Regulation of Map Kinase by Protein Motions

描述（由申请人提供）：本提案的目标是研究在响应S蛋白激酶激活中发生的情绪变化，并探索它们与酶功能的相关性。通过氢交换质谱（HX-MS）和其他测量方法对MAP^kinase^ ERK2的分析揭示了三种情况，即酶的扰动涉及(i)酶激活，（ii）结合位点之间的变构通信，以及（iii）诱变导致长距离蛋白质构象迁移的局部变化。现有的证据表明了一种新的信号转导控制模型，其中蛋白质动力学调节控制ERK2的催化和变构功能。本提案的具体目标将检验这一假设，并检查相关MAP激酶的行为。Sp. Aim 1将通过HX-MS、NMR和FRET分析ERK2中的蛋白质动力学，以确定由激酶磷酸化和激活诱导的铰链柔韧性变化的功能后果。目的2将验证MAP激酶对接基序在各自的结合袋之间发生变构相互作用的假设，并使用等温量热法、HX-MS和NMR探索变构调节的功能后果。Sp. Aim 3将通过位点定向诱变、HX-MS和NMR研究ERK2 n端结构域突变调节激活唇柔韧性的机制。Sp. Aim 4将扩展我们的HX-MS分析，以记录其他蛋白激酶的激活依赖性构象迁移。这些目标的完成将为理解蛋白激酶如何通过控制构象迁移来优化功能提供一个独特的窗口。创新技术将应用于这个问题，包括高场溶液核磁共振和氢交换质谱。这些实验将：(i)记录大型酶的内部运动调节，（ii）证明蛋白质运动在控制激酶酶功能中的作用，（iii）提高我们对远距离蛋白质运动如何控制的理解，（iv）记录信号分子调节的新机制。

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the motional changes that occur in response^S protein kinase activation, and explore their relevance to enzyme function. Analysis of the MAP^kinase^ ERK2, by hydrogen exchange mass spectrometry (HX-MS) and other measurements reveal three situations in which enzyme perturbations involving (i) enzyme activation, (ii) allosteric communication between binding sites, and (iii) mutagenesis lead to localized changes in protein conformational mobility over long distances. The available evidence suggests a novel model for signal transduction control, in which the regulation of protein dynamics controls catalytic and allosteric functions in ERK2. Specific aims in this proposal will test this hypothesis, and examine behavior of related MAP kinases. Sp. Aim 1 will analyze protein dynamics in ERK2 by HX-MS, NMR and FRET in order to determine the functional consequence of flexibility changes at the hinge that are induced by kinase phosphorylation and activation. Sp. Aim 2 will test the hypothesis that MAP kinase docking motifs interact allosterically between their respective binding pockets, and explore the functional consequences of allosteric regulation, using isothermal calorimetry, HX-MS, and NMR. Sp. Aim 3 will investigate the mechanism by which mutations in the N-terminal domain of ERK2 regulate flexibility at the activation lip, by site-directed mutagenesis, HX-MS and NMR. Sp. Aim 4 will extend our HX-MS analyses to document activation-dependent conformational mobility in other protein kinases. Completion of these aims will provide a unique window for understanding how protein kinases have evolved to optimize function by controlling conformational mobility. Innovative technologies will be applied to this problem, including high field solution NMR and hydrogen exchange mass spectrometry. The experiments will: (i) document the regulation of internal motions in a large enzyme, (ii) demonstrate the role of protein motions in controlling kinase enzymatic function, (iii) improve our understanding of how protein motions are controlled over long distances, and (iv) document new mechanisms for regulation of signaling molecules.