Membrane Proteins. David S. Goodsell Joanna R. Long 6740 February 6, 2006

Transcription

1 Membrane Proteins David S. Goodsell 999 Joanna R. Long 6740 February 6, 2006

2 Homework: ) The structure of the potassium channel: Molecular basis of K+ conduction and selectivity, Doyle et al., Science 280, (998). 2) Structural determinants of water permeation through aquaporin- Murata et al., Nature 407, (2000). General Reference: Branden & Tooze, Introduction to Protein Structure, 2 nd Ed. Voet & Voet, Biochemistry, 3 rd Ed.

3 Fluid mosaic model

4 Importance of Membrane Proteins Why we care: ~/3 of human proteins are membrane associated Less <% have solved structures Structures are generally worse than 3Å resolution Structures are generally of detergent-solubilized proteins crystallized using several tricks Environment is important for function

5 Types of Membrane Proteins Functional: Structural: + unsolved classes?

6 Methods Sequence prediction: works well for transmembrane helices 2D crystals: EM, cryoem, low resolution 3D crystals: X-ray: tough, high resolution Solution state NMR: small size Solid state NMR: complexity of data, bright future

7 Sequence Prediction Hydrophobic index: Several different scales have been developed Hydrophobicity in the lipid bilayer

8 Peptide interactions with bilayers

9 Membrane protein folding

10 Motifs in membrane proteins Most hydrophobic amino acids on the outside facing fatty acid chains Interiors of TM proteins similar to interiors of soluble proteins Commonly use gly, small sidechains for coiled coils High preponderance of prolines in helices Not completely understood

11 From Hargrave Rhodopsin

12 EM reconstruction First experimental method to identify transmembrane helices In 975, Henderson and Unwin reconstructed bacteriorhodopsin In 997, Paul Hargrave and others did a cryo- EM reconstruction of rhodopsin

13 X-ray crystallography Bacteriorhodopsin First high-resolution membrane structure: photosynthetic reaction center (Deisenhofer, Michel: Nobel Prize, 989) Porins Rhodopsin (2000), first GPCR K+ channel (MacKinnon: Nobel Prize, 2003) F ATPase (Walker: Nobel Prize, 998) Aquaporin (Agre: Nobel Prize, 2003) Partial structures of monotopic membrane proteins (ie integrins) Numbers are growing but still <99 unique structures in PDB or <0.% of the structures deposited

14 Bacteriorhodopsin Validated the EM low resolution work Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., Spudich, J. L.: Crystal Structure of Sensory Rhodopsin II at 2.4 Angstroms: Insights Into Color Tuning and Transducer Interaction Science 293 pp. 499 (200)

15 Photosynthetic Reaction Center First 3D membrane protein structure solved Nobel Prize in Chemistry in 988 (Johann Deisenhofer, Robert Huber, Hartmut Michel )

16 Maltoporin Porins are all β- sheet and span the membrane. Found in Gramnegative outer membranes Difficult to predict from sequence

17 Rhodopsin: X-ray First structure of a GPCR Basis of new generation of modeling other GPCRs Palczewski et al., Crystal structure of Rhodopsin: A G- protein-coupled receptor, Science 289, pp (2000)

18 GPCRs: Major drug targets

19 Potassium Channel First structure of an ion channel. Explains ion selectivity K+/Na+ selectivity > 0,000 K+ :0 8 ions/sec Doyle et al., 998 MacKinnon: Nobel Prize 2003

20 K+ channel The structure has K ions in it. Negative charges on both ends of channel Too narrow for hydrated K to go through The energetics of stripping H 2 O from K is compensated by good molecular interactions with channel: selectivity.

21 K+ channel

22 K+ channel

23 K+ channel ~50% occupancy in each position. Suggests sites and 3 or 2 and 4 occupied at any one time. JMB, (2003)

24 F F 0 ATPase δ α β α ATP ADP + P i β α β Motor with significant soluble (F ) and membrane-associated (F 0 ) parts. 4 H + b b γ ε F ATPase has been solved by X-ray (Nobel Prize). a c 2 F 0 has been solved (modeled) by NMR and other methods Entire complex still not solved 4 H +

25 F ATPase Stalk rotates with passage of H, and the α and β subunits produce ATP from ADP. Alternatively, hydrolysis of ATP to ADP will cause H to flow the other direction. Nobel Prize in Chemistry in 997 (Walker)

26 Solution NMR with mixed solvents was used to solve high-resolution structures of a single c subunit. Structures were solved in different ph. Girvin et al., Nature

27 The c subunit was then modeled using NMR and other data ph conformational changes suggest how complex rotates and translocates H. Girvin et al.

28 Aquaporin Conducts water across membranes at a rate of 3x0 9 molecules/sec Does not conduct ions or solutes Does not conduct H+ Nobel Prize 2003 Agre Murata et al., Nature 2000

29 Aquaporin

30 Aquaporin The 2 Asn residues in the water pore form an H-bond with the central water. The orbital overlap with the water would twist it and force it out of the H-bond chain. Thus, H-bond is lost, and this is about the energy barrier measured for water translocation.

31 Integrins: Cryoelectron Microscopy Can trap functional state Inherently low-resolution

32 Mapping Xray to cryoem

33 Helix-Helix Interactions Key to activation via dimerization of monotopic membrane proteins Gly critical for packing (GXXXG motif) Interhelical hydrogen bonding drives oligomerization (Neu receptor tyrosine kinase constitutively activated by V664 Glu or Gln) Little high resolution structural data available

34 Others in the butterfly collection Photosystem II Calcium ATPase Monoamine oxidase Cytochrome bc and b6f complexes Inward rectifier potassium channels Lipid flippase Alpha-Hemolysin Outer Membrane Receptor (OMR)

35 Caveats Xray structures require crystallization detergent solubilization NOT lipid bilayer Most structures are partial or of inactive forms Monotopic membrane proteins especially lacking How should we think about the lipid bilayer and its effects on protein structures?

36 The lipids Lipids are soluble in organic (ie methanol, chloroform) but sparingly soluble in water Components: Fatty acids---carboxylic acids with a hydrocarbon sidechain Triacylglycerols---energy storage; not in biological membranes (major component of adipose tissue Glycerophospholipids---major component of cell membranes Sphingolipids---major component of cell membranes Cholesterol---sterol

37 Outside Inside

38 POPC Gel: chains move; no fast rotation around long axis Fluid: onset of fast rotation biologically relevant phase Chol POPC POPE DPhPC PDHAPC

39 Lipid / Protein Interactions Lipid modifications

40

41 Cholesterol and the Golgi Apparatus, M.S. Bretscher & S. Munro, Science 26:280

42 Adding cholesterol Thickening of bilayer Ordering of acyl chains Membrane is less permeable Phase separation Does addition of cholesterol change hydrophobic matching of lipid and protein? Do changes in membrane elasticity affect TM helix interactions? Do rafts simply sequester proteins or do they change their functional state? Can proteins function in non-native lipid environments? T. Baumgart, S.T. Hess, W.W. Webb Nature 425:82

43 Looking at interactions between transmembrane helices Functional arguments for studying complex lipid environments Rhodopsin (GPCR) meta I / meta II states dependent on cholesterol, ω-3 fatty acid levels nachr (ion channel) inactive in the absence of cholesterol (Chol) and dioleoylphosphatidic acid (DOPA) Integrin activation / clustering constitutive activation corresponds to raft localization Stillwell & Wassall, 2003 Figure. Proposed role for DHA phospholipids in favoring the formation of lipid rafts and segregation of membrane proteins

44 Signaling in Rafts

45 Tissue specific lipid composition differences Fatty Acid Distribution Name 6:0 6: 8: 0 8: 8: 2 8: 3 20: 0 20: 20: 2 20: 3 20: 4 22: 0 22: 6 23: 0 24: 0 24: Othe r Brain Cerebroside Brain PC Brain PE Brain PS Brain Lyso PS Brain SM Heart CA Heart PC Heart PE Liver PC Liver PE Liver PI Liver Lyso PI

46 Proteins can alter lipid phase properties Lung tissue has a surface area of ~300 cm 2 per cm 3 of tissue High surface area high curvature Lung surfactant reduces surface tension at the air liquid interface. Surfactant is comprised of lipids (90% by weight) and proteins (0%) N. Engl. J. Med. 347:24 (2002) The main cause of respiratory distress in premature infants and acute distress in adults is lack of or the breakdown of lung surfactant proteins Biochim. Biophys. Acta 467:255 (2000)