SUBUNIT EXCHANGE OF ALPHA-A-CRYSTALLIN

alpha-Crystallin, the major protein in the mammalian lens, is a molecu lar chaperone that can bind denaturing proteins and prevent their aggr egation, Like other structurally related small heat shock proteins, ea ch alpha-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluor escence resonance energy transfer to monitor the rapid exchange of rec ombinant cu-crystallin subunits. We labeled alpha A-crystallin with st ilbene iodoacetamide ido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfoni c acid), which serves as an energy donor and with lucifer yellow iodoa cetamide, which serves as an energy acceptor. Upon mixing the two popu lations of labeled alpha A-crystallin, we observed a reversible, time- dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence, Thi s result is indicative of an exchange reaction that brings the fluores cent alpha A-crystallin subunits close to each other. We further showe d that the exchange reaction is strongly dependent on temperature, wit h a rate constant of 0.075 min(-1) at 37 degrees C and an activation e nergy of 60 kcal/mol, The subunit exchange is independent of pH and ca lcium concentration but decreases at low and high ionic strength, sugg esting the involvement of both ionic and hydrophobic interactions, It is also markedly reduced by the binding of large denatured proteins, T he degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of sev eral alpha A-crystallin subunits with multiple binding sites of the de naturing protein. Our findings reveal a dynamic organization of alpha A-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.