Folding of subtilisin BPN': role of the pro-sequence.
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.
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
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Eder
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Rheinnecker
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Fersht
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005798:Genes, Bacterial; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D004269:DNA, Bacterial; D004792:Enzyme Precursors; D006146:Guanidines; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C057107:prosubtilisin; D013381:Subtilisins; D019791:Guanidine