Biochemistry and Biophysics - UNC School of Medicine
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Andrew Lee, PhD
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RESEARCH INTERESTS:
Structural Biophysics and Protein NMR Spectroscopy
Nearly all biological processes are driven by the same fundamental event: protein conformational changes. The mechanics that govern how proteins “morph” their three-dimensional structures into alternative conformations need to be understood in order to understand the biochemical basis of protein function. Conformational changes require that proteins possess 1) the energetic stability to maintain a well-defined “native state structure”, and 2) the inherent flexibility enabling them to move through conformational space on relatively fast timescales (< 1 ms). Our research is aimed at atomic resolution characterizations of the structural and dynamic properties of proteins and their interactions with other proteins, ligands, and small molecule drugs. Towards this goal, we are making extensive use of modern heteronuclear NMR spectroscopy and other experimental and theoretical tools. NMR spectroscopy is ideal for our studies, as hundreds of structural and motional “spin probes” are uniformly distributed throughout any given protein. We have a general interest in applying NMR towards a variety of problems in biophysics and structural biology.
Research Projects
We are particularly interested in the biophysical effects of mutations and other perturbations on the structure, dynamics, and internal communication within proteins and protein complexes. In the globular protein eglin c, a modest V->A mutation in its hydrophobic core reveals a network of contiguous side chains that are dynamically coupled (see Figure, V->A mutation shown in yellow) even though many of these residues are not in contact with the mutated valine (in fact, as much as 12 Å away). We are characterizing the structural, dynamic, and thermodynamic effects of mutations in eglin c for the purpose of identifying functionally important residues that are not evident from structural information alone. In a second project area, we are using this NMR-based strategy to identify key networks of residues in PDZ (PSD-95/Discs large/ZO-1) domains, which are ~100 residue modules that are essential in intracellular signaling and synaptic complexes. From these studies of fast side-chain mobilities, residues are found to form continuous pathways, connecting distal sites that may have allosteric-like influence on peptide binding. Is allosteric behavior confined to proteins that have large conformational changes? Additional projects in the lab range from studies of functional dynamics in enzymes to more traditional structural determination of novel proteins. We are working on several enzymes (e.g. viral proteases, sulfotransferases) towards the goal of understanding the role of dynamics in enzyme catalysis. Another goal is to understand the mechanism of drug resistance conferred by mutations in many of these enzymes. We are also carrying out collaborative projects that involve the determination of structures of proteins involved in DNA repair. Methods We are using a variety of biophysical and biochemical approaches to assist us in understanding protein function and evolution at the site-specific level. A major tool in the lab is high-resolution NMR spectroscopy. All NMR projects typically involve the use of 1H, 13C, and 15N nuclei for NMR signal assignment and structure determination, and 15N, 13C, and 2H nuclei are being used for monitoring atomic resolution dynamics as measured by spin relaxation methods. Residual dipolar couplings (RDC’s) are measured to provide unique angular structural information. We also use the following techniques in our research: fluorescence spectroscopy for monitoring protein-ligand binding, protein unfolding, and enzyme kinetics; molecular dynamics (MD) simulations for characterizing motional details on the ps-ns timescale; and calorimetry and additional methods for thermodynamic and kinetic measurements.RECENT PUBLICATIONS:
Law, A.B., Fuentes, E.J., and Lee, A.L. Conservation of side-chain dynamics within a protein family. J. Am. Chem. Soc. (2009), 131, 6322-23.
Mauldin, R.V., Carroll, M.J., and Lee, A.L. Dynamic dysfunction in dihydrofolate reductase results from antifolate drug binding: modulation of dynamics within a structural state. Structure (2009), 17, 386-394.
Whitley, M.J. and Lee, A.L. Frameworks for understanding long-range intra-protein communication, Current Protein and Peptide Science (2009), 10, 116-127.
Whitley, M.J., Zhang, J., and Lee, A.L. Hydrophobic core mutations in CI2 globally perturb fast side-chain dynamics similarly without regard to position. Biochemistry (2008), 47, 8566-8576.
Boyer, J.A. and Lee, A.L. Monitoring aromatic ps-ns dynamics in proteins via 13C relaxation: Expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c. Biochemistry (2008), 47, 4876-4886.
DeRose, E.F., Clarkson, M.W., Gilmore, S.A., Galban, C.J., Tripathy, A., Havener, J.M., Mueller, G.A., Ramsden, D.A., London, R.E., and Lee, A.L. The solution structure of polymerase μ’s BRCT domain reveals an element essential for its role in nonhomologous end joining. Biochemistry (2007), 46, 12100-12110.
Fuentes, E.J., Gilmore, S.A., Mauldin, R.V., and Lee, A.L. Evaluation of energetic and dynamic coupling networks in a PDZ domain protein. Journal of Molecular Biology (2006), 364, 337-351.
Clarkson, M.W., Gilmore, S.A., Edgell, M.H., and Lee, A.L. Dynamic coupling and allosteric behavior in a non-allosteric protein. Biochemistry (2006), 45, 7693-7699.
Hu, H., Hermans, J., and Lee, A.L. Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR. J. Biomol. NMR (2005), 32, 151-162.
Clarkson, M.W. and Lee, A.L. Long-range dynamic effects of point mutations propagate through side chains in the serine protease inhibitor eglin c. Biochemistry (2004) 43, 12448-12458.
Ohnishi, S., Lee, A.L., Edgell, M.H., and Shortle, D. Direct Demonstration of Structural Similarity Between Native and Denatured Eglin C. Biochemistry (2004) 43, 4064-4070.
Fuentes, E.J., Der, C.J., and Lee, A.L. Ligand-Dependent Dynamics and Intramolecular Signaling in a PDZ Domain. J. Mol. Biol. (2004), 335, 1105-1115.
Hu, H., Clarkson, M.W., Hermans, J., and Lee, A.L. Increased Rigidity of Eglin c at Acidic pH: Evidence from NMR Spin-Relaxation and MD Simulations. Biochemistry (2003), 42, 13856-13868.