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Regulation of Map Kinase by Protein Motions

蛋白质运动对图谱激酶的调节

基本信息

批准号：
7746483
负责人：
NATALIE G. AHN
金额：
$ 24.6万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7746483
关键词：
Activation Analysis Affect Allosteric Regulation Amides Arts Behavior Binding Binding Sites Biochemical Biochemistry Biological Biological Process Biophysics C-terminal Calorimetry Catalysis Chemicals Communication Coupling Data Docking Enzyme Activation Enzymes Fluorescence Resonance Energy Transfer Goals Hydrogen Lead Ligand Binding Ligands Lip structure MAPK1 gene MAPK14 gene MAPK8 gene Maps Mass Spectrum Analysis Measurement Measures Mediating Mitogen-Activated Protein Kinases Modeling Molecular Molecular Conformation Molecular Probes Motion Mutagenesis Mutation N-terminal Pattern Peptides Phosphorylation Phosphotransferases Protein Binding Protein Dynamics Protein Kinase Proteins Regulation Relaxation Residual state Role Signal Transduction Signal Transduction Pathway Signaling Molecule Site-Directed Mutagenesis Solutions Structure Techniques Technology Testing Vertebral column flexibility improved innovative technologies novel peptide deformylase protein function research study response

项目摘要

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.

这项提议的目标是研究在响应蛋白质时发生的运动变化。激酶激活，并探讨它们与酶功能的相关性。MAP^KEK^的分析 ERK2，通过氢交换质谱仪(HX-MS)和其他测量揭示了三个涉及(I)酶激活，(Ii)变构的酶扰动的情况结合位点之间的通讯，和(Iii)突变导致蛋白质的局部变化远距离的构象迁移率。现有的证据表明了一种新的模式信号转导控制，其中蛋白质动力学的调节控制催化和 ERK2中的变构功能。这项提案中的具体目标将检验这一假设，并检查相关贴图激活器的行为。SP.AIM 1将通过HX-MS分析ERK2中的蛋白质动力学，核磁共振和FRET，以确定铰链柔性变化的功能后果这是由激酶的磷酸化和激活所诱导的。SP.目标2将检验这一假设 MAP激酶对接基序在其各自的结合口袋之间变构相互作用，并且使用等温量热法、HX-MS、和核磁共振。SP.目标3将研究N-末端结构域突变的机制 ERK2通过定点突变、HX-MS和核磁共振来调节激活LIP的灵活性。SP. AIM 4将扩展我们的HX-MS分析，以记录依赖于激活的构象迁移率其他蛋白激酶。完成这些目标将为理解提供一个独特的窗口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，包括高场解决方案核磁共振和氢交换质谱仪。这些实验将：(I)记录对大酶中的内部运动，(Ii)展示了蛋白质运动在控制激酶中的作用酶的功能，(Iii)提高我们对蛋白质运动是如何长期控制的理解以及(Iv)记录调节信号分子的新机制。