Protein Dynamics in Enzymatic Catalysis

Proteins have highly dynamic structures, despite the beautiful appearance of static snapshots obtained from crystallographic models. We are investigating the role of protein motions in enzymatic catalysis. A question of intense current scrutiny is whether such motions are purely stochastic, simply serving to move the system towards conformational substates that are catalytically competent, or do protein motions couple more directly to the reaction coordinate? We focus on the dynamical nature of hydride transfer in model systems, lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR). We ask what types of atomic motion take place, how are the motions defined structurally, on what time scales do they occur, and how do these motions affect function?

Our approach employs laser induced temperature jump and time-resolved, isotope-edited IR or time-resolved fluorescence spectroscopy to follow atomic motion from 10 ps to any slower time. For example, we have followed the fast relaxation kinetics of inner complex interconversion, LDH/NADH/pyruvate <-> LDH/NAD+/lactate as shown in the figure to the right. We are also investigating mutations of the active site residues that change kcat and kcat/Km in known ways and in ways thought to be influenced by the protein dynamics. Relative motions between substrate and protein are characterized using isotope-edited time resolved IR absorption studies of specific substrate groups: for example, the C=O stretch of pyruvate and the antisymmetric stretch mode of the carboxylate group of pyruvate and lactate. Hydride and proton transfer kinetics are monitored using the changes in NADH emission near 450 nm as NAD+ converts to NADH, and the changes in pyruvate's C=O stretch frequency and stretch modes of lactate as lactate converts to pyruvate. Kinetic isotope effects for the chemical processes provide further insight into the protein dynamics.

Funding

NIH P01 GM068036-01, "Protein Dynamics in Enzymatic Catalysis," (Callender, Dyer, Schramm, Schwartz co-P.I.s; 6/04-5/14

Recent Publications

“Direct Observation and Control of Ultrafast Photoinduced Twisted Intramolecular Charge Transfer (TICT) in Triphenyl-Methane Dyes” G. Li, D. Magana and R. B. Dyer J. Phys. Chem. B, 2012, 116(41): 12590–12596. PMC3475756
“Photoinduced Electron Transfer in Folic Acid Investigated by Ultrafast Infrared Spectroscopy,” G. Li, D. Magana, and R. B. Dyer, J. Phys. Chem. B, 2012, 116, 3467-3475. PMC3311227
"Time-resolved spectroscopic studies of protein dynamics," R. Callender; R. B. Dyer, Encyclopedia of Biophysics, M. Deshpande, Editor, 2012, in press.
"Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase," Deng, H., D.V. Vu, K. Clinch, R. Desamero, R.B. Dyer, and R. Callender, J. Phys. Chem. B, 2011, 115 (23), pp 7670–7678.
"On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase," Deng, H., Brewer, S., Vu, D. M., Clinch, K., Callender, R., Dyer, R. B., Biophys. J. 2008, 95, 804-813.
"A simple and economical method for the production of 13C, 18O-labeled Fmoc-amino acids with high levels of enrichment: applications to isotope-edited IR studies of proteins," J. Marecek, B. Song, S. Brewer, J. Belyea, R. B. Dyer and D. P. Raleigh, Org. Lett. 2007, 9, 4935-4937.
"Microfluidic Flow-Flash: Method for Investigating Protein Dynamics," M. W. Toepke, S. H. Brewer, D. M. Vu, K. D. Rector, J. E. Morgan, R. B. Gennis, P. J. A. Kenis, and R. B. Dyer, Anal. Chem. 2007, 79, 122-128.
"Structural Transformations in the Dynamics of Michaelis Complex Formation in Lactate Dehydrogenase," S. McClendon, D. M. Vu, K. Clinch, R. H. Callender, R. B. Dyer, Biophys. J. 2005, 89, L7-L9.