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
q
CMC
Mass
[lipid]/[detergt]
(mM)
(kD)
-
25
23.0
DPC
-
1
19.0
DHPC
-
14
12.3
CHAPSO
-
8
6.0
-
2
50.0
-
0.05
64.6
DMPC/DHPC
0.15
10
21.7
POPC/DHPC
0.15
10
23.4
POPC/DHPC
0.30
8
31.8
POPC/DHPC
0.50
7
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
0.58±0.04
SOSUI[11] c
89.1%
57.1%
5.0
5.0
0.44±0.04
DAS-TMfilter[20] d
90.7%
64.3%
6.5
5.5
0.58±0.03
TOP-PRED[1] e
92.6%
60.0%
4.5
4.6
0.45±0.02
TMHMM[6] f
91.0%
65.7%
4.5
4.5
0.44±0.02
Phobius[7] g
91.8%
71.4%
4.6
4.4
0.44±0.04
MemBrain h
97.9%
87.1%
3.2
3.1
0.35±0.02
a
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 YEβ
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 SecYEβ
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
Y
Y
Y
Y
phosphorylation
activation
of other
proteins
Example: TCR/CD3 signaling
α β
ε γ
Y
Y
α β
ε δ
ζζ
ε γ
Y
Y
Y Y Y
phosphorylation
ε δ
ζζ
Y
Y
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
β2-adrenergic GPCR
adrenalin
Crystallization of β2-adrenergic GPCR
V. Cherezov et al., Science 318, 1258 -1265 (2007)
Ligand-binding characterization and comparison
to rhodopsin