Structure/function analysis of alpha A crystallins

描述：脊椎动物小热休克蛋白α -晶体蛋白通过结合变性蛋白并阻止其聚集来帮助预防晶状体混浊（白内障）。许多研究试图确定影响这种伴侣样活性的a-晶体蛋白的结构特征。比较自然进化的a-结晶蛋白和其他来自不同物种的小热休克蛋白对我们对a-结晶蛋白结构的理解有很大贡献，但直到最近，还没有研究比较非哺乳动物脊椎动物的伴侣蛋白样活性。重要证据表明，aa -晶体蛋白在不同生理温度的鱼类之间表现出不同的伴侣能力。这种伴侣蛋白样活性的变化反映在氨基酸序列的替换上，这可能决定了这些aa -晶体蛋白与非天然蛋白结合方式的差异。在本研究中，多种生理温度不同的硬骨鱼将被用作模型组，以确定影响aA-crystallin阻止蛋白质聚集能力的氨基酸变异。具体而言，将对生理温度为-2℃至42℃的6个物种的α -结晶蛋白基因进行克隆和测序。将对推断出的氨基酸序列进行比对，以确定物种之间的氨基酸差异。克隆的基因将被用来制造重组的α -晶体蛋白，在15度到40度的温度下检测它们的伴侣样活性。氨基酸序列的变化将与伴侣样活性的变化相关联，以确定那些可能影响α -晶体蛋白结合非天然蛋白质的能力的氨基酸。这项研究将通过确定参与抑制蛋白质聚集的特定氨基酸，增加我们对α -晶体蛋白在预防白内障中的作用的理解。这些数据将为未来的定点诱变研究提供基础，这些研究可以直接测试已鉴定的氨基酸取代对伴侣蛋白样活性的影响。

DESCRIPTION: The vertebrate small heat shock protein alphaA-crystallin helps prevent lens opacity (cataract) by binding to denaturing proteins and preventing their aggregation. Many studies have attempted to identify the structural features of a-crystallins that influence this chaperone-like activity. Comparisons of naturally evolved a-crystallins and other small heat shock proteins from diverse species have contributed greatly to our understanding of a-crystallin structure, but until recently no studies had compared chaperone-like activity from non-mammalian vertebrates. Significant evidence points to aA-crystallin exhibiting different chaperone capabilities between fish species with different physiological temperatures. This variation in chaperone-like activity is reflected in amino acid sequence substitutions, which are likely to dictate the differences in how these aA-crystallins bind non-native protein. In this study, multiple bony fish species differing in physiological temperature will be used as a model group to identify amino acid variations that affect aA-crystallin's ability to prevent protein aggregation. Specifically, the alphaA-crystallin genes from six species ranging in physiological temperature from -2 degrees to 42 degrees C will be cloned and sequenced. Deduced amino acid sequences will be aligned to identify amino acid differences between the species. The cloned genes will be used to make recombinant alphaA-crystallins that will be assayed for their chaperone-like activity at temperatures from 15 degrees to 40 degrees C. Changes in amino acid sequence will be correlated with changes in chaperone-like activity to identify those amino acids that could affect alphaA-crystallin's ability to bind non-native proteins. This study will add to our understanding of alphaA-crystallin's role in preventing cataract by identifying specific amino acids involved in the suppression of protein aggregation. These data will provide the foundation for future site-directed mutagenesis studies that could directly test the effect of identified amino acid substitutions on chaperone-like activity.