title: Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.
abstract: The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biotechnology, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Mansfeld
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: B W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: O R
authors.initials: OR
authors.lastname: Veltman
authors.forename: B
authors.initials: B
authors.lastname: Van den Burg
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: R
authors.initials: R
authors.lastname: Ulbrich-Hofmann
authors.forename: V G
authors.initials: VG
authors.lastname: Eijsink
mesh_terms: D004220:Disulfides; D010450:Endopeptidases / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006207:Half-Life; D006358:Hot Temperature; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*
chemical_list: D004220:Disulfides; D010450:Endopeptidases