Rhodanese folding is controlled by the partitioning of its folding intermediates.

Rhodanese is used widely as a model for protein folding, since the enzyme as usually studied refolds poorly unless the process is assisted. Here, the influence of the partitioning of the folding intermediates of bovine rhodanese on the efficiency of its refolding has been investigated. 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 concentrations of glycerol. The concentration dependence of the protein folding kinetics indicates that associative processes occur during renaturation. It is suggested that, during enzyme refolding, rhodanese undergoes 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") is in equilibrium with I' and is prone to slow irreversible aggregation. Stabilization of I" against irreversible aggregation by glycerol results in increased yield of the protein refolding and a complex temperature dependence of the protein renaturation. The nature of the I" type intermediate has been investigated. Based on the fact that extensive hydrophobic surfaces are exposed during formation of the intermediates, it is suggested that partial dissociation of the two structural domains of rhodanese is an early event in unfolding. Interactions of different folding intermediates of rhodanese with the chaperonin GroEL were investigated, and the results suggest that the more extensively unfolded intermediates bind tighter than those that appear later on the rhodanese refolding pathway.