The proposal describes four specific aims which will test the ultrafast responses of native rhodopsin and rhodopsin analogues using visible, near, and mid infrared spectroscopy. The first specific aim concerns isotope effects on the ultrafast processes involved in isomerization. The candidate will utilize an ultrafast laser system with 35 fs resolution to monitor the dynamics of the initially-produced intermediates involved in the primary processes of isomerization. Theses experiments will test the possibility that local charge density surrounding retinal controls the dynamics of isomerization. If this is so, then a proton transfer, which may precede retinal isomerization, would change the charge density in the vicinity of retinal and affect isomerization dynamics. The candidate should be able to determine whether a proton shift or a change in hydrogen bonding strength has occurred by correlating the dynamics of isomerization, using visible light pulses, and proton shifting, using infrared light pulses. These experiments are will-designed and should provide valuable information. In the second aim, the candidate, using short excitation/probe pulses, will concentrate on following the isomerization process in real time in the spectral region to the red of 650 nm and to the blue of 480 nm. Recent results on bacteriorhodopsin, which has substantial homology with rhodopsin, indicate that important information concerning the kinetic signatures of intermediates crucial to the initial isomerization process may be contained in these spectral regions. These well- designed experiments should provide this information, as well as determine from which electronically excited state isomerization occurs. In the third specific aim, the candidate will use techniques developed to monitor conformational changes of retinal during isomerization. Specifically, she will use measurements of the vibrational characteristics and dipole moment changes of retinal to track isomerization in real time. The experiments are well-designed and should provide a clearer picture of the dynamics of isomerization as seen from changes in the anisotropy signal and vibrational spectra. In addition, these results may provide insight into the relative importance of the individual motions of specific substituents of retinal of the initial isomerization process. In the fourth specific aim, the candidate will use site directed mutagenesis to modify specific amino acid residues that reside within the cofactor binding pocket in rhodopsin. These experiments could reveal whether particular sites on the opsin interact with the cofactor. In fact, the candidate provides reasonable hypotheses with respect to the possible important of two specific opsin amino acid residues that she proposes to test. However, she does not explain how mutations of these amino acid residues will be obtained, except to state that various named individuals will develop these rhodopsin mutants or supply the resources to do so. These individuals have not provided letters of collaboration for this proposal, however. It is therefore not clear whether this last specific aim will be successfully performed.
