LENS PROTEINS--CHANGES DUE TO CATARACTOGENIC AGENTS

这项建议的长期目标是阐明 赋予这些蛋白质的晶状体蛋白的特征和相互作用 稳定性和透明度，并通过以下方式了解原因和机制 哪种结构不稳定会导致蛋白质的聚集和 白内障形成过程中透明度的丧失。这样做的具体目的是 项目期为： 1.检验假设在生理浓度下，晶状体 晶体蛋白相互作用经历构象重新调整从 通常的β-折叠到扩展的互连的多分子组装 提供短期订单和透明度。研究包括：(A)体外研究 从稀释到生理的分子相互作用的测量 集中精神。(B)量热法，以确定体系的热力学 进程。牛晶状体和人类晶状体中的蛋白质将用于这一点 学习。 2.验证α-蛋白分子伴侣活性假说 并根据晶体蛋白的透明度来定义这一功能 镜头。研究包括：(A)热力学和构象方面 利用光谱技术研究伴侣蛋白与靶蛋白的相互作用。(B) 伴侣蛋白-靶蛋白相互作用的机制。 3.确定蛋白质修饰的程度 白内障的发生及其在结构不稳定中的作用 在白内障形成过程中。研究包括：(A)广泛的物理-- 新鲜获得的人白内障的化学和生化分析 镜头。(B)糖基化、色素沉着程度的测定， 谷胱甘肽、蛋白质-二硫键和蛋白质谷胱甘肽混合 白内障晶体蛋白和晶状体中二硫键和交联键的形成 评估这些修饰的蛋白质在其 白内障形成过程中的结构不稳定。 除了适当的化学、生化和光谱 吸收、荧光、圆二向色性(CD)和 傅里叶变换红外(FTIR)，该项目还将使用(A)高 分辨率差示扫描量热法(DSC)(B)核磁 松弛分散(NMRD)和(C)免疫技术 目标。

The long term objectives of this proposal are to elucidate the structural features and interactions of the crystallins that endow these proteins with stability and transparency and to understand the cause and mechanism by which structural destabilization leads to aggregation of the proteins and loss of transparency during cataractogenesis. The Specific Aims of this project period are: 1. To test the hypothesis that at physiological concentration lens crystallins interact to undergo a conformational readjustment from the usual beta-sheet to an extended interconnected multimolecular assembly to provide short-range order and transparency. Studies include: (a) In vitro measurements of molecular interactions from dilute to physiological concentration. (b) Calorimetry to determine the thermodynamics of the process. Proteins from both bovine and human lens will be used for this study. 2. To test the hypothesis of molecular chaperone activity of alpha- crystallin and to define this function in terms of the transparency of the lens. Studies include: (a) Thermodynamics and conformational aspects of chaperone-target protein interactions using spectroscopic techniques. (b) Mechanism for chaperone-target protein interactions. 3. To determine the extent of protein modifications during cataractogenesis and to define their role in the structural destabilization during cataract formation. Studies include; (a) Extensive physico- chemical and biochemical analysis of freshly obtained human cataractous lens. (b) Determination of the level of glycation, pigmentation, glutathione, protein-protein disulfide and protein glutathione mixed disulfide and crosslink formation in cataractous lens crystallins and assessment of the role played by these modified proteins in their structural destabilization during cataract formation. In addition to appropriate chemical, biochemical and spectroscopic techniques, such as, absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FTIR), the project will also use (a) high resolution differential scanning calorimetry (DSC) (b) nuclear magnetic relaxation dispersion (NMRD) and (c) immunological techniques to accomplish the aims.