Subunit Exchange of Small Heat Shock Proteins

Abstract

αA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30–40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between αA-crystallin and other proteins. We found that Hsp27 and αB-crystallin readily exchanged with fluorescence-labeled αA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of αA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts αA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange 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 further showed that the exchange rate is independent of αA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of αA-crystallin, consisting of small multimers of αA-crystallin subunits in a dynamic equilibrium with the oligomeric complex.