This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Part of this new collaborative research project is developing novel single molecule fluorescence experiments on membrane proteins which are vital components of the cell physiology. The large, alpha-helical class of membrane proteins includes cell-surface receptors, ion channels, transporters and redox proteins. Many have a single transmembrane (TM) helix that homo-oligomerizes or associates with other TM helices to form bundles. These TM assemblies are of critical importance in a variety of biological situations and also have advantages for the study of folding in membranes. Integral membrane proteins are estimated to account for nearly one-quarter of all coding sequences in higher organisms, and more than half of all commercial drugs target this class of proteins. Their 3D structures have been studied by NMR on proteins anchored in phospholipid bilayers. The mechanisms of association and dissociation of membrane proteins in micelles, vescicles and lipid bilayers are not yet clarified. The sensitivity to probe location, temperature, detergents, cholesterol and lipid bilayer characteristics at the single monomer and dimer levels in the case of single molecule spectroscopy will yield unique information on the helix interactions in the membrane. Work in the single molecule area should provide particularly fertile and significant avenues for investigations on fluorescently labeled TM helices to expose both equilibrium dynamics and the parameters of the structures of coupled residues in addition to properties of the membranes themselves. Structural dynamics can be revealed by lifetimes, FRET, polarization anisotropies and correlation spectra of fluorescent probes like the Nile red dye we used successfully in previous studies.
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