Protein architecture and enzyme dynamics on timescales from fs to ms.

从 fs 到 ms 时间尺度上的蛋白质结构和酶动力学。

• 批准号: 8906879
• 负责人: RICHARD BRIAN DYER
• 金额: $ 48.26万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8906879
• 关键词: Affect; Architecture; Binding; Catalysis; Catalytic Domain; Chemistry; Collaborations; Complex; Coupled; Coupling; Dependence; Detection; Development; Devices; Dihydrofolate Reductase; Enzymes; Equilibrium; Fluorescence; Genetic Anticipation; Goals; Hydrogen Bonding; Image; Isotope Labeling; Isotopes; Label; Laboratories; Lactate Dehydrogenase; Measurement; Measures; Methods; Microfluidics; Modeling; Motion; Mutation; Nucleic Acids; Organism; Pathway interactions; Pharmaceutical Preparations; Population; Positioning Attribute; Production; Protein Dynamics; Proteins; Protons; Pump; Purine-Nucleoside Phosphorylase; Reaction; Research; Role; Shapes; Solutions; Solvents; Specificity; Spectrum Analysis; Structural Protein; Structure; System; Temperature; Testing; Time; Water; Work; base; design; drug development; enzyme activity; improved; infrared spectroscopy; inhibitor/antagonist; innovation; millisecond; mutant; novel strategies; programs; protein structure; protonation; research study; small molecule; theories

This project will elucidate the role of protein dynamics in enzyme function on all time scales, with the overall goal of developing enzyme design principles based on dynamics. We have pioneered new approaches to elucidate enzyme dynamics using structurally specific approaches, including isotope edited infrared spectroscopy coupled with ultrafast reaction initiation (T-jump or pH jump) and with fast microfluidics mixing methods. On the femtosecond time scale, we seek a deeper understanding of the fast atomic motions required to move the system over the transition barrier to achieve chemistry. On slower time scales (ps - ms) we seek to elucidate the conformational changes associated with substrate binding, catalytic site reorganization and product release. We focus on three enzymes, dihydrofolate reductase (DHFR) and in close collaboration with the Callender and Schramm labs respectively, lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP). The work is supported by theory and computation in the Schwartz group. The project has three specific aims: (1) Determine the conformational dynamics that control DHFR catalytic activity. This aim will test the hypothesis that the conformational dynamics of the Met20 loop act as a master control of DHFR activity by modulating the barriers to proton and hydride transfer. We will determine the effects of mutations discovered in our lab that perturb the H-bonding network on the proton transfer dynamics and any coupled protein dynamics, using pH jump methods and time-resolved IR spectroscopy. (2) Determine the protein structural dynamics that control the formation of the Michaelis complex in LDH. This aim will test the hypothesis that the Michaelis sub-state distribution and catalytic efficiency of LDH are controlled by the energy landscape of the catalytically important loop motions. We plan to determine how these loop motions are related to the sub-state distribution and the extent to which the protein conformational distribution is collapsed in the observed sub-states using ultrafast mixing, coupled with T-jump experiments in the Callender lab. Calculations by the Schwartz group will identify the dominant Michaelis configurations and measure the dynamics of transitions between them. These calculations will enable the interpretation of the dynamics observed in our experiments. (3) Investigate the relationships between protein structural dynamics, pathways for energy flow and allostery in enzymes. This aim will test the hypothesis that allostery requires pathways for energy flow to reach a specific target that depend on the protein structure and its dynamics. We have developed ultrafast, pump-probe IR spectroscopy to probe the specific pathways of energy flow in enzymes. We will apply these methods to characterize the dynamics of energy flow in DHFR, LDH and PNP, and how it depends on inhibitor binding and the distribution of the conformational sub-states of the enzymes. We expect tight binding dynamic inhibitors to have very different energy flow dynamics than less efficient inhibitors that cause conformational collapse.

这个项目将在所有时间尺度上阐明蛋白质动力学在酶功能中的作用，总体上 开发基于动力学的酶设计原理的目标。我们已经开创了新的方法来 使用结构特异性方法，包括同位素编辑红外，来阐明酶的动力学 光谱与超快反应引发(T跳跃或pH跳跃)和快速微流控混合的耦合 方法：研究方法。在飞秒时间尺度上，我们寻求对快速原子运动的更深层次的理解 使系统越过过渡势垒以实现化学作用所需的。在较慢的时间尺度上(PS- MS)我们试图阐明与底物结合、催化位置相关的构象变化 重组和产品发布。我们专注于三种酶，二氢叶酸还原酶(DHFR)和In 分别与Callender和Schramm实验室密切合作，乳酸脱氢酶(LDH)和 嘌呤核苷磷酸化酶(PNP)。这项工作得到了施瓦茨的理论和计算的支持 一群人。该项目有三个具体目标：(1)确定控制DHFR的构象动力学 催化活性。这一目标将检验Met20环的构象动力学作为 通过调节质子和氢化物转移的障碍来控制dhfr的活性。我们会 确定在我们实验室中发现的扰乱质子氢键网络的突变的影响 利用pH跃迁方法和时间分辨红外光谱研究转移动力学和任何偶联蛋白质动力学 光谱学。(2)确定控制米氏形成的蛋白质结构动力学 乳酸脱氢酶复合体。这一目标将检验米氏亚态分布和催化的假设 LDH的效率由催化重要的环路运动的能量景观控制。我们计划 为了确定这些环路运动与子状态分布的关系，以及 蛋白质的构象分布在观察到的亚态中利用超快混合，耦合 在卡伦德实验室进行T-JUMP实验。施瓦茨小组的计算将确定主导的 Michaelis构型，并测量它们之间转换的动态。这些计算将 使我们能够解释在实验中观察到的动力学。(3)调查关系 在蛋白质结构动力学、能量流动途径和酶的变构之间。这一目标将考验 变构需要能量流到达特定目标的路径的假设取决于 蛋白质结构及其动力学。我们已经开发了超快、泵浦-探测红外光谱来探测 酶中能量流动的特定路径。我们将应用这些方法来描述 DHFR、LDH和PNP中的能量流动以及它如何依赖于抑制剂的结合和分布 酶的构象亚态。我们预计紧密结合的动态抑制剂将有非常不同的 能量流动动力学比导致构象崩塌的低效抑制剂。