Support is requested for a continuation of this laboratory's structural studies of staphylococcal nuclease. In the current grant period the structures of the wild-type enzyme, and of a ternary complex of it with Ca2+ and with the nucleotide inhibitor 3',5'-deoxy-thymidine bisphosphate have been refined at high resolution to crystallographic R-values well below 0.18, forming the basis for the study of many mutant crystals; highlights are listed. The structure of the key catalytic mutant Glu-43- >Asp has been determined and refined. The structure of the mutant Val-66- >Lys has also been solved. Uniquely, this lysine is fully buried in a hydrophobic cavity. Solution studies of this mutant have allowed determination of the free energy needed to bury a charge, and of the dielectric constant within a protein. Studies of a mutant in which alanine is placed between residues 126 and 127 in the terminal helix reveal that the insertion is accommodated by a unique bulge or alpha-aneurism. Finally, in collaboration with Albert Mildvan, the structure of nuclease complexed with an authentic substrate dTpdA, and with the inactive ion La3+ has been determined by NMR and computer-assisted docking. Current specific aims focus on two areas: folding/stability and catalysis. Key catalytic studies include structure determinations of complexes of nuclease with the transition-state analogue vanadate+thymidine, and with La3+ and dTpdA. The mutant des-44-49, in which a flexible loop adjacent to the active site has been deleted appears to have a much more rigid active site, and forms the basis of proposed studies to determine why certain ions are active and others not. Stability studies will also include extension of the Val-66->Lys work to hyper-stable forms of nuclease in which the solution studies can be carried out over a wider range of conditions. Work will also proceed on a series of single and double insertion mutants placed at many sites in the molecule, and on a highly unstable quadruple mutant, known as tet 3, Data on tet 3 are being collected at a series of temperatures, in the hope that regions whose mobility increases rapidly with temperature will be revealed. A series of computational experiments is also planned.
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