The long range goal of the parent proposal, upon which the FIRCA proposal is based, is to elucidate molecular mechanisms of membrane excitation related to visual transduction. The experimental approach outlined in the parent proposal was based on a new technique for determining structure and dynamics in membrane proteins termed site-directed spin labeling. In this technique, single cysteine residues are placed at selected positions in the protein sequence using site-directed mutagenesis, and subsequently reacted with sulfhydryl specific nitroxide spin labels. Analysis of the electron paramagnetic resonance (EPR) spectrum of an attached nitroxide yields much information about local structure and dynamics, while a sufficiently large set of such labeled mutants can provide the structure of protein at the level of backbone folding. The FIRCA proposal is an important extension of the parent grant in that it provides for the development of important new tools for analyzing the attached nitroxides in terms of structure and dynamics, namely Saturation recovery (SR) and Pulsed Electron- electron Double Resonance (P-ELDOR) spectroscopies. These time- domain techniques will open a new dimension of experiments to obtain direct structural information on membrane proteins. An important part of the proposal will be to advance the state-of-the- art in time-domain techniques by construction of a bimodal loop-gap resonator for pulse techniques.
