Membrane proteins are responsible for many essential biological functions, including as the largest and most important class of drug receptors. More than 20% of the proteins encoded in the human genome are helical membrane proteins, and many human diseases result from mutations in these proteins. The structures of membrane proteins will provide information essential for understanding their functions and designing new drugs. However, structure determination of this class of proteins is problematic for current methods, and the development of new methods is urgently needed. The overall goal is to develop a generally applicable method for determining the three-dimensional structures of helical membrane proteins in phospholipid bilayers. The immediate goals are to develop solid-state NMR of aligned samples and to apply this approach to determine the structure of a family of mercury transport membrane proteins with two, three, and four trans-membrane helices. This research will bridge between the initial examples with one trans-membrane helix whose structures have been determined with this approach and larger more complex proteins with multiple membrane spanning helices. The ultimate goal is to apply this technology to G-protein coupled receptors (GPCRs) that have seven trans-membrane helices. This interdisciplinary research involves protein expression, isotopic labeling and purification;preparation of highly aligned phospholipid bilayer samples;experimental NMR spectroscopy;and the calculation of protein structures. Solid-state NMR of aligned samples is a promising approach for the following reasons: 1.) The proteins are in fully hydrated phospholipid bilayers under physiological conditions. 2.) There are no fundamental size limitations to solid-state NMR spectroscopy and the proposed studies set the stage for future applications to larger membrane proteins. 3.) All backbone and side chain resonances can be resolved and assigned in proteins obtained by expression. 4.) The measured orientation constraints yield structures with atomic resolution. 5.) An integrated view of protein structure and dynamics can be obtained.
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