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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CAMP激活蛋白激酶A(PKA)是一个多步骤的过程，包括cAMP与R亚基上的两个不同位置的顺序结合，以及由此产生的R结构变化序列，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，cAMP诱导的R亚基的结构变化在C亚基释放之前发生，但人们对激活过程中发生的结构变化的性质知之甚少。利用通过快速接入机制获得的SSRL波束时间，我们在2007年7月进行了时间分辨SAXS中试实验，以确定将PKA与cAMP和cAMP类似物混合后发生的溶液结构变化的大致时间过程。这些研究是与Hiro Tsuruta的团队合作进行的，他们使用了BL4-2上的停流仪。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，这些变化在~750ms内基本完成。Rg和Dmax的总体变化与我们之前在犹他大学使用稳态SAXS仪器观察到的结果相当。在我们成功的初步实验的基础上，我们希望利用BL4-2的TRSAXS能力对PKA结构变化的动力学进行更详细和更广泛的研究。具体地说，我们希望以更高的时间分辨率更详细地分析结构变化(在初步研究中，我们最短的时间积分为255毫秒)，并将我们的观察扩展到包括R和C亚单位的解离(我们在初步研究中选择了防止R-C解离的条件，以便只观察cAMP诱导的构象变化)。这些实验将对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.