Reduced stability and enhanced surface hydrophobicity drive the binding of apo-aconitase with GroEL during chaperone assisted refolding.

Apo-aconitase, the Fe(4)S(4) cluster free form of TCA cycle enzyme aconitase, binds with GroEL and dissociates itself upon maturation through insertion of the cluster. It is not clearly established as to why apo-protein binds with GroEL. In order to explore the possibility that stability is a factor responsible for the aggregation of apo-form at low ionic strengths and hence it associates with GroEL to avoid the unfavorable event, we carried out the unfolding studies with holo- and apo-aconitase. By probing the unfolding process through the changes in secondary structural element, exposed surface hydrophobicity, and the microenvironment around tryptophan residues, we were able to establish the relevant changes associated with the event. Apparent guanidine hydrochloride concentration required for unfolding of 50% of aconitase indicates that aconitase is destabilized in the absence of the Fe(4)S(4) cluster. The destabilization of the apo-aconitase was further reflected through its three times higher rate of unfolding as compared to the holo-protein. It was also observed that the apo-form has higher surface hydrophobicity than the holo-form. Hence, the lower ground state stability and higher solvent exposed hydrophobic surface of the apo-form makes it aggregation prone. Based on the present observation and earlier findings, we propose that binding of apo-aconitase to GroEL not only rescues it from the aggregation, but also assists in the final stage of maturation by orienting the cluster insertion site of GroEL bound apo-protein. This information sheds new light on the potential role of GroEL in the biosynthetic pathway of the metallo proteins.