LENS PROTEINS--CHANGES DUE TO CATARACTOGENIC AGENTS

本建议的长期目标是阐明 赋予这些蛋白质的晶体蛋白的特征和相互作用 稳定性和透明度，并了解原因和机制， 该结构不稳定导致蛋白质的聚集， 在白内障形成过程中失去透明度。 具体目标是 项目周期为： 1. 为了检验在生理浓度下透镜 晶体蛋白相互作用，从晶体蛋白中进行构象重新调整， 通常的β-折叠到扩展的互连多分子组装， 提供短期的秩序和透明度。 研究包括：（a）体外 从稀释到生理的分子相互作用的测量 浓度. (b)量热法来确定的热力学 过程 牛和人透镜的蛋白质将用于此 study. 2. 为了验证α-淀粉样蛋白分子伴侣活性的假设， 晶体蛋白，并根据晶体蛋白的透明度来定义这种功能。 透镜。 研究包括：（a）热力学和构象方面的 使用光谱技术的伴侣蛋白-靶蛋白相互作用。 （B） 分子伴侣与靶蛋白相互作用的机制。 3. 为了确定蛋白质修饰的程度， 白内障的发生，并确定其在结构不稳定的作用， 在白内障形成过程中。 研究包括：（a）广泛的物理- 新鲜获得的人类白内障的化学和生物化学分析 透镜。 (b)测定糖化水平、色素沉着， 谷胱甘肽、蛋白质-蛋白质二硫化物和蛋白质谷胱甘肽混合物 白内障透镜晶体蛋白中二硫化物和交联形成， 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、生物化学和光谱分析外， 技术，例如吸收、荧光、圆二色性（CD）和 傅里叶变换红外（FTIR），该项目还将使用（一）高 分辨率差示扫描量热法（DSC）（B）核磁 松弛分散（匪RD）和（c）免疫技术，以实现 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.