Mechanisms of Spectral Tuning in Blue Cone Visual Pigments: VISIBLE AND RAMAN SPECTROSCOPY OF BLUE-SHIFTED RHODOPSIN MUTANTS

Steven W Lin,Gerd G Kochendoerfer,Kate S Carroll,Dorothy Wang,Richard A Mathies,Thomas P Sakmar

doi:10.1074/jbc.273.38.24583

Steven W Lin, Gerd G Kochendoerfer + Show 4 more

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https://doi.org/10.1074/jbc.273.38.24583

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Abstract

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.

Highlights

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket
In the blue rod pigment (␭max ϳ430 nm) from the toad Bufo marinus, it was shown that the Schiff base CϭN stretching frequency of the 9-cis species of its chromophore was similar to the frequency measured for a model retinal-PSB molecule stabilized by a chloride counterion in methanol [38]
Given the small shifts induced by Leu86, Cys87, Thr124, and Cys299, we infer that the majority of the opsin shift between rhodopsin and the blue-rho pigment, totaling 2600 to 2700 cmϪ1, is caused by six substitutions as follows: G90S, A117G, E122L, W265Y, A292S, and A295S

Summary

Introduction

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. Two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments. Its ␭max can range from ϳ420 to 560 nm in visual pigments due to interactions with residues in the binding site of the protein, which modulate the ground-excited electronic (S0-S1) transition energy of the retinal PSB. The replacement of seven of these residues in the green cone sequence by those in the red cone sequence is necessary to shift the ␭max of the hybrid green pigment all the way to 560 nm [21], but three specific substitutions of polar amino acids at TM helices 4 and 6

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