MECHANISMS OF SPECTRAL TUNING IN BLUE CONE VISUAL PIGMENTS - VISIBLE AND RAMAN-SPECTROSCOPY OF BLUE-SHIFTED RHODOPSIN MUTANTS

Spectral tuning by visual pigments involves the modulation of the phys ical properties of the chromophore (Il-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 t he required green-to blue opsin shift. The simultaneous substitution o f nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W 265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nn, accounting for 2,830 cm(-1), or 80%, of the opsin shift be tween rhodopsin and the blue cone pigment. Raman spectroscopy of mutan t pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered, An increase in t he number of dipolar side chains near the protonated Schiff base of re tinal increases the ground-excited state energy gap via long range dip ole-dipole Coulomb interaction. In addition, the W265Y substitution ca uses a decrease in solvent polarizability near the chromophore ring st ructure. Finally, two substitutions on transmembrane helix 3 (A117G an d E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the pro tonated 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 major ity of the opsin shift in blue visual pigments.