We plan to determine by NMR the solution structures of various fragments of the yeast transcriptional activator GAL4 and their complexes with DNA. We will study the fragment of 1-65 that binds DNA as a dimer but is monomeric in solution as well as a fragment of ca. 94 residues that also binds DNA as a dimer but is dimeric in solution. We will attempt to identify a fragment of GAL4 that binds stably to a 10 base pair DNA fragment containing a UAS(G) half-site, in order to determine the structure of a protein-DNA complex. We also plan to study GAL4 fragments of about 200 residues, the minimum size for a protein with full transcriptional activation properties. In all these structural analyses we will focus on the geometry of the metal-thiolate cluster, on the protein DNA interface, and on the dimer interface. We will use the technique of isotope labeling in order to enhance the power of the NMR approaches. We also will direct our attention to studies of water molecules trapped in the interfaces, and we will try to characterize their mobility. The structures determined by NMR will be compared with results obtained from X-ray crystallography in the Harrison laboratory. The NMR and the X-ray groups will continue to interact and communicate freely, to take advantage of the complementary information from the two techniques, to speed up structure determination, and to recognize technique-related artifacts. Structure calculations, protein DNA docking, and energy refinements will be made using software provided by Core D.
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