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: 8708869
• 负责人: Rachel Wagner Martin
• 金额: $ 30.8万
• 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/8708869
• 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; biophysical techniques; 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）：晶体蛋白通过提供明确定义的折射率梯度来保持眼睛透镜的透明度，是一组迷人的医学相关蛋白质。与大多数其他不断降解的蛋白质相比，这些蛋白质的周转率非常低，必须在一生中保持完整。考虑到它们在透镜中的极高浓度（超过400 mg/mL），这一点甚至更加显著。晶体蛋白的主要类型可以分为结构型（？）或增溶（？）。白内障是世界范围内致盲的主要原因，其产生于结构性晶体蛋白聚集或相分离，导致透镜不透明。随着时间的推移，当晶状体蛋白被化学修饰时，晶状体蛋白发生降解，通常是在被UV光损坏时通过脱酰胺或截短，或者在糖尿病的情况下通过糖化。除了与年龄相关的白内障，几个已知的点突变导致遗传性青少年发病的白内障。尽管这些蛋白质具有医学和生物物理相关性，但缺乏关于天然状态下和白内障相关聚集体中形成的大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。 本提案的目的是阐明白内障形成的分子基础。具体的分子靶点是？S-晶状体蛋白，眼透镜的主要结构成分。新的方法将被开发，以调查相关的结构因素？S-晶状体蛋白的稳定性和溶解度，主要在固态NMR中。噬菌体展示将用于鉴定特异性结合错误折叠的晶状体蛋白变体的肽，并发现新的易于聚集的变体。这些肽结合剂将用于初步的NMR实验，沿着的方式，以全面的结构测定。肽结合剂和变体晶状体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晶状体蛋白残基并提供初步结构信息。将设计和建造一种新型高场1H，13 C，2 H，15 N固态NMR探针，以进行目前用现有探针无法进行的2 H检测实验。新的实验利用这种独特的仪器将开发调查晶体蛋白聚集体和其他固体，但高度移动的样品。我们还将利用固态核磁共振的最新进展，以确定野生型？高浓度的S-晶状体蛋白，与人类先天性白内障相关的G18 V变体的聚集体，以及使用噬菌体展示发现的潜在其他变体。G18 V变异体已被证明是生物学相关的，因此将作为我们的调查在健康和白内障状态的眼睛透镜蛋白的结构/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，并可能导致新的预防和治疗策略。