membrane proteins - university of floridamsg.mbi.ufl.edu/bch6746/lecture11.pdfimportance of membrane...
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Membrane Proteins
Joanna R. Long6740
February 6, 2006
© David S. Goodsell 1999
General Reference:
Branden & Tooze, Introduction to Protein Structure, 2nd Ed.
Voet & Voet, Biochemistry, 3rd Ed.
Homework:
1) “The structure of the potassium channel: Molecular basis of K+ conduction and selectivity”, Doyle et al., Science 280, 69-77 (1998).
2) “Structural determinants of water permeation through aquaporin-1” Murata et al., Nature 407, 599-605 (2000).
Fluid mosaic model
Importance of Membrane Proteins
Why we care:•~1/3 of human proteins are membrane associated
•Less <1% 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
Types of Membrane ProteinsFunctional:
Structural:
+ unsolved classes?
• 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
Methods
Sequence PredictionHydrophobic index: Several different
scales have been developed Hydrophobicity in the lipid bilayer
Peptide interactions with bilayers
Membrane protein folding
• 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
Motifs in membrane proteins
Rhodopsin
From Hargrave
• First experimental method to identify transmembrane helices
• In 1975, Henderson and Unwin reconstructed bacteriorhodopsin
• In 1997, Paul Hargrave and others did a cryo-EM reconstruction of rhodopsin
EM reconstruction
• Bacteriorhodopsin• First high-resolution membrane structure:
photosynthetic reaction center (Deisenhofer, Michel: Nobel Prize, 1989)
• Porins• Rhodopsin (2000), first GPCR• K+ channel (MacKinnon: Nobel Prize, 2003)• F1 ATPase (Walker: Nobel Prize, 1998)• Aquaporin (Agre: Nobel Prize, 2003) • Partial structures of monotopic membrane proteins (ie
integrins)• Numbers are growing but still <99 unique structures in
PDB or <0.1% of the structures deposited
X-ray crystallography
Bacteriorhodopsin
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. 1499 (2001)
Validated the EM low resolution work
Photosynthetic Reaction Center
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
•First 3D membrane protein structure solved
•Nobel Prize in Chemistry in 1988 (Johann Deisenhofer, Robert Huber, Hartmut Michel )
Maltoporin•Porins are all β-sheet and span the membrane.
•Found in Gram-negative outer membranes
•Difficult to predict from sequence
Rhodopsin: X-ray
•First structure of a GPCR
•Basis of new generation of modeling other GPCRsPalczewski et al., “Crystal structure of Rhodopsin: A G-protein-coupled receptor”, Science 289, pp 739-745 (2000)
GPCRs: Major drug targets
Potassium Channel
•First structure of an ion channel.
•Explains ion selectivity
•K+/Na+ selectivity > 10,000
•K+ :108 ions/sec
Doyle et al., 1998
MacKinnon: Nobel Prize 2003
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 H2O from K is compensated by good molecular interactions with channel: selectivity.
K+ channel
K+ channel
K+ channel
~50% occupancy in each position. Suggests sites 1 and 3 or 2 and 4 occupied at any one time.
JMB, 333 965-975 (2003)
F1F0 ATPaseδ α
ADP + Pi
ATPα
α
β
ββ
4 H+
a
4 H+
b bγ
α
ε
c12
•Motor with significant soluble (F1) and membrane-associated (F0) parts.
•F1 ATPase has been solved by X-ray (Nobel Prize).
•F0 has been solved (modeled) by NMR and other methods
•Entire complex still not solved
F1 ATPase•Stalk rotates with passage of 1H, and the α and βsubunits produce ATP from ADP.
•Alternatively, hydrolysis of ATP to ADP will cause 1H to flow the other direction.
•Nobel Prize in Chemistry in 1997 (Walker)
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
The c subunit was then modeled using NMR and other data
pH conformational changes suggest how complex rotates and translocates 1H.
Girvin et al.
Nobel Prize 2003 Agre
Murata et al., Nature 2000
Conducts water across membranes at a rate of 3x109 molecules/sec
Does not conduct ions or solutes
Does not conduct H+
Aquaporin
Aquaporin
AquaporinThe 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, 1 H-bond is lost, and this is about the energy barrier measured for water translocation.
Integrins: Cryoelectron Microscopy
•Can trap functional state•Inherently low-resolution
Mapping Xray to cryoEM
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 kinaseconstitutively activated by V664 Glu or Gln)
•Little high resolution structural data available
Photosystem II
Cytochrome bc1 and b6f complexes
Calcium ATPase
Lipid flippaseInward rectifier potassium channels
Monoamine oxidase
Alpha-Hemolysin
Outer Membrane Receptor (OMR)
Others in the butterfly collection
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 bilayerand its effects on protein structures?
The lipidsLipids are soluble in organic (ie methanol, chloroform) but sparingly soluble in waterComponents:• 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
“Outside”
“Inside”
POPC
Gel: chains move; no fast rotation around long axisFluid: onset of fast rotation biologically relevant phase
Chol POPC DPhPCPOPE PDHAPC
Lipid / Protein Interactions
Lipid modifications
“Cholesterol and the Golgi Apparatus”, M.S. Bretscher & S. Munro, Science 261:1280
•Thickening of bilayer•Ordering of acyl chains•Membrane is less permeable•Phase separation
Adding cholesterol
T. Baumgart, S.T. Hess, W.W. WebbNature 425:821
• 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?
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 / clusteringconstitutive activation corresponds to raft localization
Looking at interactions between transmembrane helices
Figure 1. Proposed role for DHA phospholipids in favoring theformation of lipid rafts and segregation of membrane proteins
Stillwell & Wassall, 2003
Signaling in Rafts
413758Liver Lyso PI
3139915483Liver PI
16324495354Liver PE
14199121329112Liver PC
103031214301Heart PE
76143136123Heart PC
1187821Heart CA
34675462Brain SM
10110772Brain Lyso PS
11821134421Brain PS
1091911121311915Brain PE
425113916131Brain PC
40922112376Brain Cerebroside
Other
24:1
24:0
23:0
22:6
22:0
20:4
20:3
20:2
20:1
20:0
18:3
18:2
18:1
18:0
16:116:0Name
Fatty Acid Distribution
Tissue specific lipid composition differences
N. Engl. J. Med. 347:2141 (2002)
•Lung tissue has a surface area of ~300 cm2 per cm3 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 (10%)
•The main cause of respiratory distress in premature infants and acute distress in adults is lack of or the breakdown of lung surfactant proteins
Proteins can alter lipid phase properties
Biochim. Biophys. Acta 1467:255 (2000)