Functional Elements in Alpha Crystallin Chaperone

白内障是世界上致盲的主要原因，是由于年龄相关的改变和眼球晶状体蛋白的聚集而发展起来的。α -晶体蛋白占成人晶状体蛋白的近40%，但其结构功能尚不完全清楚。a-晶体蛋白的伴侣样活性被认为在保持晶状体透明度方面起着核心作用。我们提出了以下具体目标，以增加我们对a-crystallin的伴侣功能及其亚基组织的理解，以实现我们了解a-crystallin结构-功能的长期目标。1)确认aA-crystallin残基70-88和aB-crystallin残基73-92是主要的伴侣蛋白位点。测定aB-crystallin中在37°C缺失54-61序列后疏水性和伴侣功能增强的氨基酸序列（结合位点）。2)确定a-晶体蛋白结合位点在¿-和？在37℃的体外伴侣蛋白测定中。找出a-¿和a-?中的交互点。配合物在人体晶状体高分子量聚集体与使用新的交联剂和质谱分析。3)通过定点荧光标记和猝灭研究确定ADH肽（YSGVCHTDLHAWHGDWPLPVK）与aA-结晶蛋白相互作用过程中的方向偏好和取向。4)识别和表征aB-aB-；利用半胱氨酸扫描诱变和化学修饰的aB-aA-和aA-aA-晶体蛋白相互作用位点。我们计划通过位点定向诱变研究和使用新的交联剂和质谱方法来实现这些特定的目标。了解a-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，可能为我们提供更好的工具来延缓或预防白内障的发生。

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.