Functional Elements in Alpha Crystallin Chaperone

白内障是世界范围内致盲的主要原因之一， 眼透镜蛋白的修饰和聚集。α-晶体蛋白占近40% 成年透镜蛋白的结构和功能尚不清楚。的 认为α-晶状体蛋白的伴侣样活性在维持透镜 透明度我们提出以下具体目标，以增进我们对 a-晶状体蛋白及其亚基组织的伴侣蛋白功能，以满足我们的长期目标， 了解晶体蛋白的结构和功能。 1)确认aA-晶状体蛋白中的残基70-88和aB-晶状体蛋白中的残基73-92是 主要的伴侣位点确定aB-晶状体蛋白中的氨基酸序列（结合位点） 这有助于增强疏水性和伴侣蛋白功能在37 ℃以下 54-61序列缺失。2)鉴定p-和y-晶状体蛋白中的a-晶状体蛋白结合位点 在37 ° C的体外分子伴侣测定中。确定a-p和a-y中的相互作用位点 复合物在人透镜高分子量聚集体中的应用， 接头和质谱分析。3)确定方向偏好， ADH肽（YSGVCHTDLHAWHGDWPLPVK）在与aA- 晶体蛋白的定点荧光标记和淬灭研究。4)识别和 使用半胱氨酸表征aB-aB-、aB-aA-和aA-aA-晶状体蛋白相互作用位点 扫描诱变和化学修饰。我们计划实现这些具体目标 通过定点诱变研究和使用新的交联剂和质量 光谱法 了解α-晶体蛋白的结构及其作用机制，包括其 与其他透镜蛋白的相互作用，可能为我们提供更好的工具，以延迟或预防 白内障形成

Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the eye lens proteins. a-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 p- and y-crystallins during an in vitro chaperone assay at 37¿C. Identify the interaction sites in a-p and a-y 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.