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)