Role of the C-terminal extensions of α-Crystallins

Abstract

Several small heat shock proteins contain a well conserved α-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of αA- and αB-crystallins, is not well understood. We have swapped the C-terminal extensions between αA- and αB-crystallins and generated two novel chimeric proteins, αABc and αBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric αB with the C-terminal extension of αA-crystallin, αBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric αA with the C-terminal extension of αB-crystallin, αABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that αBAc exhibits more solvent-exposed hydrophobic pockets than αA, αB, or αABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of αBAc differs from that of αB-crystallin whereas that of αABc overlaps with that of αA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.