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

摘要 眼晶状体晶体蛋白，它通过提供一个明确的 折射率梯度，是一组具有重要科学价值和医学价值的蛋白质。与之形成鲜明对比的是 大多数其他不断被降解和循环的蛋白质，晶体蛋白的含量很低 周转率，必须保持终身不变。考虑到它们的极高水平，这一点更加引人注目 凝聚力集中在镜头上。白内障是世界范围内导致失明的主要原因，当结构性白内障 晶体蛋白聚集或相分离，使晶状体不透明。随着时间的推移，蛋白质会发生降解 当晶体蛋白被化学修饰时，通常是通过去酰胺化或在被紫外线破坏时截断 光，或在糖尿病的情况下通过糖基化。此外，几个已知的点突变会导致遗传性 青少年发病的白内障。由于晶体蛋白在医学和生物物理方面的相关性，因此有必要 关于在自然状态和白内障中形成的大型复合体的详细结构信息- 相关聚合。晶体蛋白聚集性的分子水平表征 指导新的治疗策略的设计需要发展仪器和方法学。 这个项目的目标是阐明导致晶体蛋白聚集的分子基础 白内障形成。晶体蛋白的主要类型可分为结构型(b/g)和增溶型。 (A)。特定的分子靶标是GS-晶体蛋白，它是眼睛晶状体的主要结构成分，它的 与α-晶体蛋白伴侣的相互作用。将开发新的核磁共振方法来研究 结构因素与GS-晶体蛋白的稳定性和溶解性有关，主要是在固态。差示同位素 多肽结合体和变体晶体蛋白的标记可用于鉴定参与改变的晶体蛋白残基 并提供了初步的结构信息。我们设计并制作了一部小说 高场1H、13C、2H、15N固态核磁共振探头，可执行以前无法进行的2H检测实验 可用探头。在这一成功的基础上，利用这一独特仪器的新实验将 将被开发用于研究晶体蛋白聚集体和其他固体但高度流动的样品。我们将继续 利用固体核磁共振的最新进展来研究野生型GS-2的分子结构和动力学。 晶体蛋白在高浓度时，也是与人类先天性白内障相关的变异体的聚集体 由紫外线照射和金属阳离子结合形成的聚集体。G18V变种是一个开始 我们在健康和白内障状态下研究眼晶状体结构/功能关系的要点 蛋白质；然而，在项目的后期阶段，工作的重点将转移到与年龄有关的模型上。 白内障，这影响到更多的患者。对这些结构的阐明将提高我们对 如何形成白内障，并指导制定防治白内障的新策略。

Abstract The eye lens crystallins, which maintain the transparency of the eye lens by providing a well-defined gradient of refractive index, are a scientifically important and medically relevant group of proteins. In contrast to most other proteins, which are constantly subject to degradation and recycling, crystallins have very low turnover and must remain intact for a lifetime. This is even more remarkable considering their extremely high concentration in the lens. Cataract, a major cause of blindness worldwide, results when the structural crystallins aggregate or phase-separate, rendering the lens opaque. Over time, protein degradation 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. Furthermore, several known point mutations cause hereditary juvenile-onset cataracts. Because of the medical and biophysical relevance of crystallins, there is a need for detailed structural information about both the large complexes formed in the native state and in the cataract- related aggregates. Molecular-level characterization of crystallin aggregation at the level of detail required to guide the design of new therapeutic strategies requires the development of instrumentation and methodology. The objective of this project is to clarify the molecular basis of the crystallin aggregation that leads to cataract formation. The major types of crystallins can be categorized as either structural (b/g) or solubilizing (a). The specific molecular target is gS-crystallin, a major structural component of the eye lens, and its interactions with the α-crystallin chaperones. New NMR methodology will be developed to investigate the structural factors related to gS-crystallin stability and solubility, primarily in the solid state. 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. We have designed and built a novel high-field 1H,13C,2H,15N solid-state NMR probe to perform 2H-detected experiments not possible with previously available probes. Building on this success, new experiments that make use of this unique instrumentation will be developed to investigate crystallin aggregates and other solid but highly mobile samples. We will continue to utilize recent advances in solid-state NMR to investigate molecular structure and dynamics in wild-type gS- crystallin at high concentration, aggregates of variants associated with congenital cataracts in humans, as well as aggregates formed by UV irradiation and binding of metal cations. The G18V variant serves as a starting point for our investigations into structure/function relationships in the healthy and cataract states of eye lens proteins; however, in the later stages of the project, the focus of the work will shift to models for age-related cataract, which affects many more patients. Elucidation of these structures will improve our understanding of how cataract formation and guide the development of novel strategies for their prevention and treatment.