Abstract
AlphaB-crystallin is a member of the small heat shock protein family and is known to have chaperone activity. Using a peptide scan approach, we previously determined that regions 42-57, 60-71, and 88-123 in alphaB-crystallin interact with alphaA-crystallin during heterooligomer formation. To further characterize the significance of the N-terminal domain of alphaB-crystallin, we prepared a deletion mutant that lacks residues (54)FLRAPSWF(61) (alphaBDelta54-61) and found that the absence of residues 54-61 in alphaB-crystallin significantly decreased the homooligomeric mass of alphaB-crystallin. The average oligomeric mass of wild-type alphaB-crystallin and of alphaBDelta54-61, calculated using multiangle light scattering, was 624 and 382 kDa, respectively. The mutant subunits aggregate to form smaller, less-compact oligomers with a 4-fold increase in subunit exchange rate. Deletion of the 54-61 region resulted in a 50% decrease in intrinsic tryptophan fluorescence. The alphaBDelta54-61 mutant showed a 2-fold increase in 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding as compared to the wild-type protein, suggesting increased hydrophobicity of the mutant protein. Accompanying the evidence of increased hydrophobicity in the deletion mutant was a 10-fold increase in antiaggregation activity. Homooligomers of 6HalphaA (750 kDa) readily exchanged subunits with alphaBDelta54-61 homooligomers at 37 degrees C, forming heterooligomers with an intermediate mass of 625 kDa. Our data suggest that residues (54)FLRAPSWF(61) contribute to the higher order assembly of alphaB-crystallin oligomers. Residues (54)FLRAPSWF(61) in alphaB-crystallin are not essential for target protein binding during chaperone action, but this region apparently has a role in the chaperone activity of native alphaB-crystallin.