# Mechanisms and dynamics of allosteric function in proteins

> **NIH NIH R35** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2022 · $77,043

## Abstract

Abstract
Biology is driven through the action of proteins. We know that structure often provides the foundation for proteins’
function, but in recent years it has become clear that protein function is also critically dependent on dynamics,
or movements of structure. How dynamics enables function is now a central question in protein biology that limits
our basic understanding of proteins, as well as applications in drug discovery and protein design. While there
are many types of functions that dynamics – or conformational flexibility – promotes, two functional archetypes
for dynamics are enzyme catalysis and allostery. The mechanistic bases for these two phenomena, pervasive
as they are, remain largely mysterious and have attracted much attention for the likely role of dynamics. The Lee
laboratory has focused on studying dynamics and allostery in proteins using NMR and other biophysical methods
for nearly 20 years. The approach outlined in this proposal is to combine investigation of natural allosteric
enzymes (Areas 1 and 2) with efforts to engineer allosteric regulation into signaling proteins using optogenetics
(Area 3). In the last five years, the lab has developed two complementary systems for NMR and biophysical
studies of dynamics and allostery that are highly amenable for addressing these mechanistic questions and,
importantly, developing approaches to study intersubunit allosteric communication. The two systems are the
enzymes chorismate mutase (CM) and thymidylate synthase (TS), both symmetric homodimers that are
functionally allosteric. CM (from yeast) is a classically allosteric protein, exhibiting all the hallmarks of traditional
allostery: sigmoidal activity curve; symmetric quaternary structure; tense (“T”) and relaxed (“R”) conformations;
and small molecule allosteric effector ligands that either up- or down-regulate activity. In contrast to CM’s positive
cooperativity, TS is negatively cooperative because it is half-the-sites reactive. Work will be on the E. coli (ecTS)
and human (hTS) forms, which, despite their similarities show very different behaviors. The human TS is the
target of anticancer drug 5-fluoro-uracil (5-FU). CM, ecTS, and hTS all have outstanding features for study by
solution NMR since they are highly soluble, stable, and yield excellent spectra. The goals for the next five years
fall into three main areas: (1) Through the use of NMR spectroscopy, molecular dynamics simulations,
calorimetry, x-ray crystallography, and biochemistry, the structural and dynamic properties of these enzymes will
be related to functional behaviors of key interest, such as: allosteric communication; how apo state conformations
compare to T and R conformations; protomer asymmetry in singly liganded states; and the nature of the transition
state. (2) We will advance the study of protein homodimers by NMR by introducing a technology for chemical
conjugation of protomers using click chemistry. Mixed labeled dimers produced this way will fac...

## Key facts

- **NIH application ID:** 10691713
- **Project number:** 3R35GM144348-01S2
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Andrew L Lee
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $77,043
- **Award type:** 3
- **Project period:** 2022-07-01 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10691713

## Citation

> US National Institutes of Health, RePORTER application 10691713, Mechanisms and dynamics of allosteric function in proteins (3R35GM144348-01S2). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10691713. Licensed CC0.

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