KINETICS OF CAMP-INDUCED STRUCTURAL CHANGES IN PROTEIN KINASE A BY TR-SAXS

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 cAMP 激活蛋白激酶 A (PKA) 是一个多步骤过程，涉及 cAMP 与 R 亚基中两个不同位点的顺序结合，以及由此产生的 R 结构变化序列，从而将 C 亚基从抑制状态释放出来。尽管最近的 SAXS 数据和 X 射线晶体结构表明，在 C 亚基释放之前，R 亚基中发生了 cAMP 诱导的大结构变化，但对激活过程中发生的结构变化的性质知之甚少。利用通过快速访问机制授予的 SSRL 波束时间，我们于 2007 年 7 月进行了试点时间分辨 SAXS 实验，以确定将 PKA 与 cAMP 和 cAMP 类似物混合后发生溶液结构变化的大致时间过程。这些研究是与 Hiro Tsuruta 团队合作使用 BL 4-2 上的停流仪器进行的。对这些数据的分析表明，使用 TR-SAXS 跟踪 cAMP 诱导的 PKA 结构变化是可行的，并且这些变化在约 750 毫秒内基本完成。 Rg 和 Dmax 发生的总体变化与我们之前使用犹他大学稳态 SAXS 仪器观察到的结果相当。基于我们成功的试点实验，我们希望利用 BL 4-2 的 TRSAXS 功能对 PKA 结构变化动力学进行更详细和更广泛的研究。具体来说，我们希望以更高的时间分辨率更详细地分析结构变化（在试点研究中，我们的最短时间积分为 255 毫秒），并将我们的观察范围扩展到包括 R 和 C 亚基的解离（我们在试点研究中选择了阻止 R-C 解离的条件，以便仅观察 cAMP 诱导的构象变化）。这些实验将为 PKA 的结构动力学提供重要的见解。

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.