Transcript Rhodopsin

Rhodopsin
Christen Eberhart
Rhodopsin Sequence
The Eye
•Rhodopsin is found in the rods
that are located in the eye
•Rods are composed of stacked
disks
•Rhodopsin is densely packed
into each disk
•Rods are responsible for black
and white vision
•Rhodopsin works best at dim
light, responsible for night vision
(too much light will saturate the
protein)
•Rod density is found greater on
the outer edges of the retina
(peripheral vision)
Opsin & Retinal
• Rhodopsin is made up
of the protein opsin
with the chromophore,
retinal, covalently
attached
• The linkage occurs at
Lys-296
Rhodopsin Structure
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348 amino acids, 40 kD
7 transmembrane alpha
helices connected by
six loops of varying
lengths
1 short alpha helix
along the cytoplasmic
membrane
2 antiparallel beta
sheets at E-II and 2
antiparallel beta sheets
at the NH2-terminal
Cys-322 and Cys-323
attach to membrane by
palmitic acid residues
Functional monomer,
however, can form
dimers
Retinal
• Retinal is a derivative of Vitamin A,
which is a derivative of betacarotene
• Isomerization of cis-retinal to transretinal by light causes a
conformation change in rhodopsin
which triggers a signal
• Rhodopsin absorbs at green-blue
light (500 nm) which makes the
protein appear reddish-purple
Retinal Binding Site
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Lys-296 covalently attaches to
retinal by a Schiff base
Glu-113, Gly-114, Ala-117, Thr118, Gly-120, and Gly-121 side
chains make a binding pocket for
the retinal
Thr-118, Tyr-268, and Ile-189
possibly could interact with the C9
methyl on the retinylidene group
Met-207, His-211, Phe-212, Tyr268, and Ala-269 surround the Bionone ring of the retinal
Tyr-43, Met-44, Leu-47, Thr-94
and Phe-293 region surrounds the
Schiff base
Ala-169 may interact with the Bionone ring in the activated state
when retinal is trans
Helical Wheel of the Short Alpha
Helix
• Hydrophobic side
chains hold helix
parallel to
membrane
• Hydrophilic side
chains provide a
key point of
contact with the
G-protein
(Transducin)
Stabilization between the Helices
•Amino acids that provide
stabilization of structure
between five helices are
shown
•Disulfide bond between
Cys110 and Cys-187
•Ala-299 peptide carbonyl
hydrogen bonds with Asn-55
and Asp-83
•Asp-83 connects by a water
molecule to Gly-120
•Asn-78 hydrogen bonds to
Ser-127, Thr-160, and Trp161
•Asn-302 bonds through a
water molecule with Asn-83
G-protein couple receptor (GPCR)
• 7 transmembrane receptor that sense
molecules outside the cell and
activate inside signal transduction
pathways and cellular responses
(Rhodopsin)
• Found only in eukaryotes
• GPCR activates a G-protein
(Transducin) by exchanging its GDP
for GTP
• GPCR is bound to a G-protein while
in its inactive state
• Once a GPCR is active, the G-protein
detaches
• G-protein is made up of 3 subunits
(Gα, Gβ, and Gγ)
• Once G-protein is activated, the Gα
subunit activates another protein
(phosphodiesterase) and detaches
from the other two subunits
Rhodopsin
Transducin: Gα is red, Gβ is
blue, and Gγ is yellow
Phototransduction Pathway
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Light activates Rhodopsin which activates Transducin by exchanging its
GDP for GTP
When active, Transducin’s alpha subunit dissociates from the other two
subunits
The active Transducin then activates a membrane bound protein called
phosphodiesterase
Phosphodiesterase hydrolyzes cGMP
The hydrolyzes of cGMP, leads to the ion channel closing and the initiation
of an action potential
To return back to the inactive state Rhodopsin Kinase phosphorylates the
cytosolic tail of rhodopsin which inhibits the activation of transducin
Arrestin then binds to the phosphorylated rhodopsin further inhibiting activity
Inactive to Active Form
• Active (all trans form of
rhodopsin) form still awaits a
crystal structure
• These pictures are based on
computer simulations
• Picture on the right: a is the
non-active form, b-d is over
time changing to the active
from
• Picture on the bottom shows
a potential binding groove
opening up in the active form
on the cytoplasmic side
Multiple Alignment Sequence
Red-highly conserved important amino acid
Blue-conserved important amino acid
Purple-important amino acid
Black-highly conserved amino acid
Grey-conserved amino acid
Retinitis Pigmentosa
• Group of genetic eye conditions
where the retina of the eye slowly
and progressively degenerates
• Night blindness occurs early on, then
tunnel vision, and then many years
later eventually blindness by the age
of 40
• Up to 150 different point mutations
can lead to RP, which causes
misfolding of the protein
• The mutant proteins then aggregate
together and disrupt the visual
phototransduction pathway
• Rhodopsin mutations make up 25%
cases of RP
References
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Choi G, et al (2002) Structural studies of metarhodopsin II, the activated
form of the G-protein coupled receptor, rhodopsin. Biochemistry, 41(23):
7318-1324.
Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry. New York: W.H.
Freeman and Co.
Krisha GA, et al (2002) Evidence that helix 8 of rhodopsin acts as a
membrane-dependent conformational switch. Biochemistry, 41 (26): 82988309.
Palczewski K, et al (2000) Crystal structure of rhodopsin: A G-protein
coupled receptor. Science, Vol. 289, pp. 733-4.
Terakita A, et al (2002) Functional interaction between bovine rhodopsin
and G protein transducin. Journal of Biological Chemistry. 277 (1): 40-46.
Helical Wheel (http://kael.net/helical.htm)
Butterfly and Flower (photograph) (http://www.allaboutvision.com)
Magnify (photograph) (http://www.flickr.com/photos/dfrighini/3240329545/)