Associate Professor of Pharmacy, UNC-CH
PHD - University of California, Berkeley
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.
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.
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.
- Carroll MJ, Mauldin RV, Gromova AV, Singleton SF, Collins EJ, and Lee AL, Evidence for dynamics in proteins as a mechanism for ligand dissociation, Nat. Chem. Biol. (2012), in press.
- Zhang J, Petit CM, King DS, and Lee AL, Phosphorylation of a PDZ domain extension modulates binding affinity and interdomain interactions in PSD-95, J. Biol. Chem. (2011), 286, 41776-41785.
- Carroll MJ, Gromova AV, Miller KR, Tang H, Wang XS, Tripathy A, Singleton SF, Collins EJ, and Lee AL, Direct detection of structurally resolved dynamics in a multi-conformation receptor-ligand complex, J. Am. Chem. Soc. (2011), 133, 6422-6428.
- Sapienza PJ, Mauldin RV, and Lee AL, Multi-timescale dynamics study of FKBP12 along the rapamycin and mTOR binding coordinate, J. Mol. Biol. (2010),405, 378-394.
- Boyer JA, Clay CJ, Luce KS, Edgell MH, Lee AL, Detection of native-state non-additivity in double mutant cycles via hydrogen exchange, J. Am. Chem. Soc. (2010), 132, 8010-8019.
- Petit CM, Zhang J, Sapienza PJ, Fuentes EJ, and Lee AL, Hidden dynamic allostery in a PDZ domain, Proc. Natl. Acad. Sci. U.S.A. (2009), 106, 18249-18254.
- Law AB, Fuentes EJ, and Lee AL, Conservation of side-chain dynamics within a protein family, J. Am. Chem. Soc. (2009), 131, 6322-33.
- Mauldin RV, Carroll MJ, and Lee AL, Dynamic dysfunction in dihydrofolate reductase results from antifolate drug binding: modulation of dynamics within a structural state, Structure (2009), 17, 386-394.
- Whitley MJ and Lee AL, Frameworks for understanding long-range intra-protein communication, Current Protein and Peptide Science (2009), 10, 116-127.
- Boyer JA and Lee AL, 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 EF, Clarkson MW, Gilmore SA, Galban CJ, Tripathy A, Havener JM, Mueller GA, Ramsden DA, London RE, and Lee AL, 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 EJ, Gilmore SA, Mauldin RV, and Lee AL, Evaluation of energetic and dynamic coupling networks in a PDZ domain protein, Journal of Molecular Biology (2006), 364, 337-351.
- Clarkson MW, Gilmore SA, Edgell MH, and Lee AL, Dynamic coupling and allosteric behavior in a non-allosteric protein, Biochemistry (2006), 45, 7693-7699.
- Clarkson MW and Lee AL, Long-range dynamic effects of point mutations propagate through side chains in the serine protease inhibitor eglin c, Biochemistry (2004) 43, 12448-12458.
201 Beard Hall
Campus Box # 7360
Chapel Hill, NC 27599