Domain swapping in human alpha A and alpha B crystalline affects oligomerisation and enhances chaperone-like activity

alpha A and alpha B crystalline, members of the small heat shock protein fa mily, prevent aggregation of proteins by their chaperone-like activity. The se two proteins, although very homologous, particularly in the C-terminal r egion, which contains the highly conserved "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target pro teins. In order to investigate the differences between alpha A and alpha B crystallins, we engineered two chimeric proteins, alpha ANBC and alpha BNAC , by swapping the N-terminal domains of alpha A and alpha B crystallins. Th e chimeras were cloned and expressed in Escherichia coli, The purified reco mbinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to st udy the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins, alpha B NAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alpha ANBC and the wild-type proteins, indicating increased accessible hydrophobic region s. The oligomeric state of alpha ANBC is comparable to wild-type alpha B ho moaggregate. However, there is a large increase in the oligomer size of the alpha BNAC chimera. Interestingly, swapping domains results in complete lo ss of chaperone-like activity of alpha ANBC, whereas alpha BNAC shows sever alfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alpha A and alpha B crystallins in sub unit oligomerization and chaperone-like activity. Domain swapping results i n an engineered protein with significantly enhanced chaperone-like activity .