Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. 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 function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle? These and other 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 formed along its reaction pathway. DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterial trimethoprim and is of major biomedical significance. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active site, on the same time scale as key events in DHFR catalysis. The NMR experiments will provide a detailed dynamic and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that mapped the active site conformational states of DHFR and its fast (ps-ns) time scale dynamics. The proposed research will provide novel insights into the coupling between protein motions and catalytic function in DHFR, as well as an understanding of how these motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075995-03
Application #
7383939
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2006-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
3
Fiscal Year
2008
Total Cost
$322,929
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Krois, Alexander S; Dyson, H Jane; Wright, Peter E (2018) Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain. Proc Natl Acad Sci U S A 115:E11302-E11310
Fenwick, R Bryn; Oyen, David; Dyson, H Jane et al. (2018) Slow Dynamics of Tryptophan-Water Networks in Proteins. J Am Chem Soc 140:675-682
Oyen, David; Fenwick, R Bryn; Aoto, Phillip C et al. (2017) Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion. J Am Chem Soc 139:11233-11240
Aoto, Phillip C; Martin, Bryan T; Wright, Peter E (2016) NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38?. Sci Rep 6:28655
Fenwick, R Bryn; Dyson, H Jane (2016) Classic Analysis of Biopolymer Dynamics Is Model Free. Biophys J 110:3-6
Fenwick, R Bryn; Oyen, David; Wright, Peter E (2016) Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics. Phys Chem Chem Phys 18:5789-98
Oyen, David; Fenwick, R Bryn; Stanfield, Robyn L et al. (2015) Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway. J Am Chem Soc 137:9459-68
Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E (2014) Side chain conformational averaging in human dihydrofolate reductase. Biochemistry 53:1134-45
Aoto, Phillip C; Fenwick, R Bryn; Kroon, Gerard J A et al. (2014) Accurate scoring of non-uniform sampling schemes for quantitative NMR. J Magn Reson 246:31-5
Fenwick, R Bryn; van den Bedem, Henry; Fraser, James S et al. (2014) Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR. Proc Natl Acad Sci U S A 111:E445-54

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