Recent developments in NMR spectroscopy and molecular biology offer exceptional promise for structural studies of biological macromolecules in non-crystalline states. As a result of these developments, the molecular weight limit for NMR structure determination has increased significantly, such that a number of important problems in structural biology now become accessible. The general objectives of this proposal are to develop a general strategy for determination of highly refined three-dimensional solution structures of proteins of 15-2OkDa molecular weight and to extend these methods to allow structural studies of protein-protein and protein-DNA interactions that are of fundamental importance in biology. Specifically, the research will focus on (i) the 3D structure and interactions of a central bacterial regulatory protein, and (ii) zinc finger-DNA interactions. Both homonuclear and heteronuclear 2D and 3D NMR methods will be used and structures will be determined using methods based upon distance geometry and restrained molecular dynamics. High resolution solution structures will be determined for the IIIglc domain (Mr 17,400) from B. subtilis in both its phosphorylated and dephosphorylated states to elucidate the structural changes that accompany phosphorylation. IIIglc plays a central role in carbohydrate transport and as a regulator of various permeases and catabolic enzymes. The structure of the complex between IIIglc and the phosophotransfer protein HPr (Mr 6000) will be investigated to elucidate the mechanism of phosphotransfer. High resolution solution structures of synthetic single zinc fingers of the TFIIIA type will be determined to provide insights into the effects of natural sequence variations on the 3D structure and stability and on interactions with DNA. The structure of a protein containing two zinc fingers will be determined to establish whether the fingers are truly independent structural motifs and to investigate the role of the linker sequence. NMR methods will be used to study the complex of TFIIIA zinc fingers with an oligonucleotide DNA duplex corresponding to the binding site in the internal control region of the 5S RNA gene. These experiments should provide new information of fundamental importance for understanding the molecular basis for sequence-specific recognition of DNA by zinc fingers of the TFIIIA-type. Such studies are of particular importance given the central role played by zinc finger proteins in transcriptional regulatory processes in both health and disease.
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