Functional Elements in Alpha Crystallin Chaperone

产品说明：白内障是世界上致盲的主要原因之一，其发展是由于与年龄相关的眼透镜蛋白质的改变和聚集。α-晶体蛋白占成人透镜蛋白的近40%，但其结构-功能尚未完全了解。α-晶状体蛋白的分子伴侣样活性被认为在维持透镜透明度中起核心作用。我们提出了以下具体目标，以增加我们对α-晶体蛋白及其亚基组织的分子伴侣功能的理解，以满足我们理解α-晶体蛋白的结构-功能的长期目标。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），这些氨基酸序列在54-61序列缺失后有助于在37 ℃下增强疏水性和伴侣蛋白功能。2)鉴定？和？中的α-晶状体蛋白结合位点在37 ℃下体外分子伴侣试验期间的晶体蛋白。识别a-<$和a-中的相互作用位点？人透镜高分子量聚集体中的复合物，使用新型交联剂和质谱分析。3)通过荧光定点标记和猝灭研究，确定ADH肽（YSGVCHTDLHAWHGDWPLPVK）在与α A-晶状体蛋白相互作用过程中的方向偏好和取向。4)使用半胱氨酸扫描诱变和化学修饰鉴定和表征aB-aB-、aB-aA-和aA-aA-晶状体蛋白相互作用位点。我们计划通过定点诱变研究和使用新型交联剂和质谱方法来实现这些特定目标。了解α-晶状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来延迟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.