Research Topics

Research Interest

The major objective of our research has been to determine protein structure and dynamics using nuclear magnetic resonance (NMR) spectroscopy, with a goal to elucidate protein function particularly relating to protein-ligand interactions.

(1) HIV-1 protease and its drug resistant mutants
Under drug pressure, multiple mutations accumulate in HIV-1 protease (PR) to confer resistance to inhibitors while maintaining catalytic activity. The mechanism of action for the resistance mutations has been explained based on the structural changes on the PR active site residues, and also on the thermodynamics effects on the secondary mutations in remote sites in the protein. However, how the mutations at different sites are structurally and dynamically coupled in the resistant PRs is not understood at the molecular level. Thus, we aim to characterize the effect of drug resistance mutations on the dynamics of PR and how dynamics relates to thermodynamics of the inhibitor interaction. (1P01 GM 109767-01, subcontract)
(2) HIV-1 reverse transcriptase (RT) inhibitor design
Inhibitors of HIV reverse transcriptase (RT) polymerase activity are routinely used for therapeutic purposes, yet no inhibitor of the protein’s ribonuclease H (RNH) activity is available for clinical application. Information on how RNH inhibitors (RNHI) interact with RT, at an atomic level, is scarce, and this may contribute to the difficulty in targeting the ribonuclease for therapeutic purposes. Only recently were crystal structures of RNH active-site inhibitors in complex with RT or an RNH fragment published, and structural analysis of non active- site RNHI has not been carried out. Part of the difficulty in understanding the atomic mechanisms of RNH inhibition relates to the conformational flexibility of RNH: the RNH domain is known to have relatively large flexible regions that are important for substrate recognition and the enzymatic reaction. Yet, how this flexibility influences inhibitor binding, and vice versa, has not been studied in a systematic manner. The goal of this project is to investigate RNHI interactions with an RNH fragment and with full-length RT, at an atomic level, in solution. For this purpose, solution NMR spectroscopy, in concert with biochemical and virological studies, will be carried out on RNH, using RNHIs as probes to detect conformational changes. (NIH R01GM105401)
(3) HIV-1 reverse transcriptase (RT) dynamics
Structural studies of the RT p66/p51 heterodimer indicate that the domain-domain configuration of p66 is different from that of the p51/p51 and p66/p66 homodimers, suggesting that significant dynamical rearrangements of the RT domains accompany heterodimer formation. Furthermore, previous biochemical results imply that significant conformational changes in RT are necessary to adopt distinct interaction modes with substrate. Despite such fundamentally important motions, RT dynamics in solution has not been well characterized at the atomic level. Thus, we aim to study RT dynamics using NMR as well as other biophysical methods.(NIH P50GM082251)

(4) Protein dynamics study using NMR 
In biomolecular studies at atomic level resolution, the accuracy of parameters derived from experimental results is critical to exploit the methods for down-stream applications, such as drug design and developing nano-machines. Nuclear Magnetic Resonance (NMR) spectroscopy provides data at atomic resolution for protein structure and dynamics and is better suited to study dynamics compared to crystallography. Currently, several NMR relaxation experiments are routinely used to characterize protein dynamics. However, parameters derived from these relaxation experiments depend on the accuracy of both experimental data and the models of motion used for analysis. We examine these methodologies to provide better quantitative information of protein dynamics using NMR.