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. Activation of protein kinase A (PKA) by cAMP is a multi-step process involving sequential binding of cAMP to two different sites in the R subunit, and a resultant sequence of structural changes in R that release the C subunit from an inhibited state. The nature of the structural changes that occur during activation are poorly understood, although recent SAXS data and x-ray crystal structures indicate large cAMP-induced structural changes in the R subunit occur before the C subunit is released. Using SSRL beamtime granted through the Rapid Access mechanism, we conducted pilot time-resolved SAXS experiments in July 2007 to determine the approximate time course of solution structural changes that occur after mixing PKA with cAMP and cAMP analogs. These studies were conducted in collaboration with Hiro Tsuruta's group using the stopped-flow instrument on BL 4-2. Analysis of these data indicate that it is feasible to use TR-SAXS to follow the cAMPinduced structural changes in PKA and that these changes are largely complete by ~750 msec. The overall changes in Rg and Dmax that occurred were comparable to what we had previously observed using the steadystate SAXS instrument at the University of Utah. Based on our successful pilot experiments, we would like to follow up with more detailed and extensive studies of the kinetics of PKA structural changes using the TRSAXS capabilities at BL 4-2. Specifically, we would like to analyze in more detail the structural changes with higher temporal resolution (our shortest time integral was 255 msec in the pilot studies), and extend our observations to include the dissociation of R and C subunits (we chose conditions in the pilot studies that prevented R-C dissociation so as to only observe cAMP-induced conformational changes). Such experiments will provide important insights into the structural dynamics of PKA.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001209-32
Application #
8362178
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
$5,753
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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