Membrane Protein Structure Overview

present and future

D.C. Rees

Caltech/HHMI

Membrane Proteins - Interests and Challengesparticipate in transport and transduction processes that mediate the flow of matter, energy and information across the membrane bilayer

are poorly characterized relative to water-soluble proteins due to experimental challenges of mimicking the membrane and water-bilayer interfaces

constitute an estimated ~20% of all proteins, yet only ~40 distinct structures are available

membrane protein structure websites:

http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html

Liu & Rost Prot. Sci. 10, 1970 (2001)15-30% total, 10-20% > 2 TM

membrane protein abundances

Membrane protein structure: Status

bioenergetic systemsphotosynthetic reaction centers (bacterial, PSI, PSII)bacteriorhodopsin (BR, HR, SR)respiratory complexes (II, III, IV and V)FDH, NarGHIlight harvesting complexesADP/ATP carrier

transportersCa+2 ATPaseAcrBABC transporters (MsbA, BtuCD)MFS (LacY, GlpT)

channels/receptorsK+ (KcsA, MthK, KirBac1.1, KvAP)mechanosensitive (MscL, MscS)aquaporins (GlpT, AQP)ClCAchRrhodopsin

-barrel 8,10,12,14,16,18,22 -strands

Structural analysis of membrane proteins

natural sources or cloning & expression (recombinant)

solubilization (detergents)

purification

crystallization

structure determination

QuickTime™ and aSGI decompressor

are needed to see this picture.

10-30 homologs

membrane protein crystallization: approaches

detergents/additives - RClipidic phases - BRFab complexes - cytochrome c oxidaseproteolysis - KcsAsequence engineering - OmpXhomologues - MscL

BtuCD structure determination

3.2 Å resolution

Locher et al. Science 296, 1091 (2002)

BtuCD: molecular architecture of an ABC transporter

periplasm

cytoplasm

BtuC BtuC

BtuC BtuD

two-fold NCS

surface polarity of membrane proteins reflectsthe solvent environment

Nagle & Tristram-Nagle, COSB 10, 474 (2000)

surface hydrophobicity profile for membrane proteins

ARBB 31, 207 (2002)

endpoints of transmembrane helices

helix tilt and helix-helix crossing angle distributions

•••••••••••••

••••••••••••••••••

•••••

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

-180 -150 -120 -90 -60 -30 0 30 60 90 120 150 180helix-helix crossing angle (˚)

observed probability

• calculated probability

left handed

right handed

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 10 20 30 40 50tilt angle (˚)

ARBB 31, 207 (2002)

average tilt = 23˚ + 10˚

-1 0 +1

Asp Ile

hydrophobicity

buriedsurface

water-soluble proteins

buried / surface

Hydrophobicities of buried/surface residuesmembrane proteins vs water-soluble proteins

TM region, 16 -helical mps

Eisenberg et al. hydrophobicity scaleFaraday Symp. Chem. Soc. 17, 109 (1982)

Science 245, 510 (1989)ARBB 31, 207 (2002)

Membrane protein - water-soluble protein comparison

membrane proteins and water soluble proteins have similarinterior apolaritiespacking densitiessurface areasstabilities (?)

and differ in surface polaritieshelix-helix packing distributionstertiary folds

what is the role of solvent in defining protein structures?

water membrane

● ●

●●

●●

●

●●

●

●

❍ ❍ ❍❍

❍

❍

❍❍

❍

❍

❍

❍

❍

0

2

4

6

8

10

12

14

16

1960 1970 1980 1990 2000year

● water-soluble proteins

❍ membrane proteins

progress in membrane protein structure determinations parallels that of water-soluble proteins with a ~25 year offset

photosynthetic reaction center

myoglobin

http://www.mpibp-frankfurt.pg.de/michel/public/memprotstruct.html

B.W. Matthews Ann. Rev. Phys. Chem. 27, 493 (1976)

“Should we designate a certain percentage of targetsto membrane proteins and/or protein complexes?”

Membrane protein challenges:

overexpression of eukaryotic proteins

solubilization (detergents)

state-specific stabilizers

YES!!!

Acknowledgments (~t-20 years to present)

RC: J. Allen, T. Yeates, A. Chirino, A. Yeh,H. Axelrod, G. Feher

FRD: T. Iverson, C. Luna-Chavez, G. Cecchini

MscL: G. Chang, R. Spencer, A. Lee, M. Barclay, R. Bass, P. Strop, J. Choe, S. Steinbacher

MscS: R. Bass, P. Strop, M. Barclay, Y. Poon

BtuCD: K. Locher, A. Lee, L. Borths

general: R. Spencer, L. DeAntonio, D. Eisenberg