Folding of Subtilisin BPN': Role of the Pro-sequence

Journal of Molecular Biology(1993)

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摘要
Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7·5 kcal/mol; folding is accelerated more than five orders of magnitude.
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关键词
folding intermediate,serine protease,protein folding
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