Regulation of Map Kinase by Protein Motions

蛋白质运动对图谱激酶的调节

• 批准号: 7197867
• 负责人: NATALIE G. AHN
• 金额: $ 24.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197867
• 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; Class; Closure; Communication; Coupling; Data; Docking; Enzyme Activation; Enzymes; Fluorescence Resonance Energy Transfer; Goals; Hydrogen; Lead; Ligand Binding; Ligands; Lip structure; Localized; 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; Pliability; 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; improved; innovative technologies; novel; peptide deformylase; protein function; research study; response

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.

描述（由申请人提供）：该提案的目的是研究响应蛋白激酶激活中发生的运动变化，并探索它们与酶功能的相关性。通过氢交换质谱法（HX-MS）和其他测量结果对MAP^激酶^ ERK2进行分析，揭示了三种情况，其中涉及（i）酶激活的酶扰动，（ii）结合位点和（iii）mutagenesens之间的变构通信导致蛋白质构象在长距离内的局部变化。现有证据表明，信号转导控制的新型模型，其中蛋白质动力学的调节控制ERK2中的催化和变构功能。该提案中的具体目的将检验该假设，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引起的铰链柔韧性变化的功能后果。 sp。 AIM 2将检验以下假设：MAP激酶对接基序在其各自的结合口袋之间相互作用，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将研究ERK2 N末端结构域的突变通过位置定向的诱变，HX-MS和NMR调节激活唇的柔韧性的机制。 sp。 AIM 4将扩展我们的HX-MS分析，以记录其他蛋白激酶中依赖激活的构象迁移率。这些目标的完成将为了解蛋白激酶如何通过控制构象移动性而发展以优化功能的独特窗口。创新技术将应用于此问题，包括高场溶液NMR和氢交换质谱法。实验将：（i）记录大型酶中内部运动的调节，（ii）证明了蛋白质运动在控制激酶酶功能中的作用，（iii）提高了我们对蛋白质运动在长距离内如何控制的理解，并且（iv）记录调节信号分子的新机制。