We propose to purchase a 900 MHz NMR spectrometer and operate it as a national user facility. The scientific experiments planned involve solution NMR and high-resolution solid state NMR. ? ? Solution NMR: the solution experiments are directed at structural problems that: are at or beyond the capabilities of presently available instrumentation. We emphasize the importance of sample preparation technologies that enable us to study the most significant biological problems rather than just well behaved model systems. Thus, most of our molecule also represent targets for development of drugs against cancer, viral infection, bacterial and immune diseases Our novel protein expression strategies have allowed us to overcome most of the problems of poor solubility and stability for nearly all projects we are working on so that the limiting hurdle of solution structure determination is shifting back from sample preparation to spectral resolution and sensitivity. Our solution NMR studies will include structures of large protein-protein and protein-nucleic acid complexes, and structures of member C proteins. The high-field spectrometer will also be utilized, to characterize protein-ligand complexes in the sear h for agents against cancer, viral infection and as immuno-suppressants. We will pursue larger structures with R and crystallography to benefit from the synergy of both techniques. ? ? Solid State NMR: S lid-state experiments will utilize a new class of dipolar recoupling techniques that have produced the initial de novo structure of a small molecule. We will extend these techniques to microcrystalline proteins. Further, the methods will permits us to address questions of the mechanism of H+ pumping in bacteriorhodopsin and to develop methods to perform structural studies of membrane proteins. The de novo structure of gas vesicles will emerge from these investigations. A second area of interest is the structure of amyloid fibrils where e plan to determine the structure of the A protein associated with Alzheimer's disease, as well as a fibril forming SH-3 domain and a-synuclein associated with Parkinson's disease. Finally, high fields will permit us to develop 17 O NMR as an important new tool for studies of biological systems.
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