CARBON-13 AND NITROGEN-15 NUCLEAR MAGNETIC RESONANCE STUDIES OF RHODOPSIN AND BACTERIORHODOPSIN (NMR)

The primary emphasis of the work presented in this thesis is the study of the structure of the bound retinylidene chromophore and its interaction with the protein binding site in the visual pigment, rhodopsin, and the related pigment, bacteriorhodopsin, by means of ('13)C and ('15)N nuclear magnetic resonance spectroscopy. The analysis of the spectra was accomplished on the basis of ('13)C and ('15)N NMR spectral studies of a series of model imines and iminium ions. The strategy adopted was to chemically synthesize retinals enriched with ('13)C at key positions and to incorporate isomerically pure retinals, isolated by preparative high pressure liquid chromatography, into the pigments by regeneration with the native apoproteins.

Spectra of rhodopsin and bacteriorhodopsin, labelled with ('13)C at retinylidene carbon positions 15, 14, 13, 11, 9, and 6, 13 (a double-labelling experiment) were obtained on the octyl-(beta)-glucoside solubilized proteins. Based on the spectra of model retinylidene imines and iminum ions in a variety of solvent environments, the majority of the nuclear magnetic resonance spectra correlate more closely with the existence of a protonated Schiff base link between the chromophore in a perturbed environment and the protein in each pigment. In addition, the ring portion of the bound retinylidene chromophore appears to possess significantly greater mobility than the polyene chain, from a T(,1) analysis of the {6,13-('13)C(,2)}-labelled pigments. The solid state (,13)C NMR spectrum of lyophilized {13-('13)C}-bacteriorhodopsin in the purple membrane was not consistent with the solution spectral results, in that a resonance characteristic of a nonprotonated imine link was distinguished; a possible explanation of this effect may be a dehydration-induced imine deprotonation, as observed by a number of other workers in the dehydration of rhodopsin. Due to the complexity of interfering protein resonances, the spectra of pigments labelled at retinylidene carbon position 14 did not permit an unambiguous assignment of the labelled resonance. The results of these studies were discussed in terms of models for wavelength regulation in the retinylidene proteins. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of school.) UMI