How proteins regulate themselves is a fundamental question in biology. Regulation of protein activity drives cell division, metabolism, signal transduction, and therapeutic intervention. The most common and versatile regulatory proteins are allosteric proteins. Allosteric proteins are distinguished by their capacity to respond to ligand binding or chemical modification at one site, and alter ligand binding or activity at a distal site. Although many allosteric proteins have been characterized structurally, and models of allostery exist, most of the details of how allosteric transitions proceed remain unknown. The long-range goal of this research is to experimentally reveal the time-resolved structural processes that constitute allosteric function. Central to the approach to be taken is the idea that proteins are highly dynamic, especially allosteric proteins, and that dynamic motions are an essential component of allosteric conformational change. Classical oligomeric allosteric proteins are too large for in-depth studies of site-specific dynamics by NMR. Therefore, the monomeric, 14 kDa bacterial response regulator CheY protein will be used as a model allosteric domain. CheY is a chemotaxis signal transduction protein that, upon phosphorylation at Asp-57, undergoes a conformational change at a distal surface that modulates binding to the flagellar motor. The ps-ns and ?s-ms timescale dynamics of CheY will be extensively characterized in the absence and presence of phosphoryl group mimic, BeF3-, using NMR relaxation methods. In separate experiments, long-range thermodynamic couplings will be mapped using high-throughput methodology. Because, recently, long-range communication has been observed in proteins that are not functionally allosteric, mechanisms similar to those in allosteric proteins may exist in non-allosteric proteins, albeit to a lesser extent. The serine protease inhibitor eglin c is a good example of this: conservative mutations in eglin c lead to long-range dynamic effects in the absence of structural change. In the proposed research, four Specific Aims fall into two main thrusts. In the first thrust, patterns of long-range coupling (or "communication") - both dynamic and thermodynamic - will be compared between the non-allosteric eglin c and the allosteric CheY. These comparisons will shed light on any basic differences in coupling networks between allosteric and non-allosteric proteins;they will also provide a test of the role of dynamics in mediating thermodynamic coupling. In the second thrust, the mechanism of intramolecular signal transduction in CheY will be investigated from an NMR dynamics perspective, using CheY's various biological states and mutations that modulate its activity. Overall, by increasing understanding of the biophysical properties and role of dynamics in allostery, this research will help to lay the foundation for the rational design of allosteric proteins and drugs.

Public Health Relevance

Allosteric conformational change in proteins lies at the heart of regulatory processes such as cell division, metabolism, signal transduction, and drug action. This research seeks to understand the dynamic underpinnings of allostery by experimentally contrasting motional dynamics in non-allosteric and allosteric proteins. The small bacterial signal transduction protein CheY will serve as a model allosteric domain. A detailed understanding of allosteric mechanisms will be needed to rationally design proteins and drugs that take advantage of allosteric principles, as well as understand mechanisms of drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066009-09
Application #
8245094
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2004-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
9
Fiscal Year
2012
Total Cost
$324,239
Indirect Cost
$103,716
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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