Analysis of Lens Crystallins and Cataractous Mutants at High Hydrostatic Pressure

描述（由申请方提供）：本提案的具体目的是确定眼透镜晶体蛋白的生物化学和生物物理特性及其在白内障发生过程中的作用。我们的主要工作将集中在高静水压力对α晶体蛋白的功能，结构和相互作用的影响。α晶体蛋白是小热休克蛋白家族的关键成员，除了它们在透镜中的关键功能之外，还涉及许多神经退行性疾病和癌症。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。在高静水压下使用各种光谱技术。然而，圆二色性（CD），一种最有用的，强大的和现成的技术，不能在高压下使用，直到我们成功地建立了一个系统，我们可以进行CD测量高达2,500巴的压力。我们的方法将使我们能够获得热力学数据，从中我们可以得到有关α晶体蛋白中空腔状态的信息。我们的假设是，α晶体蛋白结构内的空腔和空隙对其稳定性和功能至关重要。具体目标是：（1-i）。使用高压CD光谱阐明天然α晶体蛋白的二级、三级和四级结构。（1-ii）。确定在高流体静压下的构象转变，将发生在α晶体蛋白与突变，导致白内障，并获得热力学数据计算的摩尔体积的变化，反映在蛋白质内的空腔的状态。（2-i）。使用胰岛素和乙醇脱氢酶作为靶蛋白，测定天然α晶状体蛋白、WT α A和B以及α A和B的磷酸化模拟物的分子伴侣样性质。（2-ii）.使用高静水压确定天然和WT α晶体蛋白与已知引起白内障的突变体γ晶体蛋白的相互作用。该项目的健康相关性：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主要候选者。该项目将为了解其复杂和动态结构以及其控制淀粉样变性的遗传能力以及其腔体提供治疗分子的潜力提供关键信息。

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.