Chemistry and biology of retinal proteins

I. Biooraganic studies on visual pigment rhodopsin. Rhodopsin is the photoreceptor responsible for dim-light vision in the retina of vertebrate species. This membrane protein has seven transmembrane alpha helices and contains an 11-cis retinal chromophore, which is attached to the epsilon-amino group of Lys 296 on helix G through a protonated Schiff base (PSB). The 11-cis → trans isomerization triggered by light irradiation initiates protein conformational changes leading to a cascade of events referred to as the visual transduction process.;The absolute sense of twist of the C12/13 bond of the retinal chromophore in bovine rhodopsin was investigated by incorporating 11,12-dihydroretinals into opsin and analyzing their circular dichroism (CD) spectra. A seven-membered ring was applied to fix the 11,12-dihydro analogs into the 11-cis conformation. CD spectra of 11,12-dihydrorhodopsins exhibit negative bisignate curves which reflects the chirality of C12/13 twist. This is the first time that exciton coupled CD has been applied to elucidate subtle details of substrate conformation within proteins.;It has been observed that 11-cis 13-desmethyl retinal and opsin leads to a transient dark activity. The understanding of the mechanisms of opsin dark activity is very limited despite its involvement in bleaching adaptation and some night blindness and retinal degeneration diseases. The mechanism of the transient dark activation has now been investigated using retinal analogs, transducin GTPgammaS binding assay, and UV/CD measurements.;II. Orientation of the retinal polyene methyl groups in bacteriorhodopsin . The conformation of the retinal chromophore, particularly the orientation of the polyene 9-Me and 13-Me is critical for the elucidation of the proton pumping mechanism of bacteriorhodopsin. Bioorganic studies using sulfate analogs and photoaffinity crosslinking experiments indicated that these methyls point toward the extracellular side, opposite to the results from biophysical measurements by other laboratories. It is possible that rotation about certain single bonds in the retinal analogs caused the misinterpretation of the chain methyl orientation in the bioorganic studies. 13C labels were incorporated into these analogs. Solid state 13C NMR measurements on bacteriorhodopsin incorporating these analogs are expected to determine the possibility of such a rotation.