Our goals are to understand (i) the molecular mechanism of the initial photochemical events in visual excitation, (ii) how rhodopsin shifts the absorption of its 11-cis retinal protonated Schiff base chromophore from 440 nm in solution to 500 nm in rod pigments, (iii) how this absorption maximum is regulated in UV, blue- and red-absorbing pigments, (iv) the mechanism of energy storage in the primary photoproduct, and (iv) how this stored energy is used to drive protein conformational changes that activate transducin. These goals will be addressed by performing time-resolved and low-temperature resonance Raman experiments, through femtosecond [(10)-15 s] transient absorption and Raman spectroscopy, and through time-resolved UV resonance Raman spectroscopy of protein structural dynamics.
Our specific aims are: (1) Resonance Raman microprobe spectra will be obtained of rhodopsin site-specific mutants and of UV, blue- and red-absorbing pigments stabilized at 77 K. By determining which mutations alter the vibrational structure of the chromophore in rhodopsin, we will identify the residues and interactions that produce the opsin shift. By examining the effect of these mutations on the vibrational spectra of the primary photoproduct, bathorhodopsin, we will identify the residues that are responsible for its unusual vibrational structure and energy storage. The UV, blue and red pigment data will enable us to further examine the molecular mechanism of the opsin shift. (2) We will obtain high-quality, time-resolved resonance Raman microchip flow spectra of the BSI, Lumi and Meta I intermediates and use vibrational analysis to determine the chromophore structure and protein interactions in these intermediates. (3) Femtosecond Raman Gain Spectroscopy (FRGS) will be developed as a new ultrafast structural technique for studying chemical and biochemical reactions. We will obtain vibrational spectra of the isomerizing retinal chromophore in rhodopsin with better than 100 fs time resolution and use these data to define the excited state photoisomerization mechanism. (4) Picosecond time-resolved Stokes and anti-Stokes resonance Raman spectroscopy will be used to study the structure and energetics of the primary photoproduct in vision to understand the mechanism of energy storage and of the isomerization process. (5) Picosecond time-resolved UV Raman studies will be performed to determine the protein structural changes that are driven by chromophore isomerization.
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