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
