An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

Abstract

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.

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

“…The formation of the disulfide bond Cys52‐Cys65 was helpful for the WF146 protease to resist autolysis, probably by decreasing the flexibility of the autolysis loop (positions 64–70). Similar proposal has also been suggested for a thermolysin‐like protease from Bacillus stearothermophilus , where the introduction of a disulfide bond into the enzyme enhanced the enzyme stability toward autolysis due to increased rigidity of local region rather than stabilization of the enzyme toward global unfolding [32,33]. In addition, it has been reported that the thermostable thermitase possessed three stabilizing Ca 2+ binding sites, of which the second one lay in the loop of residues 59 to 65 (thermitase numbering) and reduced the local mobility of the accessible loop (Fig.…”

In Vitro Evolved Non-Aggregating and Thermostable Lipase: Structural and Thermodynamic Investigation

“…40 Anchoring a loop in a neutral protease from Bacillus stearothermophilus, whose local unfolding is responsible for formation of an intermediate, to the rest of protein by a disulfide bridge prevented formation of the intermediate. 41 Stabilization of a loop in cytochrome c, whose local unfolding causes formation of intermediate and aggregation in the presence of benzyl alcohol, caused reduction in aggregation. 42 Recently, roles of native protein structure and amino acid sequence in aggregation were also highlighted.…”

The role of calcium ions in the stability and instability of a thermolysin‐like protease

“…Using a variety of stabilization strategies, highly stable variants of TLP‐ste could be engineered 14, 56. One particularly successful example was the introduction of a designed disulfide bridge (through mutations G8C & N60C) that covalently linked the N‐terminal β‐strand to one of the other strands in the N‐terminal three‐strand β‐sheet 28, 58. Other mutations in the region were also successful for stabilization of TLP‐ste (e.g.…”