Crystallin aggregation and stabilization

标题：晶体蛋白聚集和稳定化 项目概要/摘要： 白内障影响美国40岁以上人群中1/6的视力， 为80.它是全球失明的主要原因。因此，人们对了解 白内障的病因和形成机制。白内障被归类为一种错误折叠的疾病 由此晶状体蛋白透镜蛋白质的降解导致其聚集和沉淀。然而，在这方面， 关键的结构和机理信息很大程度上是因为很难通过实验获得 关于聚集蛋白质的结构信息，甚至更难表征中间体， 负责降水。在这个建议中，我们将使用一种新的组合，二维红外光谱，同位素 标记和质谱分析，以揭示有关结构和动力学机制的细节， 晶体蛋白聚集体和伴侣蛋白B-晶体蛋白抑制沉淀的方式。使用 表达的蛋白质连接到半合成的D-晶状体蛋白，我们将同位素标记其各个结构域， 它们的结构和动力学可以通过2DIR光谱来监测。利用UVB光模拟共价键 从太阳辐射的损害和启动聚集，我们将监测动力学和结构的 当它们形成时沉淀。从我们最初的出版物中我们知道，酸诱导的D-晶状体蛋白变性 仅导致淀粉样纤维形成，C-末端结构域形成纤维核心，而N-末端结构域不形成纤维核心。 正如以前所认为的那样。相反，UVB-变性的初步结果表明，共价 损伤导致纤维状和无定形聚集体。很明显，在不同的途径之间 这取决于蛋白质损伤的类型。一旦这些途径的特点，我们将研究如何， 被伴侣蛋白B-晶状体蛋白修饰。对于许多蛋白质来说，B-晶状体蛋白是一种更好的伴侣蛋白。 与纤维状聚集体相比，白内障沉积物似乎主要是无定形聚集体， 这意味着晶体蛋白的分子伴侣机制是完全不同的。最后，体外 将针对从人晶状体收集的体内蛋白质提取物测试机制。我们想知道 晶状体蛋白的天然发生的损伤或组成改变了晶状体蛋白的体外机制和/或结构， 沉淀物。该提案中几乎每一步都显示了初步结果。我们的新方法 使用半合成和二维红外光谱提供了分子水平的见解，这是重要的 致力于了解B-晶体蛋白的分子伴侣机制的科学家的大社区， 白内障形成。

Title: Crystallin aggregation and stabilization Project Summary/Abstract: Cataracts affect the vision of 1 in 6 people over the age of 40 in the United States and most people by the age of 80. It is the leading cause of blindness worldwide. As a result, there is much interest in understanding the cause of cataracts and the mechanism by which they form. Cataracts are classified as a misfolding disease whereby degradation of the crystallin lens proteins leads to their aggregation and precipitation. However, critical structural and mechanistic information is lacking largely because it is experimentally difficult to obtain structural information about aggregated proteins and even more difficult to characterize intermediates that are responsible for precipitation. In this proposal, we will use a novel combination of 2D IR spectroscopy, isotope labeling, and mass spectrometry to uncover details about the structure and kinetic mechanism by which ¿D- crystallin aggregates and the way in which the chaperone protein ¿B-crystallin inhibits precipitation. Using expressed protein ligation to semi-synthesize ¿D-crystallin, we will isotope label its individual domains so that their structures and kinetics can be monitored by 2D IR spectroscopy. Using UVB light to mimic covalent damage from solar radiation and initiate aggregation, we will monitor the kinetics and structures of the precipitates as they form. We know from our initial publications that acid-induced denaturation of ¿D-crystallin leads solely to amyloid fiber formation with the C-terminal domain forming the fibril core, not the N-terminal domain as was previously thought. In contrast, preliminary results on UVB-denaturation reveal that covalent damage leads to both fibrillar and amorphous aggregates. Clearly, there is a competition between pathways that depends on the type of protein damage. Once these pathways are characterized, we will study how they are modified by the chaperone protein ¿B-crystallin. For many proteins ¿B-crystallin is a better chaperone against amorphous than fibrillar aggregates, but cataract deposits appear to be mostly amorphous aggregates, implying that the chaperone mechanism is quite different for the crystallin proteins. Finally, the in vitro mechanisms will be tested against in vivo protein extracts collected from human lenses. We want to know if naturally occurring damage or composition of the crystallins alters the in vitro mechanisms and/or structure of the precipitates. Preliminary results are shown for nearly every step in this proposal. Our novel approach of using semi-synthesis and 2D IR spectroscopy is providing molecular-level insights that are important to the large community of scientists devoted to understanding the chaperone mechanisms of ¿B-crystallin and cataract formation.