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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036358-05
Application #
3290172
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1986-12-01
Project End
1991-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Garcia-Moreno, B; Dwyer, J J; Gittis, A G et al. (1997) Experimental measurement of the effective dielectric in the hydrophobic core of a protein. Biophys Chem 64:211-24
Green, S M; Gittis, A G; Meeker, A K et al. (1995) One-step evolution of a dimer from a monomeric protein. Nat Struct Biol 2:746-51
Loll, P J; Quirk, S; Lattman, E E et al. (1995) X-ray crystal structures of staphylococcal nuclease complexed with the competitive inhibitor cobalt(II) and nucleotide. Biochemistry 34:4316-24
Abeygunawardana, C; Weber, D J; Gittis, A G et al. (1995) Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase. Biochemistry 34:14997-5005
Libson, A M; Gittis, A G; Lattman, E E (1994) Crystal structures of the binary Ca2+ and pdTp complexes and the ternary complex of the Asp21-->Glu mutant of staphylococcal nuclease. Implications for catalysis and ligand binding. Biochemistry 33:8007-16
Keefe, L J; Quirk, S; Gittis, A et al. (1994) Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease. Protein Sci 3:391-401
Weber, D J; Serpersu, E H; Gittis, A G et al. (1993) NMR docking of the competitive inhibitor thymidine 3',5'-diphosphate into the X-ray structure of staphylococcal nuclease. Proteins 17:20-35
Keefe, L J; Sondek, J; Shortle, D et al. (1993) The alpha aneurism: a structural motif revealed in an insertion mutant of staphylococcal nuclease. Proc Natl Acad Sci U S A 90:3275-9
Gittis, A G; Stites, W E; Lattman, E E (1993) The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. J Mol Biol 232:718-24
Weber, D J; Gittis, A G; Mullen, G P et al. (1992) NMR docking of a substrate into the X-ray structure of staphylococcal nuclease. Proteins 13:275-87

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