Domain swapping in human αA and αB crystallins affects oligomerization and enhances chaperone-like activity

Abstract

αA and αB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "α-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between αA and αB crystallins, we engineered two chimeric proteins, αANBC and αBNAC, by swapping the N-terminal domains of αA and αB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. αBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the αANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of αANBC is comparable to wild-type αB homoaggregate. However, there is a large increase in the oligomer size of the αBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of αANBC, whereas αBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of αA and αB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.