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  • Title: Spectroscopic evidence for altered chromophore--protein interactions in low-temperature photoproducts of the visual pigment responsible for congenital night blindness.
    Author: Fahmy K, Zvyaga TA, Sakmar TP, Siebert F.
    Journal: Biochemistry; 1996 Nov 26; 35(47):15065-73. PubMed ID: 8942673.
    Abstract:
    The replacement of Gly90 by Asp in human rhodopsin causes congenital night blindness. It has been suggested that the molecular origin for the trait is an altered electrostatic environment of the protonated retinal Schiff base chromophore. We have investigated the corresponding recombinant bovine rhodopsin mutant G90D, as well as the related mutants E113A and G90D/E113A, using spectroscopy at low temperature. This allows the assessment of chromophore-protein interactions under conditions where conformational changes are mainly restricted to the retinal-binding site. Each of the mutant pigments formed bathorhodopsin- and isorhodopsin-like intermediates, but the concomitant visible absorption changes reflected differences in the electrostatic environment of the protonated Schiff base in each pigment. Fourier transform infrared-difference spectroscopy revealed effects on the chromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal of an electrostatic perturbation near C12 of the retinal chromophore in all three mutants. A comparison of the UV-visible and infrared-difference spectra of the mutant pigments strongly suggests that Glu113 is stably protonated in G90D. The corresponding carbonyl-stretching mode is assigned to a band at 1727 cm-1. In contrast to the case in native bathorhodopsin, the all-trans-retinal chromophores in the primary photoproducts of the mutant pigments are essentially relaxed. The peptide carbonyl vibrational changes in mutants G90D and G90D/ E113A suggest that this is due to a more flexible retinal-binding site. Therefore, the steric strain exerted on the chromophore in native bathorhodopsin may be caused by electrostatic forces that specifically involve glutamate 113.
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