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 disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 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 (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees 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 critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.