The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains may regulate function; at the same time, the fact is also highlighted that surface hydrophobicity is a liability in a chaperone since heating strengthens hydrophobic interactions and can potentially promote self-aggregation. Thus, it would appear that the role of the N-terminal domain in alpha-crystallin is to facilitate the creation of a porous, hollow structural framework of greater than or equal to24 subunits in which solubility is effected through increase in the ratio of exposed surface area to buried volume. Trimers of interacting C-terminal domains anchored to this superstructure, and positioned within its interior, might allow hydrophobic surfaces to remain accessible to substrates without compromising solubility. (C) 2004 Elsevier Inc. All rights reserved.