Analysis of Lens Crystallins and Cataractous Mutants at High Hydrostatic Pressure

描述(申请人提供)：这项建议的具体目的是确定眼晶状体晶体蛋白的生化和生物物理性质及其在白内障形成过程中的作用。我们的主要工作将集中在高压对α晶体蛋白的功能、结构和相互作用的影响上。α晶体蛋白是小型热休克蛋白家族的关键成员，除了在晶状体中发挥关键功能外，还与许多神经退行性疾病和癌症有关。高静水压目前被用作研究蛋白质聚集和淀粉样变性的有价值的工具。在高静水压下使用了各种光谱技术。然而，圆二色谱(CD)是一种最有用、最强大和最容易获得的技术，在我们成功地建立了一个可以测量压力高达2500巴的CD的系统之前，不能在高压下使用。我们的方法将使我们能够获得热力学数据，从中我们可以获得关于α晶体中空穴状态的信息。我们的假设是，α晶体蛋白结构中的空洞和空洞对其稳定性和功能是必不可少的。具体目标是：(1-i)。用高压圆二色谱研究天然α晶体蛋白的二级、三级和四级结构。(1-II)。在高静水压力下，确定阿尔法晶体蛋白中将发生的构象转变以及导致白内障的突变，并获得热力学数据，用于计算反映蛋白质内空腔状态的摩尔体积变化。(2-i)。以胰岛素和酒精脱氢酶为靶蛋白，确定天然α晶体蛋白、WTαA和B以及αA和B的磷酸化模拟物的伴侣性质。(2-II)。使用高静水压，确定天然和WTα晶状体蛋白与已知可导致白内障的突变伽马晶状体蛋白的相互作用。该项目与健康相关：α晶体蛋白参与白内障形成和其他疾病，特别是其控制聚集的伴侣特性使其成为用作治疗剂的首选候选药物。该项目将增加关键信息，以了解其复杂和动态的结构，以及它控制淀粉样变性的遗传能力，以及它的空洞输送治疗分子的潜力。

DESCRIPTION (provided by applicant): The specific aims of this proposal are to determine the biochemical and biophysical properties of the eye lens crystallins and their involvement in the processes of cataractogenesis. Our main effort will focus on the effect of high hydrostatic pressure on the function, structure, and interactions of alpha crystallins. The alpha crystallins are key members of the small heat-shock protein family that in addition to their critical function in the lens, are also involved in many neurodegenerative diseases and cancers. High hydrostatic pressure is currently being used as a valuable tool for studying protein aggregation and amyloidosis. A variety of spectroscopical techniques are used at high hydrostatic pressure. However, circular dichroism (CD), a most useful, powerful and readily available technique could not be used at high pressure until we were successful in building a system by which we can perform CD measurements up to pressure of 2,500 bars. Our approach will allow us to obtain thermodynamic data from which we can get information about the state of cavities in alpha crystallin. Our hypothesis is that cavities and voids within the structure of alpha crystallin are essential to its stability and function. The specific aims are: (1-i). Elucidate the secondary, tertiary, and quaternary structure of native alpha crystallins using high pressure CD spectroscopy. (1-ii). Determine under high hydrostatic pressures the conformational transitions that will occur in the alpha crystallins with the mutations that cause cataract, and obtain thermodynamic data for calculating changes in molar volume that reflect the state of the cavities within the protein. (2-i). Determine the chaperone-like properties of the native alpha crystallin, WT alpha A and B and phosphorylated mimics of alpha A and B using insulin and alcohol dehydrogenase as target proteins. (2-ii). Determine using high hydrostatic pressure, the interactions of native and WT alpha crystallins with mutant gamma crystallins that are known to cause cataract. Health relatedness of the project: The involvement of alpha crystallin in cataractogenesis and other diseases, and especially its chaperone properties for controlling aggregation make it a prime candidate to be used as a therapeutic agent. This project will add critical information for understanding its complex and dynamic structure and it's inherit capabilit to control amyloidosis as well as the potential of its cavities to deliver therapeutic molecules.