Andrew Lee

Research: Structural biophysics and protein NMR spectroscopy

Andrew Lee

Professor of Pharmacy
Joint Appointment in Biochemistry and Biophysics
PhD - University of California, Berkeley

4109 Marsico Hall
CB# 7363
Chapel Hill, NC 27599-7260

Lee Lab Website


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.  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 heteronuclear NMR spectroscopy and other experimental and theoretical tools.  NMR spectroscopy is ideal for our studies, as hundreds – even thousands – of structural and motional “spin probes” are uniformly distributed throughout any given protein.  We have a general interest in the role of dynamics in protein function and allostery.

Enzyme Dynamics and Allostery – Thymidylate Synthase

We 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/or allostery. Most 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 that is needed for DNA replication. TS is more complex than many of the enzymes studied to date by NMR, as it has a multi-state 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 non-reactivity to the other subunit. This is essentially a form of high negative cooperativity. We are using novel NMR approaches to study cooperative effects that traverse the dimer interface. In summary, TS is a fascinating, large enzyme that it interesting from both the perspective of catalysis and allostery.

Insights into allosteric mechanisms:  CheY and PDZ domains

Allostery and the role of dynamics can also be studied in smaller, even monomeric proteins. This is highlighted by comparing the different strategies of two small allosteric proteins:  Chemotaxis protein Y (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”.

Dynamics of small molecule (or drug) binding

Many 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.

NMR and other methods

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.

REPRESENTATIVE PUBLICATIONS PubMed(click for Full Publication List)

  • 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.
  • 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.
  • 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. 
  • 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.
  • 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, 6322-23.
  • 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.
  • Clarkson MW, Gilmore SA, Edgell MH, and Lee AL, Dynamic coupling and allosteric behavior in a non-allosteric protein, Biochemistry (2006), 45, 7693-7699.

Lab Contact: 

Lab Rooms: 4109 Marsico Hall
Lab Phone: (919) 966-7821
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