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.
