Subunit exchange of small heat shock proteins - Analysis of oligomer formation of alpha A-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations

alpha A-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30-40 identical subunits. Its qu aternary structure is highly dynamic, with subunits capable of freely and r apidly exchanging between oligomers. We report here the development of a fl uorescence resonance energy transfer method for measuring structural compat ibility between alpha A-crystallin and other proteins. We found that Hsp27 and alpha B-crystallin readily exchanged with fluorescence-labeled alpha A- crystallin, but not with other proteins structurally unrelated to sHsps. Tr uncation of 19 residues from the N terminus or 10 residues from the C termi nus of alpha A-crystallin did not significantly change its subunit organiza tion or exchange rate constant. In contrast, removal of the first 56 or mor e residues converts alpha A-crystallin into a predominantly small multimeri c form consisting of three or four subunits, with a concomitant loss of exc hange activity. These findings suggest residues 20-56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We furth er showed that the exchange rate is independent of alpha A-crystallin conce ntration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of alpha A-crystallin, consisting of small multimers of alpha A-crystallin subunits in a dynamic equilibrium with the oligomeric complex.