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An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding
Author(s) -
Dürrschmidt Peter,
Mansfeld Johanna,
UlbrichHofmann Renate
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04593.x
Subject(s) - chemistry , calcium , guanidine , protease , crystallography , kinetics , biophysics , stereochemistry , biochemistry , enzyme , organic chemistry , biology , physics , quantum mechanics
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 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 ( k obs ) were evaluated by chevron plots (ln k obs vs. GdnHCl concentration). The k obs of unfolding showed a difference of nearly six orders of magnitude (ΔΔG # = 33.5 kJ·mol −1 at 25 °C) between calcium saturation (at 100 m m CaCl 2 ) and complete removal of calcium ions (in the presence of 100 m m 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 m m . 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 ΔΔG # is 13.2 kJ·mol −1 at 25 °C and 5 m m CaCl 2 . 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 m m , or the introduction of the disulfide bridge G8C/N60C.