Measuring static and dynamic electric fields in proteins

描述（由申请人提供）：本申请的长期科学目标是追求一个研究领域，该领域超越了蛋白质的传统结构和生化分析，以更定量地了解支配蛋白质功能的能量学。电场是控制蛋白质结构和功能的能量景观的一个组成部分，但很少有蛋白质电场的实验测量。由Steve Boxer的研究小组开发的称为VSE光谱学的光谱技术将用于对两个生物系统中的电场进行系统表征。具体目标1涉及比较醛糖和醛还原酶的电场，这两种酶与糖尿病有关。这将揭示这些密切相关的酶的活性位点如何被静电调节到不同的功能，以及如何利用这些差异来设计更有效和更特异的醛糖还原酶抑制剂。具体目标2涉及研究小的GT3 Ras，它在大量的人类癌症中发生突变。由于Ras是一种高度动态的蛋白质，这项工作将增强我们对蛋白质动力学如何影响静电的理解，这是一个相对知之甚少的问题。VSE光谱学需要将腈基掺入到感兴趣的蛋白质中，以作为电场的实验报告物。先前已经开发了两种方法用于将腈掺入蛋白质中，并且这些方法将用于制备在不同位点具有探针的蛋白质，从而允许在感兴趣的蛋白质的大区域上测量静电。由于Boxer实验室开创了VSE光谱技术，它将为执行这些实验提供理想的环境。公共卫生相关性：许多疾病是由特定生物过程的相对微妙的扰动引起的-例如，癌症涉及改变某些蛋白质性质的突变的获得。对这些蛋白质的物理性质的全面了解将揭示疾病中出现的异常行为的起源，并使我们能够利用这些性质获得治疗益处。本文提出的关于醛糖还原酶和Ras的工作将提高我们对这些蛋白质参与疾病的理解，并导致干预疾病过程的新方法。

DESCRIPTION (provided by applicant): The long-term scientific objectives of this application are to pursue an area of research that moves beyond a traditional structural and biochemical analysis of proteins to a more quantitative understanding of the energetics that govern protein function. Electric fields are an integral component of the energy landscapes that govern protein structure and function but very few experimental measurements of electric fields in proteins have been made. A spectroscopic technique called VSE Spectroscopy, developed by Steve Boxer's research group, will be used to perform a systematic characterization of the electric fields in two biological systems. Specific aim 1 involves comparing the electric fields in aldose and aldehyde reductase, two enzymes implicated in diabetes. This will reveal how the active sites of these closely related enzymes are electrostatically tuned to different functions, and how these differences could be used to design more potent and more specific inhibitors of aldose reductase. Specific Aim 2 involves studying the small GTPase Ras, which is mutated in a large number of human cancers. Since Ras is a highly dynamic protein, this work will enhance our understanding of how electrostatics is influenced by protein dynamics, a question about which relatively little is known. VSE Spectroscopy requires the incorporation of a nitrile group into the protein of interest to serve as an experimental reporter of electric field. Two methods have been previously developed for the incorporation of nitriles into proteins and these methods will be used to prepare proteins with probes at different sites, allowing the electrostatics to be measured over large regions of the proteins of interest. Since the Boxer lab pioneered the technique of VSE Spectroscopyit will provide an ideal environment for performing these experiments. PUBLIC HEALTH RELEVANCE: Many diseases are caused by relatively subtle perturbations to specific biological processes- for instance, cancer involves the acquisition of mutations that alter the properties of certain proteins. A comprehensive understanding of the physical properties of such proteins will reveal the origin of the aberrant behavior that arises in disease and allow us to exploit these properties for therapeutic benefit. The work proposed here with aldose reductase and Ras will improve our understanding of the involvement of these proteins in disease and lead to new ways to intervene in the disease process.