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Research: Protein dynamics, allostery, NMR spectroscopy, click chemistry, protein biophysics, thymidylate synthase

Professor of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy

Joint Appointment in Biochemistry and Biophysics

(PhD – PhD – UC Berkeley)

Accepting Rotation Students


Protein dynamics and NMR spectroscopy

Nearly all biological processes are driven by the same fundamental event: protein conformational changes. The dynamic nature of proteins is now widely appreciated but not well understood since dynamics are not evident from static structural images. Our research is aimed at atomic resolution characterizations of protein dynamics and how dynamics contribute to function, especially allosteric communication and regulation. Towards this goal, we employ protein NMR spectroscopy in conjunction with other chemical biology and biophysical tools. NMR is ideal for our studies, as hundreds – even thousands – of structural and motional “spin probes” are uniformly distributed throughout any given protein.

Research Projects

Homodimers for study of protein allostery, I – Chorismate Mutase

From our work on thymidylate synthase (below), we have learned that homodimers offer a powerful and attractive approach to study how allostery works in proteins. Despite many decades of biochemical and structural work on allosteric systems (enzymes, receptors, etc.) and the fact that allostery underlies biological regulation, the basic details of how allostery works are not understood. We have worked out an approach for NMR study of symmetric homodimers that has great promise for observing the dynamic processes underlying allosteric function. Part of the approach puts click chemistry to use to create covalently linked “mixed labeled dimers” (MLDs), which provide clear signals to track allosteric movements. The enzyme chorismate mutase (CM, in the aromatic amino acid biosynthesis pathway) is an ideal system for developing this approach since it possesses all the hallmarks of classical allostery, including tense (“T”) and relaxed (“R”) allosteric conformations. NMR observation of individual residues can be correlated with allosteric function detected in enzymatic assays. Measuring specific relaxation rates of chemical groups – such as with methyl-TROSY for larger proteins – captures the dynamics of these different groups to provide molecular insight.

Homodimers for study of protein allostery, II – Thymidylate Synthase

The other homodimeric enzyme of interest in the lab is thymidylate synthase (TS). Like CM, it is also symmetric and allosteric (and about the same size, ~60 kDa), but whereas CM displays positive cooperativity between its two active sites, TS activity is negatively cooperative (specifically, “half-the-sites-reactive”). Further, its dimer interface is a beta-sheet structure whereas CM is all alpha-helical; thus the two systems provide an interesting contrast. It was on TS that we initially developed the mixed-labeled-dimer (MLD) approach now being pursued on both systems. Most recently we showed using NMR that rapid motions on the picosecond-nanosecond timescale provide an entropically driven mechanism for cooperative binding of substrate (for human TS), and that, non-intuitively, these functional motions in hTS are entirely dependent on a completely disordered N-terminal tail of ~25 amino acids. How this can occur will be the subject of further work.

Other past and present projects

Past work on the lab focused on the dynamic and allosteric properties of smaller protein domains (e.g. PDZ domains) and regulatory proteins or enzymes (CheY, DHFR). One of our current interests is in understanding the dynamic properties and conformational changes taking place in engineered allosteric proteins that respond to light – ‘optogenetic’ proteins.

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. With the larger systems now under study (CM, TS), methyl-TROSY based experiments have become indispensable.

REPRESENTATIVE PUBLICATIONS PubMed (click for Full Publication List)

Lab Contact:

Lab Rooms: 4109 Marsico Hall

Lab Phone: 919-966-7821


Picture of Andrew Lee PhD Joint Professor of Biochemistry and Biophysics and Pharmacy
  • Phone Number

    919-966-7821 (Office Phone)

  • Address

    Lineberger Cancer Cnter

    450 West Drive, A. Lee lab

    CB# 7295

    Chapel Hill, NC 27599-7295