-ATPase from skeletal muscle Depolarisation Nerve Muscle Cell Synapse Sarcoplasmic Reticulum Ca2+ Plasma membrane T system 2+ Ca Ca2+ 2+ Ca channel 2+ Ca ATP ADP + Pi Ca2+ TnC Actin Myosin

8 Effect of Lipid headgroup Calcium ATPase R P Skeletal muscle sarcoplasmic reticulum ATP ADP + Pi

9 Calcium ATPase Relative ATPase Activity Fatty Acyl Chains = leoyl (C18:1) 1 SR: 70% PC 20% PE 10% PS/PI PC PE PS PA SR

10 The importance of Annular Lipid The Lipid Headgroup Region Why is activity headgroup dependant? Why does the SR contain anionic lipids that support low ATPase activity? The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

11 E2Tg Calcium-ATPase E1Ca2 Headgroup region R63 M1 hydrophobic core Headgroup region R63 M1

12 The importance of Annular Lipid The Lipid Headgroup Region Why is activity headgroup dependant? Why does the SR contain anionic lipids that support low ATPase activity? The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

13 Calcium ATPase activity SR: 70% PC 20% PE 10% PS/PI Fractional activity di(c18:1)ps di(c18:1)pc di(c18:1)pa Mole Fraction anionic lipid

14 ATP hydrolysis Calcium accumulation (nmoles/mg protein) Accumulation of calcium by reconstituted Ca-ATPase in sealed vesicles Ca x leak 5000 PC + 10% cardiolipin PC + 10% PA PC Time (secs) 1000

15 Slippage and Passive Leak Slippage Passive leak Ca E1 Ca out Ca out E1Ca2 E1Ca2 ATP Ca E2 in E2Pi passive leak E2P ATP hydrolysis transport in mm Ca E2PCa2 Ca out slippage Passive Leak Slippage Ca accumulated (nmoles/mg) Time (sec) Time (sec)

16 Slippage and Leak ATP hydrolysis Slippage Ca Ca out Ca in E1 out E1Ca2 E1Ca2 ATP Ca E2 E2P E2Pi passive leak slippage transport in E2PCa2 mm Ca Ca out Slippage Ca accumulated (nmoles/mg) 5000 PC + 10% cardiolipin 4000 Increasing slippage PC+10%PA 1000 PC Time (secs) 1000

17 in slippage cytoplasm surface transport

18 Nerve Depolarisation Synapse Sarcoplasmic Reticulum Ca2+ Muscle Cell Plasma membrane T system 2+ Ca Ca2+ Ca2+channel Ca2+ ATP ADP + Pi Ca2+ TnC Actin Myosin Role of SR: accumulate Ca, leading to muscle relaxation thermogenesis

19 Ca E1 Heat generation out E1Ca2 E1Ca2 ATP Ca E2 E2Pi E2P Slippage = heat generation transport in E2PCa2 Ca out 1200 DPC Heat (mcal/mg) 1000 Fast slippage DPC + 20% DPA 400 Slow slippage ATP Time (min)

20 Sarcoplasmic reticulum contains sarcolipin Ca accumulation (nmoles/mg protein) MERSTRELCLNFTVVLITVILIWLLVRSYQY 2000 ATP hydrolysis SLN:ATPase slippage : mm Ca 5: :1 20: Time (secs) Sarcolipin inceases slippage

21 Nerve Depolarisation Muscle Cell Synapse Sarcoplasmic Reticulum Ca2+ Plasma membrane T system 2+ Ca Ca2+ 2+ Ca channel Ca2+ ATP ADP + Pi Ca2+ TnC Actin Myosin Roles of SR: accumulate Ca Thermogenesis Sarcolipin Anionic Lipid

22 The importance of Annular Lipid The Lipid Headgroup Region Important for function Nature of binding site on protein The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

23 Lipid Binding Specific Non-specific Lys Y39 K35 G256 PC Trp R31 PE Photosynthetic reaction centre MD simulation of helix in PC bilayer (M. Sansom) PC

24 The importance of Annular Lipid The Lipid Headgroup Region Important for function The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

25 How efficient is hydrophobic matching between the bilayer and a membrane protein? The use of Trp as a reporter group polar hydrophobic bacteriorhodopsin

26 How efficient is hydrophobic matching? Trp fluorescence spectra 1.5 Intensity W Free Trp Wavelength (nm)

27 K+ channel KcsA How efficient is hydrophobic matching 27 Å C18 16 Å C10 38 Å C24

28 Reconstituted KcsA Phosphatidylcholine Chain Length C C18 Free Trp 1.0 C Wavelength (nm)

29 Tryptophan residues do not change their environment with changing bilayer thickness C24 C18 C10 Hydrophobic matching is highly efficient Either lipid distorts to match protein or protein distorts to match lipid or both

30 Hydrophobic Mismatch Bilayer Distortion to Match Protein Bilayer too thick Compress Hydrophobic thickness ptimal matching Work Stretch Bilayer too thin

31 Fluorescence Quenching Short range requires contact between fluorophore and quencher R exciting light P Br Br Br Trp * emission of fluorescence light excited state Br exciting light heat * 100 % heat Quencher (Br)

32 Fluorescence quenching in mixtures of brominated and non-brominated lipid R R P P Br-Lipid matches High quenching W WW Br W W Br Br Br Non-Br-Lipid matches Low quenching Hydrophobic matching W W W WW

33 Binding of lipid to a membrane protein Exchange at a set of annular sites B A 1.0 K 0.8 Prot.A + B 0.6 Prot.B + A F/F o 0.4 K is binding constant for B relative to A Mole Fraction Brominated Phospholipid [A is di(br2c18:0)pc]

34 Relative Lipid Binding Constants Relative binding constant [relative to di(br2c18:0)pc] Fatty Acyl Chain Length mpf KcsA 34 Å

35 Hydrophobic mismatch protein distortion Change in helix tilt: changes at the ends of the helices Thick bilayer Thin bilayer Changes in helices change in protein activity

36 Calcium ATPase R Changing Bilayer thickness P Distortion of protein Change in activity ATP ADP + Pi

37 ATPase activity (IU/mg) 4 Ca-2+ATPase PC Me3N P Fatty acyl chain 22 24

38 Diacylglycerol kinase of E. coli Periplasm Cytoplasm Diacylglycerol + ATP Phosphatidic acid + ADP

39 Diacylglycerol kinase Dihexanoylglycerol + ATP Phosphatidic acid + ADP Activity (IU/mg) 60 PC Chain length 22 24

40 Hydrophobic mismatch Change in helix tilt Changes at ends of helices Change in helix packing Calcium ATPase

41 Helix-Helix Interactions KcsA channel L105 V106 A29 Knobs-into-hole packing

42 Fluorescence quenching Trp containing peptide Dibromo-tyrosine containing peptide W Q DibromoTyr 1.0 PC Fluorescence 0.8 Dimer formation Flu. quench C C C Mole fraction brominated peptide 0.04 W Q

43 Unitary free energies of helix-helix interaction Go = -RT ln K 0.5 kj mol-1per C atom W Q DibromoTyr Go (kj mol-1) L22-Q22 6 Dimer formation Flu. quench Fatty acyl chain length W Q

44 Hydrophobic matching between the bilayer and a membrane protein is very efficient How can we measure the hydrophobic thickness of a membrane protein? Can we use Trp mutagenesis + Fluorescence? polar hydrophobic bacteriorhodopsin

45 Do Trp residues anchor transmembrane α-helices into the membrane? Transmembrane domain C N Trp Ile Val Leu KcsA Tyr Trp Phe

46 Trp at ends of helices are not conserved KcsA Pore of Kv

47 Effects of Trp mutagenesis on activity Diacylglycerol kinase of E. coli Periplasm * 48 * 52 W117 W112 * 96 W47 W18 W25 Cytoplasm 29 * 68 * * charged residues Diacylglycerol + ATP Phosphatidic acid + ADP

48 Use of Trp fluorescence to define hydrophobic core of the mechanosensitive channel of large conductance MscL D68 D36 periplasm L69 M2 F80 D16 L92 cytoplasm

49 Use of Trp fluorescence to define hydrophobic core of MscL 344 external residues Emission Max DPC interface Residue Number

50 L69 D68 25 A V91 L92 R11 Y94 D16

51 The importance of Annular Lipid The Lipid Headgroup Region Important for function The Hydrophobic core Hydrophobic thickness The importance of non-annular lipid

52 KcsA non-annular lipid anionic annular lipid Anionic lipid essential for activity In the absence of anionic lipid is the non-annular site empty?

53 Anionic lipid 3 Trp KcsA Ro = 8Å not quenched W68 K+ 2 Trp W67 non-annular quenched nonannular lipid W87 annular quenched

54 non-annular KcsA Trp quenching annular DPC/BrPC 0.8 F/F o W87 (annular) DPC/BrPA Brominated Phospholipid W87+W67 (annular + non-annular) 1.0 The non-annular site can only be occupied by anionic lipid

55 KcsA R64 R89 Anionic lipid

56 Lipid-Protein Interactions Annular lipid Thickness: Lipid headgroup: Nonannular lipid High specificity Lipid co-factor tilt of helices/helix ends packing of helices molecular interactions

57 Lipid-Protein Interactions Malcolm East KcsA: Ian Williamson Simon Alvis Ca2+-ATPase Anthony Starling Sanja Mall MscL: Andy Powl mpf: Aisling Keeffe Diacylglycerol kinase Elizabeth Clark Model Helices Sanja Mall