Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant

In vitro folding of mature subtilisin is extremely slow. The isolated plo-d omain greatly accelerates in vitro folding of subtilisin in a bimolecular r eaction whose product is a tight complex between folded subtilisin and fold ed pro-domain. In our studies of subtilisin, we are trying to answer two ba sic questions: why does subtilisin fold slowly without the pro-domain and w hat does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants go verning individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro foldin g experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular fol ding reaction of subtilisin and proR9 occurs in two steps: an initial bindi ng of proRB to unfolded subtilisin, followed by isomerization of the initia l complex into the native complex. The central findings are as follows. Fir st, the independently stable proR9 folds subtilisin much faster than the pr edominantly unfolded wild-type pro-domain. Second, at micromolar concentrat ions of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native confo rmation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin. which form the initial complex with proR 9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the nat ive form slowly and the remaining 23% isomerizes mon rapidly (1.5 s(-1)). T he slow-folding population may be unfolded subtilisin with the trans form o f proline 168, which must isomerize to the cis form during refolding. Third , in the absence of proline isomerization, the rate of subtilisin folding i s rapid and at [proRB] less than or equal to 20 mu M is limited by the rate at which the proR9 forms a collision complex with unfolded subtilisin. Wit hout proline isomerization, the rate of the isomerization of the initial co llision complex to the folded complex is >3 s(-1). The implications of thes e results concerning: why subtilisin folds slowly without the prodomain are discussed.