Engineering the independent folding of the subtilisin BPN' pro-domain: correlation of pro-domain stability with the rate of subtilisin folding.

The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions. The instability of the isolated pro-domain suggests that there may be a thermodynamic linkage between the stability of the pro-domain and its ability to facilitate subtilisin folding. On the basis of the X-ray crystal structure of the pro-domain subtilisin complex, we have designed stabilizing mutations in three areas of the pro-domain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-helix 53-61 (K57E). These amino acid positions were selected because they do not contact subtilisin in the complex and because they appear to be in regions of the structure which are not well packed in the wild type pro-domain. Since none of the mutations directly contact subtilisin, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes subtilisin folding. By sequentially introducing the three stabilizing mutations, the equilibrium for folding the pro-domain was shifted from 97% unfolded to 65% folded. By measuring the ability of these mutants to fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin folding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined for the double mutant, Q40L-K57E, which is 50% folded: P + Su if (30 800 M-1 s-1, 0.04 s-1) PSI if (0.07 s-1, <0.005 s-1) PS. PSI is an intermediate complex which accumulates in the course of the reaction, and PS is the fully folded complex. The more stable the pro-domain, the faster the folding reaction up to the point at which the isomerization of the intermediate into the fully folded complex becomes the rate-limiting step in the folding process.