It is proposed to bring single-crystal x-ray diffraction analysis to bear upon a variety of mutants of staphylococcal nuclease (SNase), in order to probe its mechanism of action and the forces that stabilize its folded state. Structures of SNase as the apoenzyme and as an inhibited, calcium-liganded complex have previously been determined by Cotton, Hazen and co-workers at atomic resolution. It is proposed to build upon these structures to achieve 6 main objectives: first, to do least-squares, coordinate refinement on one or both of these forms to produce a structure accurate by contemporary standards; a refined structure is necessary for more detailed study of mechanism and in the use of difference Fourier methods to determine the mutant structures; second, in collaboration with John Gerlt, to determine the structures of existing crystals of a variety of mechanism-probing mutants of SNase, particularly those in which the putative general base Glu-43 and proton donor Arg-87 are changed to other amino acids. These are only the first in a carefully planned set of mechanism-probing mutants that the Gerlt laboratory will be sharing with us; third, to crystallize and study abortive complexes of SNase with substrate (perhaps by replacing Ca(II) with Nd(III)), since the exact mode of substrate binding to SNase is not clear from the inhibited crystal structure; fourth, to crystallize and study complexes of SNase with the putative transition state analogues uridine - VO++and thymidine - VO++; fifth, in collaboration with David Shortle, to study existing crystals of mutants (initially Val-23-Phe) that appear to destabilize the folded state of SNase, and of the second-site suppressors His-124- Leu or Arg that re-stabilize many different mutant molecules; last, in collaboration with Mario Amzel and Mark Snow, to determine the structures of mutants whose structures they have predicted using new algorithms, and to work with them to improve these algorithms. In addition, the structures of a vareity of single-site alterations of SNase will provide an important data base for protein structural studies of all kinds.
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