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: 10371986
• 负责人: Rachel Wagner Martin
• 金额: $ 30.25万
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371986
• 关键词: Address; Adolescent; Affect; Area; Back; Binding; Biophysics; Blindness; Cataract; Cations; Chemicals; Complex; Crystalline Lens; Crystallins; Data; Deposition; Development; Diabetes Mellitus; Disease; Divalent Cations; Engineering; Eye Lens Protein; Future; Human; Hydrogels; Inherited; Investigation; Ions; Isotope Labeling; Label; Medical; Metals; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Structure; Molecular Target; Mutation; Patients; Peptides; Phase; Point Mutation; Prevention; Proteins; Publishing; Recycling; Refractive Indices; Relaxation; Sampling; Scheme; Solid; Solubility; Solvents; Structural Models; Structural Protein; Structure; Structure-Activity Relationship; Techniques; Time; Ultraviolet Rays; Variant; Work; age related; alpha-Crystallins; biophysical techniques; congenital cataract; deamidation; design; experimental study; glycation; improved; inhibitor; innovation; insight; instrumentation; intermolecular interaction; lens; lens cortex; molecular dynamics; monomer; novel; novel strategies; novel therapeutic intervention; prevent; protein degradation; protein structure; solid state; solid state nuclear magnetic resonance; success; ultraviolet damage; ultraviolet irradiation

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.

抽象的 眼镜透镜结晶蛋白，通过提供明确的定义，可以保持眼镜的透明度 折射率的梯度是科学重要且与医学相关的蛋白质群体。与 大多数其他蛋白质不断受到降解和回收利用，结晶蛋白的蛋白质非常低 营业额，必须保持完整一生。考虑到他们的高度，这更为显着 透镜中的浓度。白内障是世界范围内失明的主要原因，当结构性 结晶蛋白聚集或分离，使镜头不透明。随着时间的流逝，蛋白质降解发生 当结晶蛋白被化学修饰时，经常被紫外线损坏时死亡或截断 在糖尿病的情况下，光或通过糖糖化。此外，几个已知点突变导致世袭 少年性白内障。由于结晶蛋白的医学和生物物理相关性，因此需要 有关本地状态和白内障中形成的大型复合物的详细结构信息 相关聚集体。在所需的细节水平上，结晶蛋白聚集的分子水平表征 指导新的治疗策略的设计需要开发仪器和方法论。 该项目的目的是阐明导致结晶蛋白聚集的分子基础 白内障形成。主要类型的结晶蛋白可以归类为结构（b/g）或可溶性化 （一个）。特定的分子靶标是GS-晶状体，这是眼镜的主要结构成分，其 与α-晶状体蛋白伴侣的相互作用。将开发新的NMR方法来研究 与GS-结晶蛋白稳定性和溶解度有关的结构因素，主要是固态。差异同位素 胡椒粘合剂和变体晶体的标记可用于识别与改变有关的结晶蛋白保留 分子间相互作用并提供初步的结构信息。我们设计并建造了一本小说 高场1H，13C，2H，15N固态NMR探针以前无法执行2H检测的实验 可用问题。在这一成功的基础上，利用这种独特仪器的新实验将 开发以研究结晶蛋白聚集体和其他固体但高度移动的样品。我们将继续 利用固态NMR的最新进展来研究野生型GS-的分子结构和动力学 高浓度的结晶蛋白，与人类先天性白内障相关的变体的聚集体 作为由紫外线照射和金属阳离子结合形成的聚集体。 G18V变体可作为起始 我们对健康和白内障状态的结构/功能关系的投资点 蛋白质；但是，在项目的后期阶段，工作的重点将转向与年龄相关的模型 白内障，会影响更多患者。阐明这些结构将提高我们对 白内障的形成和指导新型策略的预防和治疗方式。

项目成果

Automated test apparatus for bench-testing the magnetic field homogeneity of NMR transceiver coils.

用于对 NMR 收发器线圈的磁场均匀性进行台架测试的自动测试装置。

• DOI: 10.1016/j.jmro.2023.100142
• 发表时间: 2024
• 期刊: Journal of magnetic resonance open
• 作者: Uribe,JoseL;Jimenez,MatthewD;Kelz,JessicaI;Liang,Jeanie;Martin,RachelW
• 通讯作者: Martin,RachelW

Spatial reorientation experiments for NMR of solids and partially oriented liquids.

固体和部分取向液体的核磁共振空间重新取向实验。

• DOI: 10.1016/j.pnmrs.2015.10.001
• 发表时间: 2015
• 期刊: Progress in nuclear magnetic resonance spectroscopy
• 影响因子: 6.1
• 作者: Martin,RachelW;Kelly,JohnE;Collier,KelseyA
• 通讯作者: Collier,KelseyA

γS-crystallin proteins from the Antarctic nototheniid toothfish: a model system for investigating differential resistance to chemical and thermal denaturation.

• DOI: 10.1021/jp509134d
• 发表时间: 2014-11-26
• 期刊: The journal of physical chemistry. B
• 作者: Kingsley CN;Bierma JC;Pham V;Martin RW
• 通讯作者: Martin RW

Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.

• DOI: 10.1016/j.str.2022.03.002
• 发表时间: 2022-05-05
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Norton-Baker, Brenna;Mehrabi, Pedram;Kwok, Ashley O.;Roskamp, Kyle W.;Rocha, Megan A.;Sprague-Piercy, Marc A.;von Stetten, David;Miller, R. J. Dwayne;Martin, Rachel W.
• 通讯作者: Martin, Rachel W.

Function and Aggregation in Structural Eye Lens Crystallins

• DOI: 10.1021/acs.accounts.0c00014
• 发表时间: 2020-04-21
• 期刊: ACCOUNTS OF CHEMICAL RESEARCH
• 影响因子: 18.3
• 作者: Roskamp, Kyle W.;Paulson, Carolyn N.;Martin, Rachel W.
• 通讯作者: Martin, Rachel W.