This project will elucidate the role of protein dynamics in enzyme function on all time scales, with the overall goal of developing enzyme design principles based on dynamics. We have pioneered new approaches to elucidate enzyme dynamics using structurally specific approaches, including isotope edited infrared spectroscopy coupled with ultrafast reaction initiation (T-jump or pH jump) and with fast microfluidics mixing methods. On the femtosecond time scale, we seek a deeper understanding of the fast atomic motions required to move the system over the transition barrier to achieve chemistry. On slower time scales (ps - ms) we seek to elucidate the conformational changes associated with substrate binding, catalytic site reorganization and product release. We focus on three enzymes, dihydrofolate reductase (DHFR) and in close collaboration with the Callender and Schramm labs respectively, lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP). The work is supported by theory and computation in the Schwartz group. The project has three specific aims: (1) Determine the conformational dynamics that control DHFR catalytic activity.
This aim will test the hypothesis that the conformational dynamics of the Met20 loop act as a master control of DHFR activity by modulating the barriers to proton and hydride transfer. We will determine the effects of mutations discovered in our lab that perturb the H-bonding network on the proton transfer dynamics and any coupled protein dynamics, using pH jump methods and time-resolved IR spectroscopy. (2) Determine the protein structural dynamics that control the formation of the Michaelis complex in LDH.
This aim will test the hypothesis that the Michaelis sub-state distribution and catalytic efficiency of LDH are controlled by the energy landscape of the catalytically important loop motions. We plan to determine how these loop motions are related to the sub-state distribution and the extent to which the protein conformational distribution is collapsed in the observed sub-states using ultrafast mixing, coupled with T-jump experiments in the Callender lab. Calculations by the Schwartz group will identify the dominant Michaelis configurations and measure the dynamics of transitions between them. These calculations will enable the interpretation of the dynamics observed in our experiments. (3) Investigate the relationships between protein structural dynamics, pathways for energy flow and allostery in enzymes.
This aim will test the hypothesis that allostery requires pathways for energy flow to reach a specific target that depend on the protein structure and its dynamics. We have developed ultrafast, pump-probe IR spectroscopy to probe the specific pathways of energy flow in enzymes. We will apply these methods to characterize the dynamics of energy flow in DHFR, LDH and PNP, and how it depends on inhibitor binding and the distribution of the conformational sub-states of the enzymes. We expect tight binding dynamic inhibitors to have very different energy flow dynamics than less efficient inhibitors that cause conformational collapse.

Public Health Relevance

Dihydrofolate reductase (DHFR) has been a target for drug development because of its essential role in nucleic acid production. Our research program is developing a detailed understanding of how protein motions on all time scales are involved in DHFR and other enzyme function, which in turn enables the development of improved inhibitors as drugs. We have developed unique, cutting edge approaches to elucidate enzyme dynamics that includes such innovations as isotope edited infrared spectroscopy and ultrafast microfluidic mixing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-VH-F (40))
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Albert Einstein College of Medicine
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Einarsdóttir, Olöf; McDonald, William; Funatogawa, Chie et al. (2015) The pathway of O?to the active site in heme-copper oxidases. Biochim Biophys Acta 1847:109-18
Reddish, Michael J; Peng, Huo-Lei; Deng, Hua et al. (2014) Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy. J Phys Chem B 118:10854-62
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Peng, Huo-Lei; Deng, Hua; Dyer, R Brian et al. (2014) Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism. Biochemistry 53:1849-57
Masterson, Jean E; Schwartz, Steven D (2014) The enzymatic reaction catalyzed by lactate dehydrogenase exhibits one dominant reaction path. Chem Phys 442:132-136
Li, Guifeng; Magana, Donny; Dyer, R Brian (2014) Anisotropic energy flow and allosteric ligand binding in albumin. Nat Commun 5:3100
Schramm, Vern L (2013) Transition States, analogues, and drug development. ACS Chem Biol 8:71-81
Masterson, Jean E; Schwartz, Steven D (2013) Changes in protein architecture and subpicosecond protein dynamics impact the reaction catalyzed by lactate dehydrogenase. J Phys Chem A 117:7107-13
Motley, Matthew W; Schramm, Vern L; Schwartz, Steven D (2013) Conformational freedom in tight binding enzymatic transition-state analogues. J Phys Chem B 117:9591-7

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