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

Lipid

Plasma Membrane

50%

50%

Axon

20%

80%

Mitochondria Membrane

75%

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

CMC

Mass

[lipid]/[detergt]

(mM)

(kD)

25

23.0

DPC

19.0

DHPC

14

12.3

CHAPSO

6.0

50.0

0.05

64.6

DMPC/DHPC

0.15

10

21.7

POPC/DHPC

0.15

10

23.4

POPC/DHPC

0.30

31.8

POPC/DHPC

0.50

60.2

-Octylglucoside

-decylmaltoside
16:0 Lyso PG (LPPG)

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

Membrane protein topology

History of membrane protein structure determination

1984

Photosynthetic reaction centre, Deisenhofer et al, JMB 1984

1990

Bacteriorhodopsin, Henderson et al, JMB 1990

1992

Porin (beta-barrel), Weiss & Schulz, JMB 1992

1998

K+ channel, Doyle et al, Science 1998

2000

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

White and Wimley, 1999

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

Program: Membrane Protein Explor

Transmembrane helix prediction

Performance comparison of various TMH predictors a
Predictor

VTMH

VP

N-score

C-score

free-R

THUMBU[16] b

85.5%

47.1%

6.9

6.7

0.580.04

SOSUI[11] c

89.1%

57.1%

5.0

5.0

0.440.04

DAS-TMfilter[20] d

90.7%

64.3%

6.5

5.5

0.580.03

TOP-PRED[1] e

92.6%

60.0%

4.5

4.6

0.450.02

TMHMM[6] f

91.0%

65.7%

4.5

4.5

0.440.02

Phobius[7] g

91.8%

71.4%

4.6

4.4

0.440.04

MemBrain h

97.9%

87.1%

3.2

3.1

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

phosphorylation

Y Y Y

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

disappeared

changed

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