Dynamic processes are implicit in the catalytic function of all enzymes. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic functions remains unanswered. These issues will be addressed using state- of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well-characterized enzyme, dihydrofolate reductase from E. coli, in all of the intermediate states implicated along its reaction pathway. DHFR is the target enzyme for anti-folate drugs such as the anti- cancer agent methotrexate and the anti-bacterial trimethoprim and is of considerable medical significance. The proposed research will provide direct experimental data on the intrinsic molecular dynamics of this important enzyme and insights into the mechanism by which conformational fluctuations are coupled to interactions with substrate, cofactor, and products in the key intermediates of the catalytic cycle. Dynamic """"""""hot spots"""""""" identified through the NMR relaxation studies will be targeted for mutagenesis (Project 3). The insights into DHFR dynamics resulting from this research will provide a basis for the theoretical studies of Project 4.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
1P01GM056879-01
Application #
6271887
Study Section
Project Start
1998-01-01
Project End
1998-12-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Venkitakrishnan, Rani P; Zaborowski, Eduardo; McElheny, Dan et al. (2004) Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry 43:16046-55
Chen, Jianhan; Brooks 3rd, Charles L; Wright, Peter E (2004) Model-free analysis of protein dynamics: assessment of accuracy and model selection protocols based on molecular dynamics simulation. J Biomol NMR 29:243-57
Schnell, Jason R; Dyson, H Jane; Wright, Peter E (2004) Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu Rev Biophys Biomol Struct 33:119-40
Schnell, Jason R; Dyson, H Jane; Wright, Peter E (2004) Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase. Biochemistry 43:374-83
Osborne, Michael J; Venkitakrishnan, Rani P; Dyson, H Jane et al. (2003) Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes. Protein Sci 12:2230-8

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