Electrostatics and Dynamics in Proteins

蛋白质的静电和动力学

• 批准号: 7924982
• 负责人: STEVEN G. BOXER
• 金额: $ 17.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924982
• 关键词: Active Sites; Address; Affect; Aldehyde Reductase; Amino Acids; Binding; Biological Models; Biotechnology; Catalysis; Cells; Charge; Complications of Diabetes Mellitus; Coupled; Cysteine; Electrons; Electrostatics; Enzymes; Frequencies; Goals; Green Fluorescent Proteins; Health; Human; Image; Isomerase; Ketosteroids; Label; Laboratories; Lead; Location; Maps; Measurement; Measures; Membrane; Membrane Potentials; Methods; Molecular Probes; Mutation; Nitriles; Nucleic Acids; Photochemistry; Positioning Attribute; Process; Protein Region; Proteins; Protons; Reaction; Relative (related person); Reporting; Research; Resolution; Sampling; Series; Site; Solvents; Spectrum Analysis; Structure; System; Techniques; Testing; Thiocyanates; Time; Variant; Work; analog; base; chromophore; design; detector; diabetes control; electric field; enzyme mechanism; inhibitor/antagonist; insight; interest; ion mobility; macromolecular assembly; macromolecule; novel; novel strategies; protein folding; protein structure function; public health relevance; research study; response; tool; vibration

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of several enzymes and green fluorescent proteins (GFPs). Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids and membranes. Variations in the magnitude and direction of electric fields can significantly affect the rates of elementary processes such as electron and proton transfer, where charge moves over a substantial distance. Similarly, the transition states for many enzyme-catalyzed reactions involve a change in the distribution of charge relative to the starting material and/or products, and the selective stabilization of charge-separated transition states is essential for catalysis. The landscape of electric fields steers the binding of substrates, inhibitors and allosteric effectors to macromolecules and directly affects binding constants. On a larger scale, electrostatic interactions affect protein folding, macromolecular interactions and the assembly of subunits into larger structures. The magnitudes of the electric fields in proteins and the variations in these fields at different sites can be enormous. While these variations and their absolute magnitudes are well appreciated by theorists, who have developed a large body of analytical, computational, and graphical methods to evaluate electrostatic potentials, it has proven to be more difficult to obtain quantitative experimental information on either local variations in electric fields in proteins or the time-dependent changes in these fields coupled to functionally-relevant changes in charge distribution. The proposed research outlines a series of approaches and targets that can address these core issues. Aim 1 outlines experiments that probe time-averaged and time-dependent electric fields in several enzymes: human aldose reductase and human aldehyde reductase (Sub-Aim 1A), both important to the control of diabetes, and ketosteroid isomerase (Sub-Aim 1B). The proposed work is focused on rigorously comparing measured and calculated fields by using vibrational Stark spectroscopy, discriminating between structurally-similar active sites by understanding electrostatic fields, and incisively probing the mechanism of an enzyme by employing electric field detectors close to the site where catalysis occurs. Aim 2 outlines measurements that probe time-dependent excited state proton transfer and electrostatics using novel GFP constructs. In part, this work continues a long-standing and high impact effort to understand the photophysics and photochemistry of GFP variants. In addition, we propose to extend this effort by using split GFPs to probe the assembly of the 2-barrel structure and by introducing unnatural amino acids at functionally interesting sites throughout the protein. PUBLIC HEALTH RELEVANCE We are investigating several proteins that have direct relevance to human health. In particular, we propose new approaches for characterizing and potentially differentiating electrostatics at the active sites of human aldose reductase and human aldehyde reductase. The former is a primary cause of the complications of diabetes, but selective inhibition of one enzyme relative to the other has, thus far, proven elusive. New experiments are also proposed for GFP which is the most widely used fluorescent protein for cell-based imaging.

项目描述（由申请人提供）：该项目的长期目标是发展探测蛋白质中的电场的光谱方法，并应用这些方法获得电场及其对几种酶和绿色荧光蛋白（gfp）活性位点功能影响的定量信息。静电相互作用影响着蛋白质、核酸和膜的结构和功能的各个方面。电场的大小和方向的变化可以显著地影响基本过程的速率，如电子和质子转移，其中电荷移动了相当大的距离。同样，许多酶催化反应的过渡态涉及相对于起始物质和/或产物的电荷分布的变化，并且电荷分离过渡态的选择性稳定对于催化是必不可少的。电场的景观引导底物、抑制剂和变构效应物与大分子的结合，并直接影响结合常数。在更大的尺度上，静电相互作用影响蛋白质折叠、大分子相互作用和亚基组装成更大的结构。蛋白质中电场的大小以及这些电场在不同位置的变化可能是巨大的。虽然这些变化和它们的绝对大小被理论家们很好地理解，他们已经开发了大量的分析、计算和图形方法来评估静电势，但事实证明，要获得定量的实验信息，无论是蛋白质中电场的局部变化，还是这些电场与电荷分布的功能相关变化相耦合的随时间变化，都是比较困难的。拟议的研究概述了一系列可以解决这些核心问题的方法和目标。目的1概述了在几种酶中探测时间平均电场和时间依赖电场的实验：人醛糖还原酶和人醛醛还原酶（Sub-Aim 1A），两者对控制糖尿病都很重要，以及酮类固醇异构酶（Sub-Aim 1B）。本文的工作重点是通过振动斯塔克光谱严格比较测量场和计算场，通过了解静电场来区分结构相似的活性位点，并通过使用靠近催化发生位点的电场探测器来深入探索酶的机制。目的2概述测量探针时间依赖的激发态质子转移和静电使用新的绿色荧光蛋白结构。在某种程度上，这项工作继续了一项长期和高影响力的努力，以了解GFP变体的光物理和光化学。此外，我们建议通过使用分裂gfp来探测2桶结构的组装，并在整个蛋白质的功能感兴趣的位点引入非天然氨基酸来扩展这一努力。我们正在研究几种与人类健康直接相关的蛋白质。特别是，我们提出了表征和潜在区分人醛糖还原酶和人醛还原酶活性位点静电的新方法。前者是糖尿病并发症的主要原因，但迄今为止，一种酶相对于另一种酶的选择性抑制被证明是难以捉摸的。GFP作为细胞成像中应用最广泛的荧光蛋白，也提出了新的实验。