Conformation and Dynamics of Cataract Mutants of human gammaD crystallin

项目摘要 白内障是一种由晶状体蛋白缺陷引起的蛋白质聚集性疾病。这个 先天性疾病是由晶体蛋白基因突变引起的，而与年龄相关的退行性疾病 这种疾病是由晶体蛋白的化学修饰引起的。白内障是导致失明的主要原因。 在世界上，每年大约有1700万例。目前，唯一可用的治疗方法是手术， 事实证明，这是成功的。然而，世界上有很大一部分人口无法接受手术， 而且，在许多情况下，手术后会出现问题。因此，对白内障形成的基本理解是 重要的是开发延缓发病或延缓进展的新疗法。我们将调查其中的动态， 白内障相关D-晶状体蛋白突变体的结构和折叠。我们的目的是阐明其结构基础 用于白内障的形成。我们假设不是蛋白质的随机结合，而是特定的折叠 中间体参与聚合。 除了提供对白内障形成过程的洞察外，我们的研究还将探索 蛋白质生物学中的基本问题。例如，导致折叠受挫的相互作用， 关于中间体为什么和如何稳定，以及导致多肽链的过程的问题 要错误地折叠和/或聚合而不是折叠到原生状态，需要直接的实验研究来获得新的 洞察力。拟议的研究将通过对野生动物的生物物理分析来解决这些悬而未决的问题。 类型和疾病相关的晶体蛋白变异体。晶体蛋白非常适合于蛋白质的详细研究。 聚集：它们很小；有大量的X射线结构可用；以及几个野生- 对天然状态的类型蛋白进行了研究。核磁共振方法将用于直接调查 折叠跃迁以获得对这些过程的能量学的新见解并阐明结构 任何其他方法无法获得的中间体的详细信息。 我们的工作将涉及允许对蛋白质进行详细的结构和动力学表征的方法， 主要是核磁共振波谱和小角X射线散射。此外，我们将把基本的生物物理学 参数与临床观察。我们计划确定三维解的结构 与白内障相关的人D-晶状体蛋白，并表征其动态行为。我们将首先关注两个方面 形成D-晶状体蛋白突变体P23T和V75D的重要白内障。前者与先天性 人类的白内障，后者是一种变种，已被证实会导致老鼠的白内障，因此， 这有助于在白内障动物模型上进行后续研究。我们还将描述该结构和 D-晶状体蛋白折叠中间体的动力学。此外，我们将调查以前是否以及如何 经鉴定，部分折叠的D-晶体蛋白中间体导致聚集。特别是，我们将确定是否 这种部分折叠的中间产物是聚集的种子。这将为发现小的 分子聚集抑制剂，这一方法已经在许多方面取得了一些结果 神经退行性蛋白沉积病。

Project Summary Cataract is a protein aggregation disease caused by crystallin protein defects in the lens. The congenital form of the disease results from crystallin gene mutations, whereas the age-related degenerative disease results after chemical modification of crystallin proteins. Cataracts are the leading cause of blindness in the world, with approximately 17 million cases per year. Currently, the only available treatment is surgery, which has proven successful. However, a significant fraction of the world population can not access surgery, and, in many cases, problems occur after surgery. Thus, a basic understanding of cataract formation is important to develop novel therapies that delay onset or slow progression. We will investigate the dynamics, structure and folding of cataract-associated ¿D-crystallin mutants. Our aim is to elucidate the structural basis for cataract formation. We hypothesize that not random association of proteins, but specific folding intermediates are involved in aggregation. In addition to providing insight into the process of cataract formation, our studies will explore fundamental questions in protein biology. For example, the interactions that cause frustration of folding, questions about why and how intermediates are stabilized, and the processes that cause a polypeptide chain to misfold and/or aggregate rather than fold into the native state require direct experimental studies to gain new insights. The proposed research will address such outstanding issues through biophysical analyses of wild- type and disease-associated crystallin variants. Crystallins are ideally suited for detailed studies of protein aggregation: they are small; numerous X-ray structures are available; and the folding kinetics for several wild- type proteins to the native state have been investigated. NMR methods will be used to directly investigate folding transitions to obtain novel insights into the energetics of these processes and to elucidate structural details of the intermediates that cannot be obtained by any other methodologies. Our work will involve methods that allow detailed structural and dynamics characterization of proteins, primarily NMR spectroscopy and small angle X-ray scattering. In addition, we will correlate basic biophysical parameters with clinical observations. We plan to determine the three dimensional solution structures of cataract associated human ¿D-crystallins and characterize their dynamic behavior. We will initially focus on two important cataract forming ¿D-crystallin mutants, P23T and V75D. The former is associated with congenital cataracts in humans and the latter is a variant that has been identified to cause cataract in mice and, thus, will lend itself to follow-up studies in an animal model of cataract. We will also characterize the structure and dynamics of ¿D-crystallin folding intermediates. Further, we will investigate whether and how a previously identified, partially folded ¿D-crystallin intermediate causes aggregation. In particular, we will establish whether such partially folded intermediates are seeds for aggregation. This will prepare the basis for discovering small molecule inhibitors of aggregation, an approach that has already yielded some results in a number of neurodegenerative protein deposition diseases.