Regulation of Map Kinase by Protein Motions

描述(申请人提供)：本建议的目的是研究在S蛋白激酶激活过程中发生的运动变化，并探讨它们与酶功能的相关性。用氢交换质谱仪(HX-MS)对MAP、KK、ERK2的分析和其他测量揭示了三种情况：(I)酶的激活，(Ii)结合位点之间的变构通讯，(Iii)突变导致蛋白质构象迁移率的局部变化。现有证据提示了一种新的信号转导控制模型，在该模型中，蛋白质动力学的调节控制着ERK2的催化和变构功能。该提案中的特定目标将检验这一假设，并检查相关MAP激酶的行为。SP.目的1将通过HX-MS、核磁共振和FRET分析ERK2中的蛋白质动力学，以确定由激酶磷酸化和激活引起的铰链柔韧性变化的功能后果。SP.目的2将利用等温量热法、HX-MS和核磁共振技术，验证MAP激酶对接基序在其各自的结合口袋之间以变构方式相互作用的假设，并探索变构调节的功能结果。SP.目的3将通过定点突变、HX-MS和核磁共振研究ERK2 N-末端结构域突变调节激活LIP的灵活性的机制。SP.AIM 4将扩展我们的HX-MS分析，以记录其他蛋白激酶中依赖于激活的构象迁移率。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率进化来优化功能提供了一个独特的窗口。创新技术将被应用于这一问题，包括高场溶液核磁共振和氢交换质谱学。这些实验将：(I)记录大型酶的内部运动的调节，(Ii)展示蛋白质运动在控制激酶酶功能中的作用，(Iii)提高我们对蛋白质运动是如何在长距离上被控制的理解，以及(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.