This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We propose to study the mechanism of the ligand induced conformational change in beta-phosphoglucomutase (bPGM) via small angle x-ray scattering (SAXS). bPGM is a representative member of the Haloalkanoate Dehalogenase Superfamily (HADSF) of phosphotransferases. The enzyme has been extensively characterized via x-ray crystallography in open and closed, ligand bound conformation. While the conformational change is well documented, the mechanism of this transition is still unknown. The binding site for the ligand of bPGM, glucose 1,6-(bis)phosphate, contains sites for both phosphoryl moieties and the sugar ring. The proposed experiments will characterize the SAXS pattern of the enzyme in the presence of increasing concentration of ligands that occupy all or part of the active site. By determining the effect of component moieties of the native ligands, we hope to identify the crucial protein-ligand interactions that induce the conformational change. In addition, the presence of any conformational intermediates can be recognized. Given sufficient resolution, we will determine the thermodynamic quantity Kclose for each of the partial ligands and compare these results to published KI or KM values. Preliminary SAXS data on the enzyme shows significant difference in signal between momentum transfer values of s=0.1-0.4 and a 1.3? difference in Rg between the unliganded and inhibitor bound enzyme, indicating that this method has sufficient resolution to discern the two states.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001209-32
Application #
8362402
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (40))
Project Start
2011-03-01
Project End
2012-02-29
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
32
Fiscal Year
2011
Total Cost
$279
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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