tailieunhanh - Báo cáo khoa học: An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

The extreme thermal stabilization achieved by the introduction of a disul-fide bond (G8C⁄N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease fromBacillus stearothermophilus[Mansfeld J, Vri-end G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997)J Biol Chem 272, 11152–11156] was attributed to the fixation of the loop region 56–69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfold-ing and autoproteolysis kinetics of pWT and G8C⁄N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS⁄PAGE | ềFEBS Journal An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding Peter Durrschmidt Johanna Mansfeld and Renate Ulbrich-Hofmann Department of Biochemistry Biotechnology Martin-Luther University Halle-Wittenberg Halle Saale Germany Keywords autoproteolysis disulfide local unfolding neutral protease stability Correspondence R. Ulbrich-Hofmann Martin-Luther-University Halle-Wittenberg Department of Biochemistry Biotechnology Institute of Biotechnology Kurt-Mothes-Strasse 3 D-06120 Halle Saale Germany Fax 49 345 5527303 Tel 49 345 5524864 E-mail ulbrich-hofmann@biochemtech. Enzymes Neutral protease from Bacillus stearother-mophilus EC . Present address IBFB Pharma GmbH Deutscher Platz 5d D-04103 Leipzig Germany Note A website is available http www. biotech Received 9 December 2004 revised 26 January 2005 accepted 2 February 2005 doi The extreme thermal stabilization achieved by the introduction of a disulfide bond G8C N60C into the cysteine-free wild-type-like mutant pWT of the neutral protease from Bacillus stearothermophilus Mansfeld J Vri-end G Dijkstra BW Veltman OR Van den Burg B Venema G Ulbrich-Hofmann R Eijsink VG 1997 J Biol Chem 272 11152-11156 was attributed to the fixation of the loop region 56-69. In this study the role of calcium ions in the guanidine hydrochloride GdnHCl -induced unfolding and autoproteolysis kinetics of pWT and G8C N60C was analyzed by fluorescence spectroscopy far-UV CD spectroscopy and SDS PAGE. First-order rate constants kobs were evaluated by chevron plots ln kobs vs. GdnHCl concentration . The kobs of unfolding showed a difference of nearly six orders of magnitude AAG kJ-mol-1 at 25 C between calcium saturation at 100 mM CaCl2 and complete removal of calcium ions in the presence of 100 mM EDTA . Analysis of the protease variant W55F indicated that calcium binding-site III situated in the