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Lee - Conformational DynamcisWe study the role of conformational dynamics in protein function, conformational changes, enzyme catalysis, drug binding, and allostery. We use a variety of biophysical and biochemical tools, especially NMR spectroscopy.


Research in the laboratory is centered on understanding the role of structural dynamics in protein function. In past decades, proteins were essentially viewed as static structures. Today, they are widely appreciated to be dynamic ensembles of interconverting structures. Such behavior can be clearly seen in proteins that undergo dramatic shape changes in different functional states. However, the effects of dynamics can also be important when the structural changes are less apparent. The ensemble nature of proteins has far-reaching implications for understanding basic natural protein functions such as ligand binding, enzyme catalysis, and allostery. An understanding of protein dynamics should lead to improvements in protein engineering and rational drug design.

Lee - CheY_allosteric_networkWe are particularly interested in how protein dynamics facilitates enzyme catalysis and allosteric communication. To maximize our understanding of these phenomena, we study multiple systems with the idea that different proteins may use different strategies for achieving catalysis and allostery. This is highlighted by comparing the different strategies of two small allosteric proteins: CheY and the third PDZ domain from PSD-95 (PDZ3). CheY, a so-called response regulator receiver domain, is the master regulatory switch for reversing the direction of the E. coli flagellum, and it undergoes a classical allosteric conformational change upon phosphorylation of an aspartate side chain. NMR studies from our lab and others indicate these proteins dynamically switch between inactive and active conformations on the microsecond-millisecond timescale, but that the actual switching mechanism involves additional states yet to be characterized (see McDonald et al., Structure, 2012, 20, 1363). In contrast to CheY, PDZ3 exhibits allosteric behavior utilizing a different kind of dynamics. From analysis of methyl spin relaxation rates, PDZ3 modulates its binding affinity to its interacting protein using an auxiliary helix, which controls the overall level of side-chain dynamics on the picosecond-nanosecond timescale. This alters the overall entropy change upon binding PDZ3’s interacting protein and is an example of “dynamic allostery.”

TS cartoon and spectrumMost recently, we have been studying the 62 kD dimeric enzyme thymidylate synthase, or TS. This enzyme is metabolically critical, performing conversion of uridine monophosphate to thymidine monophosphate. TS is more complex than many of the enzymes studied to date by NMR as it has a multistate reaction coordinate linking substrates and products. This affords an opportunity to track how the dynamics throughout TS change as the enzyme populates different intermediate steps in catalysis. In addition to this interesting reaction mechanism, TS is known to be “half the sites reactive,” meaning that catalytic activity in one subunit imparts nonreactivity to the other subunit. This is essentially a form of high negative cooperativity. Our NMR studies of TS will also focus on this intersubunit negative cooperativity and the structural and dynamic features associated with it. In summary, TS is a fascinating, large enzyme that it interesting from both the perspective of catalysis and allostery.

Lee - Drug Binding DynamicsMany proteins serve as drug receptors. In such cases the dynamics may contribute to drug-binding affinity or residence times, both of which are important metrics for efficacious drugs. From our NMR studies on dihydrofolate reductase (DHFR), we have observed that conformational dynamics can correlate with inhibitor dissociation rates, and we have even observed “dynamic ligands” that rapidly switch their conformations while bound to the protein. We remain interested in this general area.

How do we characterize protein dynamics?  Our preferred method is heteronuclear NMR spectroscopy, which is uniquely suited to study both structure and dynamics in proteins and other biological macromolecules. A major advantage of NMR is that spectroscopic probes are distributed uniformly throughout the biomolecule, such as NH or CH atom pairs, providing large amounts of molecular information. To gain information on protein dynamics, NMR spin relaxation is highly sensitive to molecular motion over a range of timescales. We look at the relaxation properties of 15N, 13C, 1H, and 2H spins located throughout the protein scaffold, and interpret these in terms of amplitudes and timescales of individual bond vectors. Slower motions on the microsecond-millisecond timescale can be detected to yield site-specific kinetic, thermodynamic, and structural information on the switching between discrete conformational states. In many cases, these NMR-relaxation dynamics are used to complement other structural data from X-ray crystallography, or thermodynamic and kinetic biophysical measurements using methods such as fluorescence spectroscopy, calorimetry, amide hydrogen exchange, and molecular dynamics simulations.

Sapienza PJ and Lee AL, Widespread perturbation of function, structure, and dynamics by a conservative single atom substitution in thymidylate synthase, Biochemistry (2016), 55, 5702-5713.

Falk BT, Sapienza PJ, and Lee AL, Chemical shift imprint of intersubunit communication in a symmetric homodimer, Proc. Natl. Acad. Sci. (2016), 113, 9533-9538.

Sapienza PJ, Li L, Williams T, Lee AL, and Carter C, An ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures, ACS Chem Biol (2016), 11, 1661-1668.

Sapienza PJ, Falk BT, and Lee AL, Bacterial thymidylate synthase binds two molecules of substrate and cofactor without cooperativity,  JACS (2015), 137, 14260-14263.

Sapienza PJ and Lee AL, Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analog, Biomolecular NMR Assignments (2014), 8, 195-199.

McDonald LR, Whitley MJ, Boyer JA, and Lee AL, Colocalization of fast and slow timescale dynamics in the allosteric signaling protein CheY, J. Mol. Biol. (2013), 425, 2372-2381.

Wang Z, Sapienza PJ, Abeysinghe T, Luzum C, Lee AL, Finer-Moore JS, Stroud RM, Kohen A, Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site, J. Am. Chem. Soc. (2013), 135, 7583-7592.

Zhang J, Lewis SM, Kuhlman B, and Lee AL, Supertertiary structure of the MAGUK core from PSD-95, Structure (2013), 21, 402-413.

Lee AL.  Dynamics and allostery (chapter), Encyclopedia of Biophysics, Roberts GCK (Ed.), Springer (2013).

McDonald LR, Boyer JA, and Lee AL, Segmental motions, not a two-state concerted switch, underlie allostery in CheY, Structure (2012), 20, 1363-1373.

Mauldin RV, Sapienza PJ, Petit CM, and Lee AL, Structure and dynamics of the G121V dihydrofolate reductase mutant:  lessons from a transition-state inhibitor complex, PLoS ONE (2012), 7(3), e33252.

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), 8, 246-252.

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.

Whitley MJ and Lee AL, Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2, PROTEINS: Structure, Function, and Bioinformatics (2011), 79, 916-24.

Sapienza PJ, Mauldin RV, and Lee AL, Multi-timescale dynamics study of FKBP12 along the rapamycin and mTOR binding coordinate, J. Mol. Biol. (2011), 405, 378-394.

Zhang J, Sapienza PJ, Ke H, Chang A, Hengel SR, Wang H, Phillips GN, and Lee AL. Crystallographic and NMR evaluation of the impact of peptide binding to the second PDZ domain of PTP1E.  Biochemistry (2010), 49, 9280-9291.

Sapienza PJ and Lee AL.  Using NMR to study fast dynamics in proteins: methods and applications, Curr. Op. Pharmacol. (2010), 10, 723-730.

Andrew L Lee, Ph.D.



Andrew Lee studies the role of conformational dynamics in protein function, conformational changes, enzyme catalysis, drug binding, and allostery. His laboratory uses a variety of biophysical and biochemical tools, especially NMR spectroscopy. NMR spectroscopy is a powerful approach that yields atomic-resolution molecular information and is uniquely sensitive to molecular fluctuations over a broad range of timescales.

Lab Alumni


Michael “Sparky” Clarkson (2005)

Josh Boyer (2009)

Randy Mauldin (2009)

Matthew Whitley (2010)

Mary Carroll/Harner (2011)

Jun Zhang (2011)

Tony Law (2011)

Leanna McDonald (2013)

Brad Falk (2017)


Ernesto Fuentes (2005)

Paul Sapienza (2011)

Chad Petit (2012)

Maria McGresham (2016)

The Lee lab is anticipating an opening for a postdoctoral position for NMR and other biophysical studies of the dynamics, structure, and solution behavior of a classically allosteric enzyme. Two projects in the lab focus on uncovering mechanisms of allosteric communication in enzyme homodimers in the 60-70 kDa range. Both project areas will involve advanced NMR spectroscopy for large proteins, including spin relaxation, as well as other biophysical methods (e.g. calorimetry), enzyme assays, protein conjugation using click chemistry, and recently developed “protein network” analysis techniques. Candidates are sought with expertise in any one or more of the areas including:  protein structural biology and biophysics, protein conjugation chemistry, enzymology and NMR spectroscopy. Interested candidates should send their CV to Dr. Lee (drewlee@unc.edu).