Spectral Tuning in Retinal Proteins
hn
N
H
H
11-cis
N
all-trans
Color Vision
hn
N
H
11-cis
H
N
all-trans
Visual Receptors
Rod
G-protein signaling pathway
Rhodopsin
Light
Cone
Spectral tuning in color visual receptors
Color is sensed by red, green and
blue rhodopsin visual receptors.
500nm
400nm
absorption spectrum
11-cis
H
600nm
Their chromophores
are exactly the same!
N
How does the protein tune its
absorption spectrum?
Spectral Tuning in bacteriorhodpsin’s
photocycle
Me
Me
Me
N
Me
Me
H
How can we change the maximal
absorption of retinal chromophore?
Me
Me
Me
Me
N
H
Me
Me
Me
Me
Me
Me
H
N
Excitation energy determines the
maximal absorption
S1
S0
Response
Me
Me
Me
13
7
Me
Me
9
11
15 N
H
Electronic Absorption
Me
Me
Me
Me
N
H
Me
p-p*
Absorption of light in the
UV-VIS region of the
spectrum is due to
excitation of electrons to
higher energy levels.
p-p* excitation in polyenes
p*
p*
DE
photon
E
p
Ground state (S0)
p
Excited state (S1)
DE (excitation energy, band gap) = hn = hc/l
p-p* excitation in polyenes
p*
p*
p*
p
p
E
p
blue-shift
red-shift
p-p* excitation in polyenes
Tuning the length of the
conjugated backbone
b-carotene
O
O
Vitamin A2 (retinal II)
Longer wavelength
Vitamin A1 (retinal I)
Short wavelength
Retinal I
Retinal II
Salmon: different retinals in
different stages of life
OPSIN SHIFT: how protein tunes the
absorption maximum of its chromophore.
Maximal absorption of protonated
retinal Schiff base in:
Water/methanol solution: 440 nm
bR: 568 nm
rod Rh: 500 nm
red receptor: 560 nm
green receptor: 530 nm
blue receptor: 426 nm
Electrostatics and opsin shift
S2
positive charge
Me
Me
+
S1
Me
+
S2
S1
N
Me
Me
H
O
S0
O
C
Asp (Glu)
counterion
S0
no protein
in protein
• The counterion stabilizes the positive charge of
the chromophore.
•The position of the counterion determines how
and how much the band gap energy changes.
Electrostatics and opsin shift
S2
positive charge
Me
Me
+
S1
Me
+
S2
S1
N
Me
Me
H
O
S0
O
C
Asp (Glu)
counterion
S0
no protein
in protein
Maximal absopriton of protonated retinal Schiff base can be changed
by D85N (Purple to blue shift)
Red shift from 568 to 605 nm at pH = 3
Dinosaurs had red-shifted visual receptors!
Howard Hughes Medical
Institute at The Rockefeller
University and Yale University
Microbial rhodopsins in
Halobacteria
purple membrane
Sensory rhodopsin I (sRI):
attractant (repellent) to orange (near UV) light
Sensory rhodopsin II (sRII):
repellent to blue-green light
phototaxis
(color vision of halobacteria)
Bacteriorhodopsin (bR):
proton pump
Halorhodopsin (hR):
chloride pump
sRII
hR
bR
500nm
sRI
600nm
Spectral Tuning in Bacterial
Rhodopsins
Sensory Rhodopsin II
(sRII)
Phototaxis
• Large blue shift of
absorption maximum
in sRII (70 nm)
Bacteriorhodopsin
(bR)
Proton pump
sRII
500nm
bR
600nm
Structures of bR and sRII
X-ray crystallography shows
that structures are very similar.
Both include protonated alltrans retinal Schiff base
orange: sRII
purple: bR
Binding Sites of bR and sRII
sRII
bR
Similar structure
• Aromatic residues.
• Hydrogen-bond network.
(counter-ion asparatates,
internal water molecules)
Mutagenic substitutions
T204A/V108M/G130S of
sRII produces only 20 nm
(30%) spectral shift.
What is the main determinant(s) of
spectral tuning?
QM/MM Calculation of spectral
shift in bR and sR-II
• Refinement of X-ray structures
by HF (retinal, 2Asp, 3H2O)
• Excitation energy calculations
for retinal
QM/MM
helix G
retinal-K205/216
D201/212
bR: purple, sRII: orange
Calculated spectra
DE(S1-S0)
Spectral shift
sRII
bR
DE(S1-S0) : 6.1 (exp. 7.2) kcal/mol
DE(S2-S0 ): 1.7 (exp. 4.0) kcal/mol
500nm
600nm
A sub-band in sRII is due to the
second excited state (S2).
Deprotonation of the Schiff base
Me
Me
Me
Me
N
H
UV vision
birds, honeybee
Me
Me
Me
Me
Me
N
Me
Strong blue shift
p atomic orbitals
Planarity is essential for
maximal overlap of p orbitals
in a double bond (p molecular
orbital)
Steric interactions and
spectral shift?
A highly twisted structure can decrease the overlap of
p orbitals and effectively decrease the length of the
conjugation, i.e., blue shift.
Summary of Mechanisms of Spectral Tuning
• Using a different chromophore with a longer or a
shorter conjugated chain
• Modifying the amino acid composition of the binding
pocket (electrostatics)
• Manipulating the distance and/or conformation of
charged/polar groups in the vicinity of retinal
• Steric interaction with the chromophore so that
some of the double bonds go out of plane (a similar
effect to using a shorter chromophore)
• Protonation state of retinal Schiff base (Strong
blue shift upon deprotonation)
Me
Me
Me
Me
N
H
Me
Me
Me
Me
Me
N
H
Me
The chromophore retinal adopts different
colors in different environments. Doesn’t it
remind you of something?
cameleon