Functional Elements in Alpha Crystallin Chaperone

Alpha Crystallin Chaperone 中的功能元素

• 批准号: 7213275
• 负责人: KRISHNA K SHARMA
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7213275
• 关键词: Accounting; Adult; Alcohol dehydrogenase; All Sites; Amino Acid Sequence; Binding; Binding Sites; Biological Assay; Blindness; Cataract; Chemicals; Complex; Crystallins; Cysteine; Event; Eye Lens Protein; Fluorescence; Goals; Grant; Heating; Human; Hydrophobicity; In Vitro; Indium; Investigation; Knowledge; Label; Methods; Modification; Molecular; Molecular Chaperones; Molecular Weight; Mutagenesis; Numbers; Peptides; Physiological; Play; Progress Reports; Proteins; Research Personnel; Role; Scanning; Site; Site-Directed Mutagenesis; Structure; Temperature; Water; age related; alpha-Crystallins; insight; lens; lens protein; lens transparency; macromolecule; mutant; novel; orientation selectivity; prevent; programs; tool

DESCRIPTION: Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the eye lens proteins. Alpha-Crystallin accounts for nearly 40% of the adult lens proteins but its structure-function is yet to be fully understood. The chaperone-like activity of a-crystallin is believed to play a central role in maintaining lens transparency. We propose the following specific aims to increase our understanding of chaperone function of a-crystallin and its subunit organization to meet our long-term goal of understanding structure-function of a-crystallin. 1) Confirm that residues 70-88 in aA-crystallin and residues 73-92 in aB-crystallin are the major chaperone sites. Determine the amino acid sequences (binding site) in aB-crystallin that contribute to the enhanced hydrophobicity and chaperone function at 37¿C following deletion of 54-61 sequence. 2) Identify the a-crystallin binding site(s) in ¿- and ?-crystallins during an in vitro chaperone assay at 37¿C. Identify the interaction sites in a-¿ and a-? complexes in human lens high-molecular-weight aggregates with the use of novel cross- linkers and mass spectrometric analysis. 3) Determine the directional preference and orientation of ADH peptide (YSGVCHTDLHAWHGDWPLPVK) during its interaction with aA- crystallin by site-directed fluorescence labeling and quenching studies. 4) Identify and characterize the aB-aB-; aB-aA- and aA-aA- crystallin interaction sites using cysteine scanning mutagenesis and chemical modification. We plan to accomplish these specific aims by site-directed mutagenesis studies and the use of novel cross-linkers and mass spectrometric methods. Understanding the structure of a-crystallin and its mechanisms of its action, including its interaction with other lens proteins, is likely to provide us better tools to delay or prevent cataractogenesis.

描述：白内障是世界上导致失明的主要原因之一，它是由于年龄相关的修饰和眼晶状体蛋白质的聚集而发展的。Alpha-Crystallin占成体晶状体蛋白的近40%，但其结构和功能尚不完全清楚。A-晶体蛋白的伴侣样活性被认为在维持晶状体透明度方面起着核心作用。为了更好地理解α-晶体蛋白的伴侣功能及其亚基结构，我们提出了以下具体目标，以满足我们理解α-晶体蛋白结构与功能的长期目标。1)确定AA-晶状体蛋白中的70-88位残基和AB-晶状体蛋白中的73-92位残基是主要的伴侣位点。确定AB-晶体蛋白中的氨基酸序列(结合部位)，这些氨基酸序列有助于在54-61序列缺失后，在37℃增强疏水性和伴侣功能。2)在37℃的体外伴侣实验中，确定α-晶状体蛋白和β-晶状体蛋白中的α-晶状体蛋白结合位点(S)。利用新型交联剂和质谱学分析人晶状体中的高分子量聚集体。3)通过定点荧光标记和猝灭研究，确定ADH多肽(YSGVCHTDLHAWHGDWPLPVK)在与AA-晶体蛋白相互作用过程中的定向偏好和定向。4)通过半胱氨酸扫描诱变和化学修饰，鉴定和表征了AB-AB-、AB-AA-和AA-AA-晶状体蛋白相互作用部位。我们计划通过定点突变研究和使用新的交联剂和质谱学方法来实现这些特定的目标。了解α-晶状体蛋白的结构及其作用机制，包括它与其他晶状体蛋白的相互作用，可能会为我们提供更好的工具来延缓或预防白内障的发生。