We propose the determination of metal transporter structures using high field and fast (>60 kHz) magic angle spinning NMR spectroscopy. Proteins provide the basis for many important biological processes. They are found as soluble protines, such as enzymes, membrane associated proteins that interact with specific locations in the cell, and transmembrane (TM) proteins that often serve as the gatekeepers of cellular compartments. Membrane proteins comprise 20 to 30 percent of all proteins, however, less than 0.1% of the structures in the protein data bank are of membrane proteins. The proposed research applies the latest developments in MAS NMR to the structure determination of membrane proteins, providing a starting point for the rational development of inhibitors. An important class of membrane proteins are metal ion transporters and symporters, which selectively move metal ions across membranes. Most of these proteins are predicted to contain about 10 TM alpha helices, and many have been functionally characterized, but 3D structural information is lacking. Although these proteins are large by MAS NMR standards, recent advances in methodology such as proton detection at 60 kHz MAS and a high magnetic field of 1GHz has made these promising targets. Even more immediate targets for structural characterization are oligomeric proteins, including human copper transporter hCTR1 and cobalt/nickel transporter CorA. Because of oligomerization, these proteins have only 2 to 3 asymetric transmembrane helices, simplifying assignment of the spectra and structure determination. The application of 60 kHz MAS at high magnetic fields of 1GHz represents a major contemporary advance in sensitivity and resolution, that will extend the upper molecular weight limit of structure determination to include many membrane proteins. We propose to develop and apply these methods to alpha helical membrane proteins of 3 to 12 TM helices.
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