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: 9312299
• 负责人: RICHARD BRIAN DYER
• 金额: $ 48.26万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9312299
• 关键词: Affect; Architecture; Binding; Catalysis; Catalytic Domain; Chemistry; Collaborations; Complex; Coupled; Coupling; Dependence; Development; Devices; Dihydrofolate Reductase; Drug Targeting; Enzyme Inhibition; Enzymes; Equilibrium; Fluorescence; Goals; Hydrogen Bonding; Isotope Labeling; Isotopes; Label; Laboratories; Lactate Dehydrogenase; Measurement; Measures; Methods; Microfluidics; Modeling; Molecular Conformation; Motion; Mutation; Nucleic Acids; Pathway interactions; Pharmaceutical Preparations; Population; Positioning Attribute; Production; Protein Conformation; Protein Dynamics; Proteins; Protons; Pump; Purine-Nucleoside Phosphorylase; Reaction; Research; Role; Shapes; Solvents; Specificity; Spectrum Analysis; Structural Protein; Structure; System; Temperature; Testing; Time; Water; Work; base; design; drug development; enzyme activity; experimental study; imaging detection; improved; infrared spectroscopy; inhibitor/antagonist; innovation; millisecond; mutant; novel strategies; programs; protein structure; protonation; 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.

这个项目将阐明蛋白质动力学在所有时间尺度上酶功能中的作用， 目标是发展基于动力学的酶设计原理。我们开创了新的方法， 使用结构特异性方法阐明酶动力学，包括同位素编辑红外光谱 与超快反应引发（T-跳跃或pH跳跃）和快速微流体混合相结合的光谱学 方法.在飞秒时间尺度上，我们寻求对快速原子运动的更深入了解 需要移动系统越过过渡势垒以实现化学反应。在较慢的时间尺度上（ps - ms），我们试图阐明与底物结合、催化位点 重组和产品发布。我们专注于三种酶，二氢叶酸还原酶（DHFR）和 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）。这项工作是支持理论和计算在施瓦茨 组本项目有三个具体目标：（1）确定控制DHFR的构象动力学 催化活性这一目的将检验Met 20环的构象动力学充当 通过调节质子和氢化物转移的障碍来控制DHFR活性。我们将 确定我们实验室发现的突变对质子氢键网络的影响 转移动力学和任何耦合的蛋白质动力学，使用pH跳跃方法和时间分辨IR 谱(2)确定控制米氏形成的蛋白质结构动力学 复合物LDH。这一目的将检验假设，即米氏子状态分布和催化 LDH的效率由催化重要环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 蛋白质构象分布是崩溃的观察到的子状态使用超快混合，耦合 卡伦德实验室的T型跳跃实验施瓦茨小组的计算将确定 米氏配置和测量它们之间的过渡的动态。这些计算将 能够解释我们实验中观察到的动力学。(3)调查关系 蛋白质结构动力学，能量流动途径和酶的变构之间的联系。这一目标将检验 变构需要能量流的途径来到达特定目标的假设， 蛋白质结构及其动力学我们已经开发出超快，泵浦探测红外光谱探测 酶中能量流动的特定途径。我们将应用这些方法来表征的动力学 DHFR，LDH和PNP中的能量流，以及它如何依赖于抑制剂结合和 酶的构象亚状态。我们预期紧密结合的动态抑制剂具有非常不同的 能量流动动力学比导致构象崩溃的效率较低的抑制剂。