Biophysical Journal 63: 1244-1255 (1992)
© 1992 the Biophysical Society

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Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier transform infrared difference spectra.

Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.

ABSTRACT

Fourier transform infrared difference spectra were measured at 30-s intervals after a complete bleach of rhodopsin (rho) samples at 20 degrees C and three different pH values. At each pH, all of the spectra could be fit globally to two exponential decay processes. Using a branched unimolecular kinetic model in which metarhodopsin II (meta II) is hydrolyzed to opsin and retinal both directly and through metarhodopsin III (meta III), we calculated rho-->meta II, rho-->meta III, and rho-->opsin difference spectra at each of the pH values and obtained estimates for the microscopic rate constants at each pH. Because of assumptions that had to be made about the branching ratio between the meta II decay pathways, some uncertainties remain in our calculated rho-->meta III difference spectrum at each pH. Nevertheless, our data covering long time ranges, especially those obtained at pH 8, place significant new constraints on the spectrum of meta III and thus on its structure. The rho-->meta II spectrum shows no significant pH dependence over the range examined (pH 5.5-8). However, the rho-->meta III and rho-->opsin spectra each include a limited subset of pH-dependent peaks, which are mostly attributable to titratable amino acid side chains. Our observations can be used to refine an earlier conclusion that the visual pigment refolds to a rhodopsin-like conformation during meta II decay (Rothschild, K.J., J. Gillespie, and W.J. DeGrip. 1987 Biophys. J. 51:345-350). Most of this refolding occurs in the same way at pH values ranging from 5.5 to 8 and whether meta II decays to meta III or opsin. Meta II displays unique spectral perturbations that are mostly attributable to a few residues, probably including three to four aspartic or glutamic acids and an arginine.

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