Electrostatics and Dynamics in Proteins

描述（由申请人提供）：本项目的长期目标是开发用于探测蛋白质电场的光谱方法，并应用这些方法获得关于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影响基本过程的速率，例如电子和质子转移，其中电荷移动相当长的距离。类似地，许多酶催化反应的过渡态涉及相对于起始材料和/或产物的电荷分布的变化，并且电荷分离的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点的变化可能是巨大的。虽然这些变化和它们的绝对大小是很好的赞赏理论家，谁已经开发了大量的分析，计算和图形方法来评估静电势，它已被证明是更难以获得定量的实验信息，无论是在蛋白质中的电场的局部变化或在这些领域的时间依赖性变化耦合到功能相关的电荷分布的变化。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概述了探测几种酶中的时间平均和时间依赖性电场的实验：人醛糖还原酶和人醛还原酶（子目的1A），这两种酶对糖尿病的控制都很重要，以及酮类类固醇异构酶（子目的1B）。拟议的工作重点是严格比较测量和计算的领域，通过使用振动斯塔克光谱，区分结构相似的活性位点之间的理解静电场，并通过采用电场探测器接近催化发生的网站，深刻探测酶的机制。目的2概述了测量，探测时间依赖性激发态质子转移和静电使用新的绿色荧光蛋白构建。在某种程度上，这项工作延续了长期以来的高影响力努力，以了解GFP变体的光物理学和光化学。此外，我们建议通过使用分裂GFP来探测2桶结构的组装，并通过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，我们提出了新的方法来表征和潜在的区别静电在活性位点的人醛糖还原酶和人醛还原酶。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已被证明是难以捉摸的。新的实验也提出了绿色荧光蛋白，这是最广泛使用的荧光蛋白为基础的细胞成像。

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.