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. (R01 GM135919, subcontract)
(2) Domain antibody characterization
We have a long-term collaboration with Dr. Dimiter Dimitrov and now with Dr. Wei Li on characterization of domain antibodies. Although domain antibody structures are well known, they behave differently upon mutations. Our major role is to characterize their designed domain antibodies using biophysics methods, such as NMR, CD, and SPR (UPMC subcontract)
(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.(P50 AI150481)
(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.