Solid-state NMR methods for investigating native and aggregated eye lens proteins

描述（由申请人提供）：晶体蛋白通过提供明确定义的折射率梯度来保持晶状体的透明度，是一组迷人且与医学相关的蛋白质。与大多数其他不断降解的蛋白质不同，这些蛋白质的周转率非常低，必须在一生中保持完整。考虑到它们在镜片中的浓度极高（超过400毫克/毫升），这一点就更加引人注目了。晶体蛋白的主要类型可分为结构型（??）和溶解型（??）。白内障是世界范围内致盲的一个主要原因，当晶体蛋白聚集或相分离，导致晶状体混浊。随着时间的推移，当晶体蛋白发生化学修饰时，就会发生降解，通常是在紫外线照射下被脱酰胺或截断，或者在糖尿病的情况下被糖基化。除了与年龄有关的白内障外，一些已知的点突变可引起遗传性青少年白内障。尽管这些蛋白具有医学和生物物理上的相关性，但缺乏关于天然状态下形成的大复合物和白内障相关聚集体的详细结构信息。确定这些结构需要新的生物物理和分析方法。本建议的目的是阐明白内障形成的分子基础。具体的分子靶点是？晶状体的主要结构成分。将开发新的方法，以调查与？s -结晶蛋白的稳定性和溶解度，主要在固态核磁共振。噬菌体展示将用于识别特异性结合错误折叠的晶体蛋白变体的肽，并发现新的易于聚集的变体本身。这些肽结合剂将用于初步的核磁共振实验，以确定完整的结构。肽结合物和变异结晶蛋白的差异同位素标记可用于鉴定参与改变的分子间相互作用的结晶蛋白残基，并提供初步的结构信息。一种新型的高场1H，13C,2H，15N固态核磁共振探针将被设计和建造，以执行目前无法用现有探针检测2H的实验。利用这种独特仪器的新实验将被开发用于研究结晶蛋白聚集体和其他固体但高流动性的样品。我们还将利用固态核磁共振的最新进展来确定野生型的分子结构？高浓度的s -晶体蛋白，与人类先天性白内障相关的G18V变体的聚集体，以及使用噬菌体展示发现的潜在其他变体。G18V变异已被证明具有生物学相关性，因此将作为我们研究健康和白内障状态下晶状体蛋白结构/功能关系的起点。阐明这些结构将提高我们对白内障形成的理解，并可能为其预防和治疗提供新的策略。

DESCRIPTION (provided by applicant): The crystallins, which maintain the transparency of the eye lens by providing a well-defined gradient of refractive index, are a fascinating and medically relevant group of proteins. In contrast to most other proteins, which are constantly subject to degradation, these proteins have very low turnover and must remain intact for a lifetime. This is even more remarkable considering their extremely high concentration in the lens (more than 400 mg/mL). The major types of crystallins can be categorized as either structural (???) or solubilizing (?). Cataract, a major cause of blindness worldwide, results when the structural crystallins aggregate or phase- separate, leading to opacity of the lens. Over time, degradation of the crystallins occurs when the crystallins become chemically modified, often by deamidation or truncation when damaged by UV light, or by glycation in the case of diabetes. In addition to age-related cataracts, several known point mutations cause hereditary juvenile-onset cataracts. Despite the medical and biophysical relevance of these proteins, there is a lack of detailed structural information about both the large complexes formed in the native state and in the cataract- related aggregates. Determining these structures will require new biophysical and analytical methods. The objective of this proposal is to clarify the molecular basis of cataract formation. The specific molecular target is ?S-crystallin, a major structural component of the eye lens. New methodology will be developed in order to investigate the structural factors related to ?S-crystallin stability and solubility, primarily in solid-state NMR. Phage display will be used to identify peptides that specifically bind to misfolded crystallin variants and to discover new aggregation-prone variants themselves. These peptide binders will be used in preliminary NMR experiments along the way to full structure determination. Differential isotope labeling of peptide binders and variant crystallins can be used to identify crystallin residues involved in altered intermolecular interactions and provide preliminary structural information. A novel high-field 1H,13C,2H,15N solid- state NMR probe will be designed and built in order to perform 2H-detected experiments currently not possible with available probes. New experiments taking advantage of this unique instrumentation will be developed to investigate crystallin aggregates and other solid but highly mobile samples. We will also make use of recent advances in solid-state NMR to determine the molecular structures of wild-type ?S-crystallin at high concentration, aggregates of the G18V variant associated with congenital cataracts in humans, and potentially other variants found using phage display. The G18V variant has been demonstrated to be biologically relevant and therefore will serve as a starting point for our investigations into structure/function relationships in the healthy and cataract states of eye lens proteins. Elucidation of these structures will improve our understanding of how cataracts form and may lead to novel strategies for their prevention and treatment.