Visual processes in vertebrates and invertebrates utilize the trans- membrane protein, rhodopsin (Rh), to store and transduce absorbed light energy. At the molecular level, the energy storage/transduction mechanism involves a series of transformations in (i) the structure of the single visible chromophore in Rh, retinal, and (ii) the interactions between retinal and its protein environment. For an in vivo protein, these processes are prerequisites for lipid conformational changes, signal amplification, and eventually the transformation, via G-protein (transducin) binding at the protein surface, into nerve signals. Many aspects of the Rh mechanisms are mimicked by the bacteriorhodopsin (BR) photocycle which is easier than Rh to study experimentally. The proposed research expands earlier work performed in the group during which the earliest (femto/picosecond) molecular processes in the room temperature, Rh photo-reaction were examined. Emphasis in the proposed work is placed on the structural changes associated with (i) the photo and batho intermediates where much of the absorbed energy is stored initially and (ii) the blue-shifted intermediate (BSI) and lumi-rhodopsin (lumi) which are involved in the energy transduction. Until this study, structural information for Rh has been limited to frozen samples in which the photo-sequence is interrupted to stabilize a particular species. The vibrational spectrum of room-temperature batho, obtained recently for the first time in this laboratory, demonstrated that low-temperature vibrational data do not accurately describe the room-temperature visual mechanism. The time-resolved coherent Raman techniques and quantitative analysis methods of non-linear optical data required to measure picosecond structural changes of a room-temperature protein were also recently developed in this laboratory. These experiments overcome the irreversibility of the Rh photo-sequence which has limited previous efforts to obtain room temperature data. Artificial (chemically- and isotopically-modified retinals) and mutant Rh pigments (provided through collaborations with leading researchers in these respective fields) will be examined to elucidate the importance of molecular motions at specific retinal bonds to the Rh photo-sequence. Proposed experiments will characterize the structures and kinetics properties assignable to the photo, batho, BSI, and lumi intermediates in the Rh photo-sequence. Experimentally, the successful development and application of picosecond time-resolved coherent anti-Stokes Raman (PTR/CARS) pioneered in this laboratory will be expanded to elucidate how changes in the retinal structure and in retinal-protein interactions control the early stages of the Rh photo-sequence. Picosecond transient absorption and fluorescence measurements will complement those of vibrational spectroscopy. The time- resolved coherent Raman techniques also will be expanded to measure dipole moment changes along specific vibrational modes through the systematic control of polarization. Efforts also will be made to measure back reactions which effectively interrupt these respective photo-reactions. Some parallel studies in BR will be undertaken to further develop and evaluate new experimental and analysis methods as well as to expand our understanding of this independently important trans-membrane, photosynthetic protein.
