Lecture 19 Membrane Protein

James Chou BCMP201 Spring 2008

Lecture outline Properties of cellular membrane, lipid bilayer, detergent micelles Membrane protein topologies Folding of proteins into membrane assisted by protein translocon Signaling across membrane: example of G protein coupled receptor

Membrane protein is one of the frontiers in structural biology

Membrane proteins account for 25-35% of genome in living organisms. There are over 17,000 structures of water-soluble proteins, but only ~150 unique structures of membrane proteins. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html Many membrane-embedded receptors, transporters, and ion channels are important therapeutic targets.

Properties of cellular membrane

The Fluid Mosaic Model of plasma membrane, Singer & Nicholson, 1972

http://www.molecularexpressions.com/cells/plasmamembrane/plasmamembrane.html

Proteins vs Lipid

Protein Plasma Membrane Axon Mitochondria Membrane 50% 20% 75%

Lipid 50% 80% 25%

Assembly of lipid bilayer

Common cartoon drawing of lipid bilayer

Membrane lipid components

Sterol

Cholesterol

Weight percent of membrane components in several types of membranes

Alberts et al., Molecular Biology of the Cell 1994

Permeability of lipid bilayer

Dynamic properties of lipid bilayer

Common cartoon drawing of lipid bilayer

Real lipid bilayer is dynamic

MD simulation from Dr. Scott Feller, Wabash College

Structure of a fluid DOPC bilayer

Time averaged distributions of the principal structural groups of the lipid. Structure determined by joint refinement of X-ray and neutron diffraction data (Wiener and White, Biophys J, 1992).

3D viewing of the simulated DOPC bilayer

MD simulation from Dr. Scott Feller, Wabash College

Other membrane properties

Rate of lateral diffusion of lipid molecules in E. coli membrane is 10-8 10-7 cm2s-1.

Rate of flip-flop of lipids (from one half of a bilayer to the other) is very slow, e.g., time for movement of 50% of lipids from one monolayer to the other is hours to days. Lipid flip-flop in real membranes is catalyzed by enzymes such as flippases.

Various lipids, detergents, metals, and proteins can induce membrane curvature

Extract membrane proteins from cell membranes using detergents

Fractionation of membranes using centrifuge

Detergent micelle formation

Property of detergent micelle

C free = conc. of detergent not in micelle

Cmic = conc. of detergent micelle

N = aggregation number
Ctotal = NCmic + C free

Critical micelle conc. (cmc) is Ctotal at which C free = NCmic

Lipid/detergent bicelles

With detergent

Without detergent

Various detergent micelles and lipid/detergent bicelles

Micelle / Bicelle q [lipid]/[detergt] -Octylglucoside DPC DHPC CHAPSO -decylmaltoside 16:0 Lyso PG (LPPG) DMPC/DHPC POPC/DHPC POPC/DHPC POPC/DHPC 0.15 0.15 0.30 0.50 CMC (mM) 25 1 14 8 2 0.05 10 10 8 7 Mass (kD) 23.0 19.0 12.3 6.0 50.0 64.6 21.7 23.4 31.8 60.2

Vinogradova et al., Biochemistry 1998; Chou et al., JBNMR 2004

Membrane protein topology

History of membrane protein structure determination

1984 1990 1992 1998 2000

Photosynthetic reaction centre, Deisenhofer et al, JMB 1984 Bacteriorhodopsin, Henderson et al, JMB 1990 Porin (beta-barrel), Weiss & Schulz, JMB 1992 K+ channel, Doyle et al, Science 1998 Rhodopsin, Palczewski et al, Nature 2000

Example of a helical membrane protein: the mitochondrial ATP/ADP carrier

Pebay-Peyroula et al, Nature 2003

Example of a beta barrel membrane protein: the outer membrane protein G (OmpG)

Subbarao and van den Berg, JMB 2006

Some definitions of membrane protein types

Type I

Type II

Type III

Hydropathy plot

http://www.vivo.colostate.edu/molkit/hydropathy/index.html

Hydrophobicity scale of amino acids

Amino Acid Ala Arg+ Asn AspAsp0 Cys Gln GluGlu0 Gly

G(interface) (kcal/mol) 0.17+0.06 0.81+0.11 0.42+0.06 1.23+0.07 -0.07+0.11 -0.24+0.06 0.58+0.08 2.02+0.11 -0.01+0.15 0.01+0.05

G(octanol) (kcal/mol) 0.50+0.12 1.81+0.13 0.85+0.12 3.64+0.17 0.43+0.13 -0.02+0.13 0.77+0.12 3.63+0.18 0.11+0.12 1.15+0.11

His+ His0 Ile Leu Lys+ Met Phe Pro Ser Thr Trp Tyr Val

0.96+0.12 0.17+0.06 -0.31+0.06 -0.56+0.04 0.99+0.11 -0.23+0.06 -1.13+0.05 0.45+0.12 0.13+0.08 0.14+0.06 -1.85+0.06 -0.94+0.06 0.07+0.05

2.33+0.11 0.11+0.06 -1.12+0.11 -1.25+0.11 2.80+0.11 -0.67+0.11 -1.71+0.11 0.14+0.11 0.46+0.11 0.25+0.11 -2.09+0.11 -0.71+0.11 -0.46+0.11

White and Wimley, 1999

Program: Membrane Protein Explor

Transmembrane helix prediction

Performance comparison of various TMH predictors a Predictor THUMBU[16] b SOSUI[11] c DAS-TMfilter[20] d TOP-PRED[1] e TMHMM[6] f Phobius[7] g MemBrain h
a

VTMH 85.5% 89.1% 90.7% 92.6% 91.0% 91.8% 97.9%

VP 47.1% 57.1% 64.3% 60.0% 65.7% 71.4% 87.1%

N-score 6.9 5.0 6.5 4.5 4.5 4.6 3.2

C-score 6.7 5.0 5.5 4.6 4.5 4.4 3.1

free- R 0.580.04 0.440.04 0.580.03 0.450.02 0.440.02 0.440.04 0.350.02

The testing dataset consists of 378 TMH segments from 70 proteins (see Supplementary Table S2). b http://sparks.informatics.iupui.edu/Softwares-Services_files/thumbup.htm [16]. c http://bp.nuap.nagoya-u.ac.jp/sosui/ [11]. d http://mendel.imp.ac.at/sat/DAS/DAS.html [20]. e http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html [1]. f http://www.cbs.dtu.dk/services/TMHMM/ [6]. g http://phobius.cgb.ki.se/ [7]. h http://chou.med.harvard.edu/bioinf/MemBrain/.

The Positive Inside Rule

cytoplasm

+ +

+ -

+ +

von Heijne, G. EMBO J 1986.

Pseudosymmetry Many helical membrane proteins (~60%) contain homologous domains with opposite membrane orientation. Example: 3D viewing of Aquaporin 1 (6 TMs), PDB code : 1J4N. Sui et al, Nature 2001.

How do membrane proteins fold into lipid bilayer?

Image from University of Oxford

Protein translocation across the plasma membrane (co-translational translocation)

(Signal Recognition Particle)

ER membrane

Rapoport, T. A., Nature 2007

Protein translocation across the plasma membrane (post-translational translocation in bacteria)

Cytosolic ATPase

Protein translocation into the plasma membrane

ribosome

3D viewing of protein translocon from the archaeon Methanococcus jannaschii, Sec YE!

van den Berg et al, Nature 2004. PDB code : 1RHZ

EM structure of protein translocon in complex with ribosome in E. coli

Mitra et al., Nature 2005

The MD simulation system

Total 106,679 atoms 251 POPC lipids 21520 H2O 5 Na+ 21 ClProtein SecYE!

111 X 109 X 106.5 A3

Gumbart and Schulten, Biophys J. 2006

MD simulation of protein translocation across the protein translocon

Force applied in Steered MD (~10 ns simulation)

MD simulation of the lateral exit of peptide from protein translocation

MD simulation of the lateral exit of peptide from protein translocation (~1 us)

Gumbart and Schulten, Biochemistry 2007

Membrane receptors Two types of membrane receptors which transduce signal across the membrane
ligand

ligand

phosphorylation

activation of other proteins

Example: TCR/CD3 signaling

" ! # % # $ && # %

" ! # $ &&

Y Y Y

Y Y Y

phosphorylation

Example: G protein coupled receptors

Seven transmembrane helices Largest family of membrane receptors 50% of medicinal targets Difficult to express and crystallize

Coupling to different receptor isoforms lead to different G protein pathways and different biological effects

General themes in heterotrimeric G protein pathways

Rockman H.A. et al (2002) Nature 415:206

Rhodopsin - the visual pigment of rod cells

Palczewski et al, Science 2000

Differences between the ground and photoactivated states

changed

disappeared

Salom et al, PNAS 2006

3D viewing of Rhodopsin

PDB code : 1F88

!2-adrenergic GPCR

adrenalin

Crystallization of !2-adrenergic GPCR

V. Cherezov et al., Science 318, 1258 -1265 (2007)

Ligand-binding characterization and comparison to rhodopsin