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

描述(申请人提供)：晶体蛋白，通过提供一个明确的折射率梯度来维持眼晶状体的透明度，是一组迷人的和医学上相关的蛋白质。与大多数经常被降解的其他蛋白质不同，这些蛋白质的周转率非常低，必须保持完好无损。考虑到它们在晶状体中的极高浓度(超过400毫克/毫升)，这一点更加值得注意。晶体蛋白的主要类型可分为结构(？)或增溶(？)。白内障是世界范围内导致失明的主要原因，当结构晶体蛋白聚集或相分离时，会导致晶状体混浊。随着时间的推移，当晶体蛋白被化学修饰时，晶体蛋白就会降解，通常是在紫外线破坏时通过去酰胺化或截断，或者在糖尿病的情况下通过糖基化。除了年龄相关性白内障，几个已知的点突变还会导致遗传性少年性白内障。尽管这些蛋白质与医学和生物物理有关，但缺乏关于天然状态下形成的大型复合体和与白内障相关的聚集体的详细结构信息。确定这些结构将需要新的生物物理和分析方法。这项建议的目的是阐明白内障形成的分子基础。特定的分子靶点是S-晶体蛋白，它是眼晶状体的主要结构成分。将开发新的方法来研究与S晶体蛋白稳定性和溶解性有关的结构因素，主要是在固体核磁共振中。噬菌体展示将用于识别与错误折叠的晶体蛋白变体特异结合的多肽，并发现新的易于聚集的变体本身。这些多肽结合剂将用于初步的核磁共振实验，从而获得完整的结构。多肽结合体和不同晶体蛋白的差异同位素标记可用于鉴定参与分子间相互作用改变的晶体蛋白残基，并提供初步的结构信息。将设计和制造一种新型的高场1H，13C，2H，15N固态核磁共振探头，以执行目前用现有探头无法进行的2H检测实验。利用这一独特仪器的新实验将被开发来研究晶体蛋白聚集体和其他固体但高度流动的样品。我们还将利用固体核磁共振的最新进展来确定高浓度的野生型S晶体蛋白、与人类先天性白内障相关的G18V变异体的聚集体，以及可能使用噬菌体展示技术发现的其他变异体的分子结构。G18V变异体已被证明具有生物学相关性，因此将作为我们研究眼晶状体蛋白在健康和白内障状态下的结构/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，并可能导致预防和治疗的新策略。

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.