Catalytic Mechanism and Strategies

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Catalytic Mechanism and Strategies

• Lecture 5 Bacteriorhodopsin and Rhodopsin

• Archeal proteins
• Bacteriorhodopsin - proton pump
• Halorhodopsin - chloride ion pump
• Sensory rhodopsin - phototaxis
• Rhodopsin - G protein-coupled receptor

2
bR and ATP Synthase - a simple energy producing
system
H
light
H
H
H
H
H
bacteriorhodopsin
ATP synthase
ADP
ATP
Proton gradient created by bR is used to drive
ATP synthesis
3
Photoreaction cycle of retinal chromophore
N-Lys
bR568

H

H
N
H
K640
Lys
N
M412
H
Lys
4
Bacteriorhodopsin Photocycle
5
Isomerization of the retinal to 13-cis
6
Protonation equilibrium between the Schiff base
and Asp-85
7
Switch and proton release to the
extra- cellular surface
8
Reprotonation of the Schiff base by Asp-96
9
Reprotonation of Asp-96, reisomerization of
the retinal to all-trans
10
Deprotonation of Asp-85
11
Conformational changes related to
function what causes steps 1 - 5?
a
12
Photoreaction cycle of retinal chromophore
Asp 96 - COOH
Proton donor
in
direction of proton pumping
N
bR568

H
W402
out
Asp 85 - COO-
W401
Proton acceptor
W406
Arg 82 - NH2
W402 water 402
W403
W404
Hydrogen-bonded network with well-defined structur
al water molecules on extracellular side of the
protein Initial direction of proton motion is
opposite to the direction of proton pumping!
Glu 194, 204
H
13
Photoreaction cycle of retinal chromophore
in
direction of proton pumping
N
bR568

H
out
Asp 96 - COOH (high pKa)

H
pKa of Schiff base drops
N
NH N H Ka HN/NH
K640, L550
Asp 85 - COO-
Arg 82 - NH2
pKa of Asp85 increases
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H2O
Photoreaction cycle
Asp 96 - COOH
in
direction of proton pumping
N
M412
out
Asp 85 - COOH
W401
W406
Arg 82 - NH2
Glu-C00- HOOC-Glu
Proton transfers to Asp85 Arg 82 swings down
toward Glu194/Glu204 Glu204 deprotonates Helix 6
moves and allows water exposure to Asp96
H
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H2O
Photoreaction cycle
Asp 96 - COO-
in
direction of proton pumping

H
N
N520
out
Asp 85 - COOH
W401
W406
Arg 82 - NH2
-OOC-Glu
Asp96 deprotonates Proton transferred to Schiff
base Retinal reisomerizes
H
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H
H2O
Photoreaction cycle
Asp 96 - COOH
in
direction of proton pumping
O640
N

H
out
Asp 85 - COOH
W401
W406
Arg 82 - NH2
Retinal reisomerizes Helix 6 moves back and
forces water out Asp96 reprotonates Asp85
deprotonates (due to return of NH) Glu204
reprotonates, Arg82 swings back toward Asp85
-OOC-Glu
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1.55 Å crystal structure reveals functional
water molecules
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The BR state
Hydrogen-bonds at active center
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Hydrogen-bonded network around water 402
20
The BR state
Hydrogen-bonds at active center
A hydrogen-bonded network links Asp-85 to
Glu-194/Glu-204
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The BR state
A chain of covalent and hydrogen-bonds links
region of retinal to Asp-96
Hydrogen-bonds at active center
A hydrogen-bonded network links Asp-85 to
Glu-194/Glu-204
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H-bond network on intracellular side is not
continuous
23
(No Transcript)
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BR
25
K
26
M1
27
M2
28
M2
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G protein-coupled receptors are involved in many
signal transduction cascades and are a major
targetof pharmaceuticals

• Visual receptors rhodopsin, cone
  pigments
• Olfactory receptors
• Hormone receptors follicle stimulating
  hormone thyrotropin
  receptor
• Neurotransmitters dopamine receptor
• Metabotropic receptors glutamate receptor

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G protein-coupled receptors
Ligand

• 7 transmembrane helices
• Ligands bind from the extracellular side
• Heterotrimeric G protein binds from the
  cytoplasmic side
• Conserved residues in transmembrane helices

b
GDP
a
g
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G Protein-Coupled Receptors
Light
Ligand

• Receptor conformational change
• upon ligand binding.
• Binding of heterotrimeric G protein.
• Exchange of GDP for GTP in a-subunit.

b
b
GDP
g
a
g
GTP
a
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Light
Each disc membrane contains many photoreceptors
Rod Cell
Outer segment contains about 2000 disc membrane


Each photoreceptor is a protein containing a
small molecule, vitamin A
Nerve impulse
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Color Vision

• 11-cis retinal is
• the same in all
• visual pigments
• Without bound
• retinal the protein
• is colorless.
• Interaction
• between
• the retinal and
• protein changes
• the color of the
• retinal.

11-cis retinal lmax 380 nm
green cone protein
green cone pigment


11-cis retinal lmax 380 nm
blue cone protein
blue cone pigment


11-cis retinal lmax 380 nm
red cone pigment
red cone protein
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Vitamin A (retinal) is the photoreactive molecule
in photoreceptors
Light
Cis, bent
N-Lys 296
11-cis retinal (inactive)
H
Trans, straight
all-trans retinal (active)
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Energy storage in rhodopsin
Light
hydrophobic
N

H
Glu113 - COO-
hydrophilic

H
N
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Energy storage in rhodopsin

H
N
N
Distance 5.0 Å

H
Distance 2.5 Å
Glu113 - COO-
W k q1 q2/er -66 kcal/ mol ( r 2.5 Å, e
2)
W k q1 q2/er -33 kcal/ mol ( r 5.0 Å, e
2)
Energy stored 33 kcal/mol
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Activation of rhodopsin
?G 40 kcal/mol
?Gstored 33 kcal/mol
11-cis-retinal
All-trans-retinal
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Activation of rhodopsin
?-electrons go into anti-bonding orbitals CC
effectively becomes C-C
No barrier to isomerization - isomerization
takes 200 fs !
Isomerization occurs in the excited electronic
state
11-cis-retinal
H
All-trans-retinal
N-Lys
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Helix interactions in GPCRs are critical.
?
GDP
?
?
Inactive
Active
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Structural model of rhodopsin
IV
II
III
Glu113
-

H
I
N
V
VII
VI
Schiff base and Glu113 form a stable salt bridge
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Structural model of rhodopsin
IV
II
III
Glu113
-
Gly121

H
I
N
V
Trp265
Tyr268
VII
VI
Shape and charge of retinal PSB is complementary
to protein binding site.
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Structural model of rhodopsin
IV
II
III
Glu122
Glu113
-
His211

H
Arg135
I
N
V
Glu247
VII
VI
Helix interactions lock the receptor in the
off-state in the dark.
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Isomerization of retinal breaks helix interactions
IV
II
III
Glu113
H
I
V
N
VII
VI