RHODANESE FOLDING IS CONTROLLED BY THE PARTITIONING OF ITS FOLDING INTERMEDIATES

Rhodanese is used widely as a model for protein folding, since the enz yme as usually studied refolds poorly unless the process is assisted. Here, the influence of the partitioning of the folding intermediates o f bovine rhodanese on the efficiency of its refolding has been investi gated. Metastable intermediates can be formed during unfolding of the enzyme. The stabilities of these intermediates and the native protein with respect to chemical unfolding can be greatly increased by high co ncentrations of glycerol. The concentration dependence of the protein folding kinetics indicates that associative processes occur during ren aturation. It is suggested that, during enzyme refolding, rhodanese un dergoes fast collapse to an intermediate state I' which partitions to at least two other states (I '' and I'''). One of these states (I''') is able to refold to the native enzyme, while the other state (I '') i s in equilibrium with I' and is prone to slow irreversible aggregation Stabilization of I '' against irreversible aggregation by glycerol re sults in increased yield of the protein refolding and a complex temper ature dependence of the protein renaturation. The nature of the I '' t ype intermediate has been investigated. Based on the fact that extensi ve hydrophobic surfaces are exposed during formation of the intermedia tes, it is suggested that partial dissociation of the two structural d omains of rhodanese is an early event in unfolding. Interactions of di fferent folding intermediates of rhodanese with the chaperonin GroEL w ere investigated, and the results suggest that the more extensively un folded intermediates bind tighter than those that appear later on the rhodanese refolding pathway. (C) 1998 Elsevier Science B.V.