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

用于研究天然和聚集的眼晶状体蛋白的固态核磁共振方法

• 批准号: 8523892
• 负责人: Rachel Wagner Martin
• 金额: $ 30.18万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8523892
• 关键词: Address; Adolescent; Area; Binding; Biochemical; Blindness; Cataract; Complex; Crystalline Lens; Crystallins; Diabetes Mellitus; Disease; Engineering; Eye Lens Protein; Future; Human; Hydrogels; In Vitro; Induced Mutation; Inherited; Investigation; Isotope Labeling; Label; Lead; Lens Opacities; Measures; Medical; Methodology; Methods; Molecular; Molecular Structure; Molecular Target; Peptide Library; Peptides; Phage Display; Phase; Point Mutation; Precipitation; Prevention; Proteins; Proxy; Refractive Indices; Relaxation; S-crystallin; Sampling; Scheme; Solid; Solubility; Structural Models; Structural Protein; Structure; Structure-Activity Relationship; Techniques; Time; Ultraviolet Rays; Variant; age related; analytical method; base; combinatorial; congenital cataract; deamidation; design; fascinate; glycation; improved; inhibitor/antagonist; innovation; insight; instrumentation; intermolecular interaction; lens; novel; novel strategies; protein aggregation; protein degradation; protein misfolding; protein structure; research study; solid state nuclear magnetic resonance; success

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.

描述（由申请人提供）：通过提供明确定义的折射率梯度来维持眼镜的透明度，这是一种引人入胜且与医学上相关的蛋白质组。与大多数其他蛋白质不断降解的蛋白质相反，这些蛋白质的周转率很低，并且一生必须保持完整。考虑到它们在镜头中的浓度极高（超过400 mg/ml），这是更引人注目的。主要类型的结晶蛋白可以归类为结构（???）或溶解（？）。白内障是世界范围内失明的主要原因，当结构晶体骨料聚集或相分离时，导致了镜头的不透明度。随着时间的流逝，当结晶蛋白被化学修饰，通常是通过紫外线损伤或在糖尿病的情况下通过糖化而被脱胺或截断时，结晶蛋白的降解发生。除了与年龄相关的白内障外，几个已知的点突变引起了遗传性少年性白内障。尽管这些蛋白质具有医学和生物物理的相关性，但缺乏有关本地状态和白内障相关骨料中形成的大型复合物的详细结构信息。确定这些结构将需要新的生物物理和分析方法。 该建议的目的是阐明白内障形成的分子基础。特定的分子靶标是眼镜的主要结构成分。将开发新的方法，以研究与S-晶状体稳定性和溶解度相关的结构因素，主要是在固态NMR中。噬菌体显示器将用于识别特异性结合的肽变种的肽，并发现新的容易发生变体的变体。这些肽结合剂将用于进行完整结构确定的初步NMR实验。肽结合剂和变体晶体的差异同位素标记可用于鉴定涉及改变分子间相互作用的结晶蛋白残基并提供初步的结构信息。将设计和构建一种新型的高场1H，13C，2H，15N固态NMR探针，以便使用可用的探针进行目前无法进行2H检测的实验。将开发利用这种独特仪器的新实验来研究结晶蛋白聚集体和其他固体但高度移动的样品。我们还将利用固态NMR的最新进展来确定高浓度下的野生型？s-晶状体的分子结构，这是与人类中先天性白内障相关的G18V变体的聚集体，以及可能使用噬菌体显示的其他变体。 G18V变体已被证明具有生物学相关性，因此将作为我们研究眼镜蛋白健康和白内障态的结构/功能关系的起点。阐明这些结构将提高我们对白内障的理解，并可能导致预防和治疗的新策略。