Crystallin aggregation and stabilization

标题：晶体蛋白聚集与稳定 项目摘要/摘要： 在美国，每6名40岁以上的人中就有1人患有白内障，大多数人的视力受此影响。 80岁。它是世界范围内导致失明的主要原因。因此，人们非常有兴趣了解 白内障的病因和形成机制。白内障被归类为一种折叠错误的疾病 晶体蛋白的降解导致晶体蛋白的聚集和沉淀。然而， 关键的结构和机械信息在很大程度上是缺乏的，因为在实验上很难获得 关于聚集蛋白质的结构信息，甚至更难表征 对降水负责。在这项提议中，我们将使用2D IR光谱、同位素 标记和质谱学以揭示有关D-的结构和动力学机制的细节。 晶状体蛋白聚集体和伴侣蛋白B-晶状体蛋白抑制沉淀的方式。vbl.使用 表达的蛋白质连接到半合成D-晶体蛋白，我们将同位素标记它的各个结构域，以便 它们的结构和动力学可以通过二维红外光谱进行监测。利用UVB光模拟共价键 太阳辐射的破坏和引发聚集，我们将监测动力学和结构 在它们形成时沉淀。我们从最初的出版物中了解到，酸诱导D-晶状体蛋白的变性 只导致淀粉样纤维的形成，C-末端结构域形成原纤核，而不是N-末端 正如之前所认为的那样，是一个领域。相反，UVB变性的初步结果显示共价 损伤导致纤维状聚集体和无定形聚集体。显然，在不同的路径之间存在竞争 这取决于蛋白质损伤的类型。一旦这些路径被表征，我们将研究它们是如何 被伴侣蛋白B-晶状体蛋白修饰。对于许多蛋白质来说，B-晶状体蛋白是更好的伴侣 抗无定形聚集体而不是纤维状聚集体，但白内障沉积物似乎主要是无定形聚集体， 这意味着，对于晶体蛋白来说，伴侣机制是非常不同的。最后，体外培养 机制将针对从人类晶状体中收集的体内蛋白质提取物进行测试。我们想知道如果 自然发生的损伤或晶体蛋白的组成改变了体外机制和/或结构 沉淀物。这项提案中的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，这些见解对 致力于了解B-晶状体蛋白和β-晶状体蛋白的伴侣机制的大型科学家社区 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.