Regulation of Map Kinase by Protein Motions

这个提议的目的是研究在反应蛋白中发生的运动变化 激酶激活，并探索它们与酶功能的相关性。MAP激酶的分析 ERK 2，通过氢交换质谱法（HX-MS）和其他测量揭示了三个 酶扰动涉及（i）酶活化，（ii）变构 结合位点之间的通信，和（iii）诱变导致蛋白质中的局部变化 长距离构象迁移率。现有的证据表明， 信号转导控制，其中蛋白质动力学的调节控制催化和 ERK 2的变构功能。本提案中的具体目标将检验这一假设， 相关MAP激酶的行为。Sp. Aim 1将通过HX-MS分析ERK 2中的蛋白动力学， NMR和FRET，以确定铰链处柔性变化的功能后果 由激酶磷酸化和激活引起的。SP. Aim 2将检验以下假设： MAP激酶对接基序在它们各自的结合口袋之间变构相互作用， 探索变构调节的功能后果，使用等温量热法，HX-MS， 和NMR。目的3将研究在N-末端结构域的突变的机制， 通过定点诱变、HX-MS和NMR，ERK 2调节激活唇处的柔性。Sp. 目的4将扩展我们的HX-MS分析，以记录活化依赖的构象迁移率， 其他蛋白激酶。这些目标的完成将为理解 蛋白激酶是如何通过控制构象迁移率来优化功能的。 创新技术将应用于这一问题，包括高场溶液NMR和 氢交换质谱法实验将：（一）记录对 在一个大的酶的内部运动，（ii）证明蛋白质运动在控制激酶的作用 酶的功能，（iii）提高我们对蛋白质运动如何长期控制的理解 距离，和（iv）文件的信号分子调节的新机制。

The goal of this proposal is to investigate the motional changes that occur in response^¿ 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.