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 compl ex between folded subtilisin and folded pro-domain. In this complex th e pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subti lisin, however, the pro-domain is 97% unfolded even under optimal fold ing conditions. The instability of the isolated pro-domain suggests th at there maybe a thermodynamic linkage between the stability of the pr o-domain and its ability to facilitate subtilisin folding. On the basi s of the X-ray crystal structure of the pro-domain subtilisin complex, we have, designed stabilizing mutations in three areas of the pro-dom ain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-he lix 53-61 (K57E). These amino acid positions were selected because the y 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 t ype pro-domain. Since none of the mutations directly contact subtilisi n, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes su btilisin folding. By sequentially introducing the three stabilizing mu tations, the equilibrium for folding the pro-domain was shifted from 9 7% unfolded to 65% folded. By-measuring the ability of these mutants t o fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin f olding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined fo r the double mutant, Q40L-K57E, which is 50% folded: P + S-u double le ft right arrow (30800 M-1 s(-1), 0.04 s(-1)) PS1 double left right arr ow (0.07 s(-1), <0.005 s(-1)) PS. PS1 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 react ion up to the point at which the isomerization of the intermediate int o the fully folded complex becomes the rate-limiting step in the foldi ng process.