title: Introduction of disulfide bonds into Bacillus subtilis neutral protease.
abstract: The effects of engineered disulfide bonds on autodigestion and thermostability of Bacillus subtilis neutral protease (NP-sub) were studied using site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the introduction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fully active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermostability, which, by using reducing agents, was shown to be caused by the introduction of the cysteines and not by the formation of the disulfides. Mutants containing one cysteine exhibited intermolecular disulfide formation at elevated temperatures, which, however, was shown not to be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of disulfide bridges is unsuitable for the stabilization of proteases. Possible explanations for this phenomenon are discussed.
authors.affiliation: Department of Molecular Genetics, Centre for Biological Sciences, Haren, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: van den Burg
authors.forename: B W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: B
authors.initials: B
authors.lastname: van der Vinne
authors.forename: B K
authors.initials: BK
authors.lastname: Stulp
authors.forename: V G
authors.initials: VG
authors.lastname: Eijsink
authors.forename: G
authors.initials: G
authors.lastname: Venema
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics*; D003553:Cystine / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D003553:Cystine; D008666:Metalloendopeptidases; C071298:bacillolysin; D003545:Cysteine