Membrane proteins and protein complexes have in general eluded detailed structural characterization since many membrane systems and large macromolecules are inaccessible to traditional structural methods such as a solution NMR and X-ray diffraction. Solid-state NMR spectroscopy has developed into a versatile probe of molecular structure and dynamics, and is ideally suited for such systems. The proposed research from four principal investigators at Yale covers a broad range of active research in biochemistry and biophysics where the problems involve membrane systems or molecular complexes that require solid-state NMR methodology. The acquisition of a high-field solid-state NMR spectrometer is essential for addressing these problems, and will extend and complement the current methods for studying biomolecular structure and dynamics at Yale. (1) The first proposal on the visual pigment rhodopsin takes advantage of low-temperature solid-state NMR methods to study the photoreaction intermediates of rhodopsin. These experiments will provide critical new information on the location of protein charges in rhodopsin, and on the mechanism of light-transduction by the retinal chromophore. (2) Solid- state NMR studies are proposed for determining the mechanism of proton translocation by ubiquinone in mitochondrial and photosynthetic membranes. The research proposal describes a newly developed method for enhancing the rates of magnetization transfer between nuclear spin pairs and measuring intermolecular distances. These methods shall be used to establish the orientation of ubiquinone in energy-transducing membranes. (3) The third proposal uses deuterium NMR methods as a sensitive probe of the structure of phospholipids that are important in membrane fusion. These studies focus on anionic lipid-cation complexes that have been implicated in the initiation of fusion, a key process in cellular function. (4) Several recent advances in solid-state NMR methods are exploited in proposed research to study the dynamics of metal-ligand exchange in the active sites of metalloproteins. Exchange processes have prevented the observation of NMR resonances in solution. (5) The fifth proposal focuses on the structure of nucleotide-protein complexes. These studies are designed to bridge the two established structural methods, solution NMR and X-ray diffraction.
