An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding Peter Durrschmidt*, Johanna Mansfeld and Renate Ulbrich-Hofmann ă 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-LutherUniversity 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 uni-halle.de Enzymes Neutral protease from Bacillus stearothermophilus (EC 3.4.24.28) *Present address IBFB Pharma GmbH, Deutscher Platz 5d, D-04103 Leipzig, Germany Note A website is available: http://www biochemtech.uni-halle.de/biotech (Received December 2004, revised 26 January 2005, accepted February 2005) 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, UlbrichHofmann 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 (DDG# ¼ 33.5 kJỈmol)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 critical region 56–69, determines the stability at calcium ion concentrations between 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 DDG# is 13.2 kJỈmol)1 at 25 °C and 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 > mm, or the introduction of the disulfide bridge G8C ⁄ N60C doi:10.1111/j.1742-4658.2005.04593.x The neutral protease from Bacillus stearothermophilus belongs to a group of metalloendopeptidases that have maximum activity at neutral pH Some members of this group are highly conserved in amino acid sequence and tertiary structure and form the group of thermolysin-like proteases (TLPs) with thermolysin as the best characterized representative [1] TLPs consist of 300– 319 amino acid residues and are organized into two domains They have one catalytic zinc ion, and between two and four stabilizing calcium ions The X-ray structures of thermolysin [2] and the neutral protease from B cereus [3] have been resolved and show great similarities Because of the high degree of sequence identity (85%) with thermolysin [4], a 3D model of the neutral protease from B stearothermophilus (Fig 1) was constructed, on the basis of the X-ray structure of thermolysin, by homology modeling [5] and has been successfully used in a number of Abbreviations Abz-AGLA-Nba, 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide; DG#, Gibbs free energy of activation; GdnHCl, guanidine hydrochloride; kobs, observed rate constant; pWT, pseudo-wild type; TLP, thermolysin-like protease FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1523 Engineered disulde bridge mimics calcium effect P Durrschmidt et al ă Fig Model of the 3D structure of the neutral protease from Bacillus stearothermophilus, including the position of the disulfide bridge in mutant G8C ⁄ N60C The model was created on the basis of the X-ray structure of thermolysin by using homology modeling with the program WHAT IF [5] The orange sphere indicates the catalytic zinc ion, and the violet spheres indicate the bound calcium ions with numbering of the binding sites The sensitive loop 56–69 is shown in red, and the disulfide bridge connecting positions and 60 is shown in yellow mutational studies with the aim of elucidating the reasons for the great stability differences between the two enzymes Extensive thermal inactivation studies have revealed that mutations in the loop region 56–69 (numbering according to thermolysin) strongly affect the thermal stability, whereas mutations in other regions have only marginal effects [6–8] These findings were consistent with our concept of the unfolding region [9,10] According to this concept, unfolding of a protein molecule starts at its weakest site, and local stabilization of this unfolding region results in global stabilization of the whole molecule This stabilization strategy was examined by site-directed immobilization of the neutral protease from B stearothermophilus [11,12], showing a distinctly stronger stabilization effect if immobilization was performed within the region 56–69 An extreme thermal stabilization was obtained by fixation of the critical loop region by an engineered disulfide bond crosslinking positions and 60 [13] This enzyme variant (G8C ⁄ N60C) was produced by the introduction of two cysteine residues into the pseudo-wild type enzyme (pWT), a mutant in which the only naturally occurring cysteine residue at position 288 was exchanged with a leucine residue without significant influence on the specific activity, thermostability [14] or spectroscopic properties of the enzyme [15] The disulfide bond produced a shift in the half-life at 92.5 °C from III > IV ‡ II Sequence alignments with thermolysin revealed four adequate calcium-binding sites also for the neutral protease from B stearothermophilus (Fig 1) The thermal inactivation strongly depends on the calcium ion concentration [7,23], and calcium binding-site III seems to play a crucial role [24] To date, almost all stability investigations of the neutral protease from B stearothermophilus and its variants have been based on thermoinactivation measurements assuming that inactivation is determined by autoproteolysis as a consequence of rate-limiting unfolding A separate kinetic analysis of conformational unfolding, however, is hampered by autoproteolysis Recently, we have shown that guanidine hydrochloride (GdnHCl) denaturation under certain conditions (low protein concentration and short incubation times) allows the analysis of global unfolding without significant interference by autoproteolysis [15] In this article we exploit GdnHCl denaturation for a kinetic analysis of unfolding and autoproteolysis of G8C ⁄ N60C and pWT In contrast to previous studies on the kinetics of thermoinactivation [13,24], this approach allows quantification of the contribution of FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS P Durrschmidt et al ă Engineered disulde bridge mimics calcium effect the engineered disulfide bond to conformational and autoproteolytical stabilization Particular attention is paid to the role of calcium ions For this reason, an enzyme variant with a mutation in the vicinity of calcium binding-site III (W55F) is included in the studies The results indicate two competing routes of autoproteolysis, one starting from locally unfolded molecules and the other starting from globally unfolded molecules Results Screening of conditions for GdnHCl-induced autoproteolysis The unfolding of nonspecific proteases, such as TLPs, is accompanied by autoproteolysis, rendering unfolding irreversible To screen conditions where autoproteolysis becomes significant in GdnHCl-induced unfolding of pWT and G8C ⁄ N60C, autodegradation of the enzymes was determined at different protein concentrations (5– 100 lgỈmL)1), GdnHCl concentrations (0–8 m) and time periods of incubation (0–3 h) The calcium concentration was kept constant at mm, a concentration commonly used in studies on the neutral protease from B stearothermophilus [8,25,26] Autoproteolysis was quantified by evaluation of the bands of intact protein in SDS ⁄ PAGE gels after staining with Coomassie brilliant blue Figure shows the autoproteolysis of pWT at 5.5 m GdnHCl As SDS ⁄ PAGE was too insensitive to permit quantification of protein degradation at protein increasing time concentrations of < 20 lgỈmL)1, autoproteolysis at lower protein concentrations was followed by measurement of the remaining activity towards casein or the synthetic substrate 2-aminobenzoyl-Ala-Gly-Leu-Ala-4nitrobenzylamide (Abz-AGLA-Nba), as described in the Experimental procedures Inactivation data obtained in this way were shown to be identical with autoproteolysis kinetics for GdnHCl £ m No dramatic autoproteolysis of pWT or G8C ⁄ N60C was detectable after incubation in GdnHCl at low enzyme concentrations (£ 20 lgỈmL)1) for up to (Fig 3) As shown recently, global unfolding of the proteases without interference by autoproteolysis can be observed under these conditions [15] For longer periods of time, however, incubation of the enzymes in GdnHCl results in autoproteolysis, as shown in Fig Autoproteolysis becomes more significant as the concentration of GdnHCl increases However, autoproteolysis decreases again at GdnHCl concentrations of >5 m for pWT and m for G8C ⁄ N60C, and almost no protein degradation was observed at 7–8 m GdnHCl Obviously, at high GdnHCl concentrations, inactivation of the enzyme by unfolding is so fast that autoproteolysis cannot occur G8C ⁄ N60C behaves similarly to pWT, but the curves in Fig are shifted towards higher concentrations of GdnHCl Autoproteolysis is independent of the protein concentration up to m GdnHCl, as concluded from degradation kinetics between and 100 lgỈmL)1 pWT (Fig 4) Hence, at GdnHCl concentrations of < m, unfolding is suggested to be the rate-limiting step for autoproteolysis In contrast, at GdnHCl concentrations of > m, the extent of autoproteolysis depends on the protein concentration between 20 and 100 lgỈmL)1 pWT (Fig 4), showing that unfolding is not rate limiting for autoproteolysis under these conditions An exact analysis of the interplay of unfolding and autoproteolysis, however, requires kinetic measurements, as described below Influence of the disulfide bridge on the kinetics of unfolding and autoproteolysis Fig Autoproteolysis of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus in 5.5 M guanidine hydrochloride (GdnHCl) The enzyme (100 lgỈmL)1) was incubated in 50 mM Tris ⁄ HCl buffer, pH 7.5, containing mM CaCl2 and 5.5 M GdnHCl After 0.5, 2, 5, 10, 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min, aliquots were removed, precipitated and separated by SDS ⁄ PAGE, as described in the Experimental procedures The first lane is a control, showing the enzyme incubated in the absence of GdnHCl FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS Unfolding of pWT and G8C ⁄ N60C at the standard calcium concentration of mm was monitored by farUV CD spectroscopy, showing conformational changes of the secondary structure, and by fluorescence spectroscopy, showing conformational changes of the tertiary structure Subsequent autoproteolysis did not disturb the measurement of unfolding kinetics because the spectra of unfolded intact proteins and completely degraded proteins did not differ significantly [15] Autoproteolysis was followed by densitometric evaluation of the 1525 Engineered disulde bridge mimics calcium effect P Durrschmidt et al ă Fig Autoproteolysis, in guanidine hydrochloride (GdnHCl), of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus and of the disulfide bond mutant G8C ⁄ N60C The enzymes (20 lgỈmL)1) were incubated in 50 mM Tris ⁄ HCl buffer, pH 7.5, containing mM CaCl2 and GdnHCl After (s), 30 (h) and h (n), nondegraded protein was quantified as described in the Experimental procedures Each value is the average from three independent experiments Fig Autoproteolysis kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus in M and 6.5 M guanidine hydrochloride (GdnHCl) at different protein concentrations pWT [100 lgỈmL)1 (s), 50 lg mL)1 (e), 20 lg mL)1 (,), lg mL)1 (h) and lg mL)1 (n)] was incubated in 50 mM Tris ⁄ HCl, pH 7.5, containing mM CaCl2 and GdnHCl After various periods of time, nondegraded protein was quantified as described in the Experimental procedures bands of intact protein in SDS ⁄ PAGE gels after staining with Coomassie brilliant blue The progress curves were fitted to a single exponential function and the resulting rate constants (kobs) were plotted in a semilogarithmic graph as a function of the GdnHCl concentration (chevron plot) (Fig 5) The unfolding rates of secondary and tertiary structure coincided for both of the enzymes at all GdnHCl concentrations The autoproteolysis rates were identical to unfolding rates for G8C ⁄ N60C over the whole range investigated (4–8 m GdnHCl) and for pWT at GdnHCl concentrations of £ m All curves coincide 1526 FEBS Journal 272 (2005) 15231534 ê 2005 FEBS P Durrschmidt et al ă Engineered disulfide bridge mimics calcium effect k¼ kB Á T ÀDG# Á e RT h ð1Þ with kB, h and R as Boltzmann’s constant, Planck’s constant and gas constant, respectively, and T and DG# as absolute temperature and Gibbs free energy of activation, respectively, the DG# of unfolding under native conditions is 110.8 ± 0.9 kJỈmol)1 for pWT and 124.0 ± 1.5 kJỈmol)1 for G8C ⁄ N60C Hence, a kinetic stabilization (DDG#) of 13.2 ± 2.4 kJỈmol)1 through the introduction of the disulfide bridge (at mm CaCl2 and 25 °C) is obtained The influence of calcium ions on the unfolding kinetics Fig Unfolding and autoproteolysis kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus and of the disulfide bond mutant G8C ⁄ N60C The enzymes were incubated in 50 mM Tris ⁄ HCl buffer, pH 7.5, containing mM CaCl2 and guanidine hydrochloride (GdnHCl) A protein concentration of 100 lgỈmL)1 was used, except for the fluorescence experiments at ‡ M GdnHCl, where the protein concentration was lgỈmL)1 Kinetic measurements of autoproteolysis, CD and fluorescence spectroscopy were performed as described in the Experimental procedures The error bars show the standard deviations of the kobs values fitted from the measuring data according to a first-order reaction at a concentration of GdnHCl of ‡ 7.5 m, where autoproteolysis no longer occurs, showing that both of the enzymes unfold at the same rate constant under these conditions However, at concentraions of GdnHCl up to 7.5 m, the disulfide-containing variant unfolds distinctly more slowly than pWT Interestingly, G8C ⁄ N60C shows a linear behavior in the chevron plot, whereas pWT shows a remarkable deviation from the expected linearity The curve for pWT consists of two linear branches with a transition at 6–7 m GdnHCl The data from Fig allow us to calculate the contribution of the disulfide bridge to the increase in the Gibbs free energy of activation of unfolding under native conditions Using the linear extrapolation model [27], the rate constant for the unfolding of G8C ⁄ N60C in the absence of denaturant is 1.09 ± 0.66 · 10)9 s)1 The corresponding rate constant for pWT, as determined experimentally by following the subsequent autoproteolysis (unfolding was too slow to be measured directly), was 2.44 ± 0.76 · 10)7 s)1 Following Eyring’s equation: FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS The influence of the calcium ion concentration on unfolding kinetics of pWT and G8C ⁄ N60C was investigated by fluorescence measurements Figure shows the chevron plots in the presence of 2, and 100 mm CaCl2, which is below, at and above the standard concentration of calcium ions used in studies on this protease [8,25,26] The observed rate constants of unfolding proved to be very sensitive to the calcium ion concentration As Fig demonstrates, decreasing the calcium ion concentration to mm results in a very distinct nonlinearity of curves in the chevron plot At mm CaCl2, even G8C ⁄ N60C shows a nonlinear correlation between ln kobs and the GdnHCl concentration, whereas at 100 mm CaCl2 the deviations from linearity in the semilogarithmic plot disappear for both of the enzymes Obviously, in chevron plots low calcium ion concentrations promote nonlinearity and high calcium ion concentrations promote linearity The influence of calcium ions on the unfolding rate constants was investigated, in greater detail, in the presence of 7.25 m GdnHCl where autoproteolysis is widely suppressed (Fig 7) At this GdnHCl concentration, the addition of calcium ions is able to change the unfolding rate constant over four orders of magnitude The addition of EDTA resulted in a further increase of the unfolding rate, showing that at least one of the four calcium ions is bound very efficiently In the presence of less than mm CaCl2, pWT and G8C ⁄ N60C unfold with the same rate constant at 7.25 m GdnHCl Only when the calcium ion concentration increases (> mm), does the disulfide-containing variant unfold more slowly Near 100 mm CaCl2, no further reduction of the rate constant was observed Even under these saturation conditions G8C ⁄ N60C proved to unfold more slowly than pWT Hence, the disulfide bridge in G8C ⁄ N60C does not 1527 Engineered disulfide bridge mimics calcium effect P Durrschmidt et al ă Fig Unfolding kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus, of the disulfide bond mutant G8C ⁄ N60C, and of the protease variant W55F, in the presence of 7.25 M guanidine hydrochloride (GdnHCl) The enzymes (5 lgỈmL)1) were incubated in 50 mM Tris ⁄ HCl, pH 7.5, containing CaCl2, as indicated, and 7.25 M GdnHCl kobs values of unfolding were determined by fluorescence measurements, as described in the Experimental procedures Fig Unfolding kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus and of the disulfide bond mutant G8C ⁄ N60C at various concentrations of CaCl2 The enzymes (5 lgỈmL)1) were incubated in 50 mM Tris ⁄ HCl, pH 7.5, containing mM (s), mM (h) or 100 mM (n) CaCl2 and the indicated concentration of guanidine hydrochloride (GdnHCl) kobs values of unfolding were determined by fluorescence measurements, as described in the Experimental procedures merely enhance the calcium affinity, but has an additional stabilizing effect The difference in the observed rate constants of unfolding between pWT and G8C ⁄ N60C at calcium ion concentrations of > mm (Fig 7) must be related to the position of the introduced disulfide bridge that is located in the vicinity of calcium binding-site III 1528 (Fig 1) To assign the contribution of bound calcium ions at calcium binding-site III, the variant W55F, carrying a mutation in the respective binding site, was included in the measurements The specific activity of W55F and the content of secondary structure measured by far-UV CD is comparable with that of pWT (results not shown) Therefore, differences in the unfolding behavior between pWT and W55F should be caused by alterations in the affinity at calcium binding-site III The rate constants of unfolding of pWT and W55F are similar for calcium ion concentrations of < mm and > 50 mm, but differ at concentrations between these values (Fig 7) This means that a higher calcium concentration is needed for occupation of calcium binding-site III in W55F Because of the saturation behavior of all three enzyme variants at >50 mm CaCl2 (Fig 7), calcium binding-site III seems to be occupied as the last one of the four binding sites From the change of the unfolding rate constant under extreme conditions of calcium availability, we were able to estimate the contribution of the incorporation of calcium ions into pWT to the Gibbs free energy of activation of unfolding under native conditions When 100 mm EDTA was added to pWT for complete removal of all bound calcium ions in the FEBS Journal 272 (2005) 15231534 ê 2005 FEBS P Durrschmidt et al ă Engineered disulfide bridge mimics calcium effect absence of GdnHCl, the rate constant of unfolding of pWT (k0Ca2+) was 3.01 ± 0.42 · 10)3Ỉs)1 In the presence of 100 mm CaCl2, where complete saturation of all four calcium-binding sites is assumed, the rate constant of unfolding (k4Ca2+) cannot be determined directly because unfolding in the absence of GdnHCl is too slow to be detectable However, the k4Ca2+ value could be obtained from the chevron plot in the presence of 100 mm CaCl2 (Fig 6), by using linear extrapolation [27], to zero GdnHCl and amounts to 4.12 ± 1.16 · 10)9Ỉs)1 The comparison of the rate constants in the presence of 100 mm EDTA and 100 mm CaCl2 reveals a deceleration of the unfolding process by almost six orders of magnitude after binding of all four calcium ions, corresponding to an increase in Gibbs free energy of activation of unfolding, at 25 °C, of 33.5 ± 1.0 kJỈmol)1 Interestingly, the extrapolated rate constant of unfolding for the disulfide-containing variant under native conditions in the presence of 100 mm CaCl2 (Fig 6) amounts to 1.76 · 10)13 s)1, which corresponds to a half-life of % 125 000 years The different unfolding rate constants for pWT and G8C ⁄ N60C under native conditions at calcium saturation (100 mm) confirm the conclusion, drawn above, that the introduction of the disulfide bridge stabilizes the molecule more than calcium ions The influence of isopropanol on the unfolding rate constant To measure unfolding without the interference of autoproteolysis, the addition of inhibitors to the enzymes was examined Inhibitors such as phosphoramidon or o-phenanthroline are fluorescent and disturb the applied spectroscopic techniques The addition of isopropanol [IC50 ¼ 2.3% (v ⁄ v)], which is known to act as an inhibitor of thermolysin [28], resulted in a marked decrease (but not complete elimination) of autoproteolysis without interfering with the spectroscopic methods In the presence of and mm CaCl2, the observed rate constants of unfolding were found to decrease in an exponential manner with increasing amounts of isopropanol At 20% (v ⁄ v) isopropanol, no further decrease of the observed rate constants of unfolding was observed, and linearity in the chevron plot was found without any differences between pWT and G8C ⁄ N60C (Fig 8) In the presence of 100 mm CaCl2, the addition of up to 20% (v ⁄ v) isopropanol had no effect on the observed rate constants These results lead to the conclusion that the different unfolding behavior of pWT and G8C ⁄ N60C is the result of autoproteolysis FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS Fig Unfolding kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus (s) and of the disulfide bond mutant G8C ⁄ N60C (d) in the presence of isopropanol The enzymes (5 lgỈmL)1) were incubated in 50 mM Tris ⁄ HCl, pH 7.5, containing 20% (v ⁄ v) isopropanol, or mM CaCl2 and guanidine hydrochloride (GdnHCl) of the indicated concentration kobs values of unfolding were determined by fluorescence measurements, as described in the Experimental procedures Discussion Autoproteolysis indicates an unfolding intermediate Regarding unfolding and autoproteolysis at the commonly used calcium ion concentration (5 mm), the 1529 Engineered disulfide bridge mimics calcium effect results show that autoproteolysis of both pWT and G8C ⁄ N60C occurs at GdnHCl concentrations of < 7.5 m (Fig 3) For pWT, unfolding is rate-limiting up to % m GnHCl (Figs and 5), whereas at higher GdnHCl concentrations (5.5–7.5 m), unfolding becomes faster than subsequent autoproteolysis (Fig 5) In this intermediate range of GdnHCl concentration, the rate of autoproteolysis depends on the protein concentration (Fig 4) For G8C ⁄ N60C in the presence of mm CaCl2, unfolding is rate-limiting at GdnHCl concentrations of < 7.5 m At very high GdnHCl concentrations (> 7.5 m) unfolding is so fast that no autoproteolysis can occur, either with pWT or with G8C ⁄ N60C Under these conditions, both variants show the same unfolding rates (Fig 5) Stability differences between pWT and G8C ⁄ N60C emerge under conditions where autoproteolysis occurs (< 7.5 m GdnHCl) These differences are connected with evident deviations from linearity in the chevron plots of unfolding of pWT (Figs and 5), which indicates that unfolding occurs in more than one step [29–31] Reduction of autoproteolysis, in the presence of 2– mm CaCl2, by the addition of isopropanol leads to a reduction of the observed rate constants of unfolding The kobs values coincide for both of the enzymes and produce straight lines in the chevron plots (Fig 8) Global unfolding, as an all-in-one step, should not be accelerated by autoproteolysis because proteolytic degradation occurs in a subsequent reaction The observation that autoproteolysis apparently promotes unfolding can be explained by the assumption that local unfolding events, such as the flexibility increase of certain regions of the protein molecule or transient fluctuations [32] that are not detectable by the applied spectroscopic methods, lead to an autoproteolytically susceptible (intermediate) state Similarly, Panchal et al [33] took advantage of autoproteolysis of the HIV protease to monitor local unfolding events by NMR Obviously, nonlinear chevron plots act as an indicator for the occurrence of the intermediate in the unfolding Calcium ions are the main contributory factor to enzyme stability The binding of calcium ions seems to be the main source of stabilization of the neutral protease from B stearothermophilus Assuming that all four binding sites for calcium ions are occupied at a saturating concentration of 100 mm CaCl2 (Fig 7), their contribution to the Gibbs free energy of activation of unfolding is 1530 P Durrschmidt et al ă 33.5 kJặmol)1 This difference in kinetic stability corresponds to the difference between a mesophilic and a thermophilic enzyme [34] From unfolding of pWT and G8C ⁄ N60C as a function of added CaCl2 in comparison to the unfolding of W55F, which is modified near binding site III within the region 56–69, it can be concluded that binding site III starts to become occupied at % mm CaCl2 and is saturated at 100 mm CaCl2 (Fig 7) This binding site is formed by residues 55, 57, 59 and 61 [8] and has obviously the lowest affinity of the four calcium-binding sites, as shown by the W55F variant (Fig 7) This finding is consistent with the results of Veltman et al [24] who showed that the thermoinactivation of the neutral protease from B stearothermophilus is dramatically changed by mutations in calcium binding-site III, whereas mutations in binding site IV have much smaller effects At low calcium concentrations (2 mm in Fig 7), calcium binding-site III is unoccupied and mutations in this region (W55F, G8C ⁄ N60C) have no effect on the unfolding kinetics Under these conditions, regions other than the unfolding region 56–69 determine the stability The disulfide bridge in G8C/N60C mimics the occupation of calcium binding-site III Similarly to the occupation of calcium binding-site III, the introduction of the disulfide bridge G8C ⁄ N60C into the enzyme abolishes the nonlinearity in the chevron plot (Fig 5), except at low calcium ion concentrations (< mm) (Fig 6) The stabilization energy of 13.2 kJỈmol)1 by the introduced disulfide bridge at mm CaCl2 reflects the high sensitivity of the crosslinked region against unfolding From the data of thermoinactivation measurements [13], where the half life at 92.5 °C was determined to 35.9 for G8C ⁄ N60C and 0.3 for pWT, the Gibbs free energy of activation at 92.5 °C can be calculated for pWT and G8C ⁄ N60C as 100.1 kJỈmol)1 and 114.6 kJỈmol)1, respectively Correspondingly, the disulfide bridge yields an increase of the kinetic stability at this temperature by 14.5 kJỈmol)1, which is similar to the value obtained here at 25 °C High stabilization effects of disulfide bridges are often observed for reversibly unfolding proteins and are mostly attributed to the restriction of the degrees of freedom in the unfolded state [35] Following this concept, disulfide bridges should not influence unfolding processes Indeed, there are only a few reports of successful stabilization against irreversible unfolding by disulfide linkage in the literature [36–38] The high stabilization effect of the disulfide bond in G8C ⁄ N60C FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS P Durrschmidt et al ă Engineered disulde bridge mimics calcium effect can best be explained by the assumption that unfolding and ⁄ or autoproteolysis via the unfolding intermediate is hampered Similarly to the calcium ions occupying calcium binding-site III, the loop region 56–69 is stabilized by the disulfide bridge connecting residues and 60 Engineered disulfide bridges producing a similar effect on protein stability as calcium ions have been also reported for subtilisin BPN¢ or alkaline protease [37,38] However, under calcium saturation (Figs and 6), the introduced disulfide bridge seems to have an additional stabilization effect Unfolding model The results of our experiments suggest the existence of two unfolding pathways for the neutral protease from B stearothermophilus, namely (a) a global route and (b) a local unfolding route, as demonstrated in Fig Following this scheme, calcium binding-site III determines the kinetic stability of pWT at the commonly used concentration of calcium ions (5 mm) Calcium binding-site III is unoccupied at low calcium ion concentrations (£ mm) and unfolding proceeds via the (locally unfolded) intermediate state that is visualized by the subsequently faster autoproteolysis (for < m GdnHCl) After reduction in the rate of autoproteolysis by the addition of isopropanol, or in the presence of > 7.5 m GdnHCl, the intermediate accumulates and only global unfolding to D is spectroscopically indicated Because the latter reaction is slower than the local unfolding process, no differences in the unfolding rate constants between pWT und G8C ⁄ N60C can be observed in these cases The unfolding intermediate is suggested to be a state with native-like properties, but with autoproteolytic susceptibility owing to higher local unfolding N N * fragments 2+ Ca N Ca2+ D global unfolding Fig Unfolding scheme The native state with unoccupied calcium binding-site III (N) unfolds via an intermediate state (N*), which is susceptible to autoproteolysis Under calcium-saturation conditions the native state (NCa2+) unfolds globally to the completely unfolded state D The introduced disulfide bridge, G8C ⁄ N60C, restricts unfolding FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS flexibility in the region of calcium binding-site III (unfolding region 56–69) The unfolding route via the intermediate can be prevented in two ways, namely (a) the addition of 100 mm CaCl2 (saturation concentration) or (b) by the introduction of the disulfide bridge G8C ⁄ N60C (for CaCl2 ‡ mm) Experimental procedures Chemicals Casein was purchased from Merck (Darmstadt, Germany), Abz-AGLA-Nba from Bachem (Heidelberg, Germany), GdnHCl from ICN Biomedicals GmbH (Eschwege, Germany), isopropanol from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany), and tris(hydroxymethyl)aminomethane (Tris) from Amersham Biosciences (Uppsala, Sweden) All other reagents were the purest ones available Enzymes All enzyme variants were produced as described previously [13,14] The mutation W55F was introduced by site-directed mutagenesis using the QuickChangeÔ site-directed mutagenesis kit (Stratagene, Heidelberg, Germany), the primers 5¢-GTTTTGCCCGGCAGCTTGTTTACCGATGGCGACA ACCAA-3¢ (forward) and 5¢-TTGGTTGTCGCCATCG GTAAACAAGCTGCCGGGCAAAAC-3¢ (reverse), and the wild-type gene of the neutral protease from B stearothermophilus, cloned into pET-28b(+) (J Mansfeld, unpublished data) As checked by the progam what if [5], the mutation W55F should not dramatically disturb the protein geometry The sequence of the mutated plasmid was verified by dideoxy sequencing before using the SalI fragment to reconstruct the pGE501 variant for expression in B subtilis Immediately before use, the enzyme solution was dialyzed against 50 mm Tris ⁄ HCl buffer, pH 7.5, containing mm CaCl2, incubated at 65 °C for and dialyzed again After this procedure the enzyme solution was homogeneous according to SDS ⁄ PAGE analysis The absence of low molecular weight fragments was checked by size-exclusion chromatography using a Superdex G-75 (10 ⁄ 30) column (Pharmacia, Uppsala, Sweden) with 50 mm Tris ⁄ HCl buffer, pH 7.5, containing mm CaCl2 and 20% (v ⁄ v) isopropanol as eluent The protein concentration was determined by using the bicinchoninic acid protein assay (Pierce, Germany) with BSA as standard Activity assay The activity towards casein as substrate was determined at 37 °C, as previously described [39,40] The activity towards 1531 Engineered disulfide bridge mimics calcium effect Abz-AGLA-Nba was measured at 25 °C at a substrate concentration of 20 lm The increase in fluorescence emission at 415 nm after excitation at 340 nm was recorded over 10 [41] Detection of autoproteolysis in the presence of GdnHCl SDS ⁄ PAGE was used to follow autoproteolysis of the enzyme at a protein concentration of ‡ 20 lgỈmL)1 To concentrate the protein and to remove GdnHCl, the samples were treated with sodium deoxycholate [15] The precipitates were dried under vacuum, dissolved in SDS sample buffer and separated on 15% (w ⁄ v) polyacrylamide gels according to Laemmli [42] The proteins were stained with Coomassie Brilliant Blue G 250 and scanned at 595 nm by using a CD 60 densitometer (Desaga, Darmstadt, Germany) The amount of intact protein was calculated from the intensity of the protein band Autoproteolysis in sample solutions containing