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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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的晶状体蛋白，通过提供明确的边界来保持晶状体的透明度折射率梯度，是一组具有科学意义和医学相关性的蛋白质。相比之下大多数其他蛋白质不断受到降解和回收，但晶状体蛋白的含量非常低周转，并且必须终生保持完好无损。考虑到其极高的镜头内的浓度。白内障是全世界失明的主要原因，当结构性失明时就会导致白内障晶状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状体蛋白被化学修饰时，通常通过脱酰胺或被紫外线损坏时截短光，或者在糖尿病的情况下通过糖化。此外，一些已知的点突变会导致遗传性青少年发病的白内障。由于晶状体蛋白的医学和生物物理相关性，因此需要有关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。在所需细节水平上对晶状体蛋白聚集进行分子水平表征指导新治疗策略的设计需要仪器和方法的开发。该项目的目的是阐明导致晶状体蛋白聚集的分子基础白内障的形成。晶状体蛋白的主要类型可分为结构型 (b/g) 或增溶型（一个）。特定的分子靶点是 gS-晶状体蛋白，它是眼晶状体的主要结构成分，其与α-晶状体蛋白伴侣的相互作用。将开发新的核磁共振方法来研究与 gS-晶状体蛋白稳定性和溶解度（主要是固态）相关的结构因素。差别同位素肽结合物和变体晶状体蛋白的标记可用于鉴定参与改变的晶状体蛋白残基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部小说高场 1H、13C、2H、15N 固态 NMR 探针可进行 2H 检测实验，这是以前不可能实现的可用的探头。在此成功的基础上，利用这种独特仪器的新实验将可用于研究晶状体蛋白聚集体和其他固体但高度流动的样品。我们将继续利用固态核磁共振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晶状体蛋白，以及与人类先天性白内障相关的变异体的聚集作为通过紫外线照射和金属阳离子结合形成的聚集体。 G18V 变体作为起始我们研究眼睛晶状体健康和白内障状态下的结构/功能关系的要点蛋白质；然而，在项目的后期阶段，工作重点将转向与年龄相关的模型白内障，影响着更多的患者。对这些结构的阐明将加深我们对白内障是如何形成的，并指导制定预防和治疗白内障的新策略。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.

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。