Abstract

This research will encompass two distinct projects in the area of biomolecular structure-function studies: 1) Construction of selected mutants of E. coli dihydrofolate reductase (DHFR); by x-ray crystallography observe the structural perturbations effected by these mutations; and characterize the catalytic properties of the mutant enzymes by steady-state and pre-steady-state kinetic methods, focusing on individual elementary steps in the pathway. The goal of this project is to answer various fundamental questions about the stereochemical mechanism that arise upon examination of crystal structures of E. coli DHFR and three other species of DHFR previously determined in this laboratory. 2) X-ray crystallographic structure determination of several lymphokines, including especially interleukin-2 and interferon- alpha. The lymphokines are a class of protein molecules that act as intercellular signals of the immune system. It is hoped to identify and examine the chemical geometry of the sites on these molecules that interact with their receptors and thereby elicit biological activity.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM010928-27
Application #
3268186
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1977-01-01
Project End
1993-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
27
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Sawaya, M R; Pelletier, H; Kumar, A et al. (1994) Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science 264:1930-5
Pelletier, H; Sawaya, M R; Kumar, A et al. (1994) Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264:1891-903
Brown, K A; Howell, E E; Kraut, J (1993) Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase. Proc Natl Acad Sci U S A 90:11753-6
David, C L; Howell, E E; Farnum, M F et al. (1992) Structure and function of alternative proton-relay mutants of dihydrofolate reductase. Biochemistry 31:9813-22
Farnum, M F; Magde, D; Howell, E E et al. (1991) Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis. Biochemistry 30:11567-79
Warren, M S; Brown, K A; Farnum, M F et al. (1991) Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis. Biochemistry 30:11092-103
Howell, E E; Booth, C; Farnum, M et al. (1990) A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase. Biochemistry 29:8561-9
Howell, E E; Warren, M S; Booth, C L et al. (1987) Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase. Biochemistry 26:8591-8
Villafranca, J E; Howell, E E; Oatley, S J et al. (1987) An engineered disulfide bond in dihydrofolate reductase. Biochemistry 26:2182-9
Howell, E E; Villafranca, J E; Warren, M S et al. (1986) Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis. Science 231:1123-8

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