Membrane-bound light-activated proteins: Study of the visual pigments and bacteriorhodopsin analogues

Date of Completion

January 2006


Chemistry, Biochemistry




The photophysical properties of the retinal-binding proteins, rhodopsin, bacteriorhodopsin and selected cone pigments are analyzed by using spectroscopy, chromophore analogues and site-directed mutagenesis. Both bacteriorhodopsin (BR) and the vertebrate visual pigments are composed of a seven α-helical transmembrane apo-protein called opsin and a retinylidene chromophore covalently linked to a lysine residue. In chapter one, a detailed background on retinal proteins is presented. A key goal of this chapter is to describe our current understanding of wavelength regulation. We note that spectral tuning involves manipulation of the chromophore geometry as well as the electrostatic environment of the binding site. Chapter two describes the photobleaching pathway and the role of the primary counterion in the blue and UV cone pigments, investigated via site-directed mutagenesis and cryogenic UV-VIS spectroscopy. Chapter three examines the photophysical properties of the BR analogue, 4-keto bacteriorhodopsin, by using time-resolved UV-Vis, FTIR spectroscopy, and computational studies. The 4-keto bacteriorhodopsin photocycle is assigned and we demonstrate that there is only one M state, in contrast to previously published models. Chapter four examines the effect of chromophore geometry in spectral tuning of M/LWS pigments. Analyses of homology models and spectral nomograms indicate that an important molecular mechanism of spectral tuning in the very long-wavelength M/LWS pigments is a change in conformation of the chromophore around the C 6-C7 bond. Conclusions and suggestions for future work are made at the end of each chapter. ^