| Abstract
| - The replacement of Gly90 by Asp in human rhodopsincauses congenital night blindness. Ithas been suggested that the molecular origin for the trait is analtered electrostatic environment of theprotonated retinal Schiff base chromophore. We have investigatedthe corresponding recombinant bovinerhodopsin mutant G90D, as well as the related mutants E113A andG90D/E113A, using spectroscopy atlow temperature. This allows the assessment ofchromophore−protein interactions under conditions whereconformational changes are mainly restricted to the retinal-bindingsite. Each of the mutant pigmentsformed bathorhodopsin- and isorhodopsin-like intermediates, but theconcomitant visible absorption changesreflected differences in the electrostatic environment of theprotonated Schiff base in each pigment. Fouriertransform infrared-difference spectroscopy revealed effects on thechromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal ofan electrostatic perturbation nearC12 of the retinal chromophore in all three mutants. Acomparison of the UV−visible and infrared-difference spectra of the mutant pigments strongly suggests thatGlu113 is stably protonated in G90D.The corresponding carbonyl-stretching mode is assigned to a bandat 1727 cm-1. In contrast to thecasein native bathorhodopsin, the all-trans-retinalchromophores in the primary photoproducts of the mutantpigments are essentially relaxed. The peptide carbonyl vibrationalchanges in mutants G90D and G90D/E113A suggest that this is due to a more flexible retinal-binding site.Therefore, the steric strain exertedon the chromophore in native bathorhodopsin may be caused byelectrostatic forces that specifically involveglutamate 113.
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