The long term goal of this project is to elucidate the mechanisms of (1) transmembrane molecular switching during signal transduction and (2) the protein transport across membranes. The proposed research explores a new electron paramagnetic resonance (EPR) approach to the investigation of the structure and dynamics of proteins. The strategy is to site-specifically place a nitroxide spin label at any selected site in a protein either using site-directed mutagenesis to replace a native residue with cysteine that provides unique labeling sites for the nitroxide spin labels, or using the synthetic method in which an unnatural amino acid with a nitroxide moiety is directly incorporated. Information on the structure and topology of a protein is obtained from EPR analysis of spin labeled mutants. Association equilibria of membrane receptors can be also studied. Moreover, time-resolved EPR spectroscopy makes it possible to resolve the sequential mechanism of the structural dynamics of spin labeled mutants. With these methods we (i) characterize protein conformational changes; (ii) identify all intermediates; (iii) interpret the kinetics in terms of structure. This unique EPR approach will be applied to two systems: (i) the intact bacterial chemotaxis receptor, a homodimeric membrane protein that is directly involved in signal transduction, for which motion of structural units during transmembrane signaling will be, characterized, defined, and time resolved; (ii) a mitochondrial signal peptide, for which the mechanism of transmembrane potential-dependent translocation will be determined to probe the biophysical principles of protein transport.
