The broad, long.term objective of this application is to determine the detailed molecular mechanism of visual receptor excitation, recovery and adaptation (light and dark) at the physical biochemical level. The chain of components we address are molecules of receptor (rhodopsin), GTP- binding protein and effector (phosphodiesterase) with their interacting protein subunits, nucleotides and Mg ions. These form a hair-triggered, single photon detecting dual amplifier cascade capable of molecular amplification of one million fold per photon within one second. The equally essential property or molecular stabilization against spontaneous activity in darkness via molecular kinetic energy barriers forms our principal focus in this grant period. We specifically propose to study individual bimolecular and trimolecular reaction kinetics and thermodynamics using absorption and fluorescence spectroscopy, dual wavelength kinetics, microcalorimetry, radioisotopic nucleotide exchange, differential centrifugation and fast filtration where membrane proteins move to and from aqueous phase. Quantitative experimental design with computer assisted data acquisition together with thermodynamic and kinetic analysis and molecular reaction modeling are essential tools for this word We plan to use specific peptide molecules which may substitute for parts or all of a specific protein reactive interface or that may block interface access to each other to identify reaction partners. These will also help us to identify the causal forces and to identify intermolecular groupings responsible for these events. Photophysical analysis of tryptophan fluorescence emission will be used to localize intramolecular regions and to determine how specific internal parts of a transduction molecule changes upon interaction with a reaction partner and how fast the change occurs. Health consequences of this research are directly related to understanding the mechanism by which mutations in the many different forms of congenital blindness can occur due to faults in the visual transduction protein machinery. Global consequences for many other sensory, hormone, neurotransmitter and growth control (cancer) mechanisms are strongly implied because of the molecular design of all of these systems is the same, differing only in the mutual recognition specificity of the individual proteins.
