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Transcript
Structure of GPCRs and G proteins
Goal of the lecture:
Understanding the structural basis of how a
GPCR activates a G protein
Heterotrimeric G protein Pathway
Clapham Nature. 1996 Jan 25;379(6563):297-299.
Ribbon Diagram of
Rhodopsin Structure
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
Two dimensional Representation of Rhodopsin
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
The environment of 11-cis retinal chromophore
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
Salient features of Rhodopsin Structure
Organization of the extracellular region serves as
the basis seven-helix bundle arrangement
11-cis retinal holds transmembrane regions in the
inactive conformation by interacting with key
residues that participate in intra-helical
interactions
Activation of Rhodopsin
Requirement of rigid-body motion of
transmembrane helices for light activation of
rhodopsin.
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Design of the Experiment
Mutate all Cys to Ser
Bring back Cys of interest
Construct double
Cys mutants
Keep Cys at 139 (helix 3)
constant
Vary 2nd Cys from 247-252
in helix 6
Put spin label on the Cys
EPR spectroscopy
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
EPR spectra of inactive (dark state)
shown as red trace
and activated (meta-rhodopsin II)
shown as yellow trace
to study interactions between loops
3 and 6
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Results from EPR Spectroscopy
Dark State: Distance between Cys at 139
and Cys at 248-251 = 12-14 Å
After illumination increases in distances
23-25 Å
Conclusion: Helices 3 and 6 move apart from
each other after activation
Biochemical Verification of EPR predicted
movement of helices
Cross link with disulfide reagent, cut with V8 protease
Run SDS-PAGE
If cross linked 1 band without DTT; 2 bands with DTT
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Crosslinking of helices 3 and 6 blocks the ability
of Rhodopsin to activate Transducin
Fluorescence assay
to measure GTPgS
binding to transducin
Farrens et al, Science. 1996 Nov
1;274(5288):768-770.
Conclusions
Helix 6 moves with respect to
Helix 3
Movement is required for
activation of transducin
Helix 6 movement causes
cytoplasmic loop3 to move
Cytoplasmic loop3 is involved
in coupling to transducin
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
G protein structure
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
Space filling model of Ga interacts with Gbg
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
The Gb interface that interacts with Ga
contains key residues required for
interaction with effectors
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
G protein residues involved in
regulation of effectors
Space filling model of Gbg.
Gb is white and Gg is pink.
The green region is the
area of Gb covered by Ga
in the heterotrimer
The smaller regions
marked by colored dashed
lines identify residues
involved in interactions
with various effectors.
Each color corresponds to
an effector
Ford et al, Science. 1998 May 22;280(5367):1271-1274.
In the heterotrimer the switch II region of Ga is
contact with Gb
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
Changes in the conformation of Ga in the GDP
vs GTP bound forms and interactions with Gb
Gb
GTPgS-Ga Red
GDP-Ga Blue
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
The Switch II region of Ga has different
conformation in the GDP and GTP bound states
GTPgS
GDP
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
The heterotrimeric G protein interacts with
the membrane and receptor
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
A structural cartoon of G protein
interaction with receptor
Hamm J Biol Chem. 1998 Jan 9;273(2):669-672.
Evolving view of receptors GPCRs exist as dimers
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Atomic Force Microscopy Picture of
mouse rod-outer segment disc membrane
Fotiadis et al, Nature. 2003 Jan 9;421(6919):127-128.
Organization of the cytoplasmic surface of
rhodopsin dimers are clearly visible
Fotiadis et al, Nature. 2003 Jan 9;421(6919):127-128.
Model of Rhodopsin
Dimer
Here phosphorylated
Rhodopsin is shown
binding to arrestin
(This would be the
Desensitized state)
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Model of rhodopsin dimer binding to one
molecule of transducin
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Receptor Dimer Activation of G proteins
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656. A movie of this molecule is
available from http://stke.sciencemag.org/cgi/content/full/sigtrans;2005/276/tr10/DC1