A variety of studies are proposed to enhance our understanding of visual transduction, the process in which light absorbed in the retina is converted to an electrical impulse signaling light detection. In the past grant period substantial progress was made using the mechanistic framework that describes visual pigment activation in order to understand how mutations affect vision and to develop concrete structural models of the intermediates along the activation pathway of pharmacologically important G-protein coupled receptors (GPCRs) which are less accessible to direct time-resolved study. Refinement of our models will be supported by characterization of photointermediates in 2D and 3D rhodopsin crystals where static structure can be determined to atomic resolution. More dynamic structural information will be obtained using polarized optical methods, with linear dichroism probing membrane properties and the oligomeric state of rhodopsin, and circular dichroism being used to probe chromophore torsion and protein structure changes during activation. The high time resolution of our methods allows all of these techniques to be applied under physiological conditions, which is critical to success because the membrane/protein interactions important in GPCR activation are extremely sensitive to the physical state of the system, changing not only quantitatively but also in some circumstances, qualitatively with temperature and membrane solubilization. Given the structural similarities of rhodopsin and other GPCRs, the resulting information is likely to also be very important for gaining a molecular level understanding of how this important class of proteins function. Inherited mutations in the protein rhodopsin are a cause of retinitis pigmentosa, a disease that affects approximately 100,000 people in the U.S. and that often causes blindness. Our studies show how mutations cause rhodopsin to change its function. A wider public health reason for rhodopsin study is that over half of all current medications act on receptors in the rhodopsin family, so by using light to study rhodopsin, we can understand how those receptors work which will help to make existing medications more effective and help to develop new ones. ? ? ?
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