The use of solution NMR to determine MP structure is now in a rapid state of growth. Recently, this approach has been employed to solve a number of new MP structures of high biological impact, demonstrating its potential in MP structural genomics. However, there is not yet a specialized center that focuses on broad development of this important emerging area of structural biophysics. We propose a Protein Structure Initiative (PSI) Center that aims to develop an efficient solution NMR pipeline for solving MP structures. This Center consists of a team of investigators who are among the most productive in the world in solving MP structures by NMR, including James Chou, Gerhard Wagner, Charles Sanders, and Volker Dotsch. The Center also includes a synthetic core for providing the materials used in MP NMR, an NMR/computational core for developing faster methods of structure determination, and an administrative core for management and dissemination of technology. To drive technology development and to test the proposed pipeline, we have selected 10 MP targets for which the structures are not known, including membrane-embedded transporters, enzymes, and receptors. These targets are polytopic helical MPs with 3-7 transmembrane helices and with sizes from 18-43 kDa. The Center will develop technologies that will have immediate and practical impact on structure determination. They include (1) cell-free expression platforms for production of MPs and for screening for NMR-feasible MP targets;(2) new detergents, bicelles and MP refolding methods;(3) non-uniform sampled high resolution 4D NOESYs;(4) new strategies for selective isotope labeling of methyl groups for acquiring long-range NOEs;(5) novel reagent for site-directed paramagnetic tagging and universal DNA-nanotube alignment media for RDC measurements;and (6) RDC-based molecular fragment replacement and structure calculation protocols. Although the phrase """"""""high-throughput"""""""" does not yet apply to MP in any technological context, we aim to establish a NMR tool package within the proposed funding period that has the capacity for systematic production of MP structures, while delivering the structures of the target MPs.
MPs constitute about 50% of drug targets, but their structures for rational drug design are generally not available due to the technical challenges involved in solving MP structures. The NMR technologies proposed in this grant would constitute a robust pipeline for systematic production of MP structures. This pipeline would nicely complement the existing crystallography-based Centers in the coverage of MP structure space.
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