In the chevron plots the disulfide bridge in G8C⁄ N60C shows a similar effect compared with pWT as the addition of calcium ions, suggest-ing that the introduced disulfide bridge fixes the r
Trang 1calcium to protect neutral protease against local unfolding Peter Du¨rrschmidt*, Johanna Mansfeld and Renate Ulbrich-Hofmann
Department of Biochemistry ⁄ Biotechnology, Martin-Luther University Halle-Wittenberg, Halle ⁄ Saale, Germany
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
thermo-lysin-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 stearothermophi-lus (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
Keywords
autoproteolysis; disulfide; local unfolding;
neutral protease; stability
Correspondence
R Ulbrich-Hofmann,
Martin-Luther-University 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
stearother-mophilus (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 9 December 2004, revised 26
January 2005, accepted 2 February 2005)
doi:10.1111/j.1742-4658.2005.04593.x
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 from Bacillus 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 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 kJÆmol)1 at 25C) 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, suggest-ing that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autopro-teolysis Owing to the presence of the disulfide bridge, the DDG# is 13.2 kJÆmol)1 at 25C 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 concen-trations of > 5 mm, or the introduction of the disulfide bridge G8C⁄ N60C
Abbreviations
Abz-AGLA-Nba, 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide; DG # , Gibbs free energy of activation; GdnHCl, guanidine hydrochloride;
k obs , observed rate constant; pWT, pseudo-wild type; TLP, thermolysin-like protease.
Trang 2mutational studies with the aim of elucidating the
rea-sons 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
tion of this unfolding region results in global
stabiliza-tion of the whole molecule This stabilizastabiliza-tion 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 8 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
posi-tion 288 was exchanged with a leucine residue without
significant influence on the specific activity,
thermosta-bility [14] or spectroscopic properties of the enzyme
[15] The disulfide bond produced a shift in the half-life
at 92.5C from <0.3 min to 35.9 min and increased
the temperature, after which half of the initial activity
is lost within 30 min (T50), by 16.7C [13] The
combi-nation of eight stabilizing mutations in the region
56–69, including the introduction of the disulfide
bridge, G8C⁄ N60C, produced an enzyme variant
(boilysin) with a half-life of 170 min at 100C [16]
Calcium ions play an important role in the stability
of TLPs From the X-ray structure of thermolysin [17,18], one double-binding site (I and II) and two sin-gle-binding sites (III and IV) have been derived [19] Data on their binding constants, however, are contra-dictory Tajima et al [20] postulated the same calcium affinity for all binding sites of thermolysin, whereas Voordouw & Roche [21] found two classes of calcium-binding sites with a lower affinity for the double-bind-ing site By usdouble-bind-ing crystal soakdouble-bind-ing studies, Weaver et al [22] showed an ordering of the affinity of the binding sites as follows: I > III > IV‡ II Sequence align-ments with thermolysin revealed four adequate cal-cium-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 cru-cial role [24]
To date, almost all stability investigations of the neutral protease from B stearothermophilus and its variants have been based on thermoinactivation meas-urements assuming that inactivation is determined
by autoproteolysis as a consequence of rate-limiting unfolding A separate kinetic analysis of
conformation-al 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 signifi-cant 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
Fig 1 Model of the 3D structure of the neutral protease from Bacillus stearothermo-philus, 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 8 and 60 is shown in yellow.
Trang 3the 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
cal-cium binding-site III (W55F) is included in the studies
The results indicate two competing routes of
auto-proteolysis, one starting from locally unfolded
mole-cules 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 lgÆmL)1), GdnHCl concentrations (0–8 m) and time
periods of incubation (0–3 h) The calcium
concentra-tion was kept constant at 5 mm, a concentraconcentra-tion
com-monly 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
brilli-ant blue Figure 2 shows the autoproteolysis of pWT at
5.5 m GdnHCl As SDS⁄ PAGE was too insensitive to
permit quantification of protein degradation at protein
concentrations of < 20 lgÆmL)1, autoproteolysis at lower protein concentrations was followed by measure-ment of the remaining activity towards casein or the synthetic substrate 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide (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£ 5 m
No dramatic autoproteolysis of pWT or G8C⁄ N60C was detectable after incubation in GdnHCl at low enzyme concentrations (£ 20 lgÆmL)1) for up to 5 min (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 3 Autoproteolysis becomes more significant as the con-centration of GdnHCl increases However, autoproteo-lysis decreases again at GdnHCl concentrations of
>5 m for pWT and 7 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 3 are shifted towards higher concentrations of GdnHCl
Autoproteolysis is independent of the protein concentration up to 5 m GdnHCl, as concluded from degradation kinetics between 1 and 100 lgÆmL)1 pWT (Fig 4) Hence, at GdnHCl concentrations of < 5 m, unfolding is suggested to be the rate-limiting step for autoproteolysis In contrast, at GdnHCl concentrations
of > 5 m, the extent of autoproteolysis depends on the protein concentration between 20 and 100 lgÆmL)1 pWT (Fig 4), showing that unfolding is not rate limit-ing for autoproteolysis under these conditions An exact analysis of the interplay of unfolding and auto-proteolysis, however, requires kinetic measurements, as described below
Influence of the disulfide bridge on the kinetics
of unfolding and autoproteolysis Unfolding of pWT and G8C⁄ N60C at the standard calcium concentration of 5 mm was monitored by
far-UV CD spectroscopy, showing conformational changes
of the secondary structure, and by fluorescence spectro-scopy, showing conformational changes of the tertiary structure Subsequent autoproteolysis did not disturb the measurement of unfolding kinetics because the spec-tra of unfolded intact proteins and completely degraded proteins did not differ significantly [15] Autoproteo-lysis was followed by densitometric evaluation of the
increasing time
Fig 2 Autoproteolysis of the pseudo-wild type (pWT) neutral
prote-ase from Bacillus stearothermophilus in 5.5 M guanidine
hydrochlo-ride (GdnHCl) The enzyme (100 lgÆmL)1) was incubated in 50 m M
Tris ⁄ HCl buffer, pH 7.5, containing 5 m M CaCl 2 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.
Trang 4bands of intact protein in SDS⁄ PAGE gels after
stain-ing with Coomassie brilliant blue The progress curves
were fitted to a single exponential function and the
resulting rate constants (kobs) were plotted in a
semi-logarithmic graph as a function of the GdnHCl
concen-tration (chevron plot) (Fig 5)
The unfolding rates of secondary and tertiary struc-ture coincided for both of the enzymes at all GdnHCl concentrations The autoproteolysis rates were identi-cal to unfolding rates for G8C⁄ N60C over the whole range investigated (4–8 m GdnHCl) and for pWT at GdnHCl concentrations of £ 5 m All curves coincide
Fig 4 Autoproteolysis kinetics of the pseudo-wild type (pWT) neutral protease from Bacillus stearothermophilus in 5 M and 6.5 M
guanidine hydrochloride (GdnHCl) at different protein concentra-tions pWT [100 lgÆmL)1 (s), 50 lg mL)1 (e), 20 lg mL)1 (,),
5 lg mL)1 (h) and 1 lg mL)1 (n)] was incubated in 50 m M
Tris ⁄ HCl, pH 7.5, containing 5 m M CaCl2and GdnHCl After various periods of time, nondegraded protein was quantified as described
in the Experimental procedures.
Fig 3 Autoproteolysis, in guanidine hydrochloride (GdnHCl), of the
pseudo-wild type (pWT) neutral protease from Bacillus
stearother-mophilus and of the disulfide bond mutant G8C ⁄ N60C The
enzymes (20 lgÆmL)1) were incubated in 50 m M Tris ⁄ HCl buffer,
pH 7.5, containing 5 m M CaCl2 and GdnHCl After 5 min (s),
30 min (h) and 3 h (n), nondegraded protein was quantified as
described in the Experimental procedures Each value is the
aver-age from three independent experiments.
Trang 5at a concentration of GdnHCl of‡ 7.5 m, where
auto-proteolysis 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 5 allow us to calculate the
con-tribution 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)9s)1
The corresponding rate constant for pWT, as
deter-mined experimentally by following the subsequent
autoproteolysis (unfolding was too slow to be
meas-ured directly), was 2.44 ± 0.76· 10)7s)1 Following
Eyring’s equation:
k¼kB T
h e
DG#
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 kJÆmol)1 for pWT and 124.0 ± 1.5 kJÆmol)1 for G8C⁄ N60C Hence, a kinetic stabilization (DDG#) of 13.2 ± 2.4 kJÆmol)1 through the introduction of the disulfide bridge (at
5 mm CaCl2and 25C) is obtained
The influence of calcium ions on the unfolding kinetics
The influence of the calcium ion concentration on unfolding kinetics of pWT and G8C⁄ N60C was inves-tigated by fluorescence measurements Figure 6 shows the chevron plots in the presence of 2, 5 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 6 demonstrates, decreasing the calcium ion concentration to 2 mm results in a very distinct nonlinearity of curves in the chevron plot At 2 mm CaCl2, even G8C⁄ N60C shows a non-linear correlation between ln kobs and the GdnHCl concentration, whereas at 100 mm CaCl2 the devia-tions from linearity in the semilogarithmic plot dis-appear for both of the enzymes Obviously, in chevron plots low calcium ion concentrations promote nonline-arity 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 concentra-tion, the addition of calcium ions is able to change the unfolding rate constant over four orders of magni-tude 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 5 mm CaCl2, pWT and G8C⁄ N60C unfold with the same rate constant at 7.25 m GdnHCl Only when the calcium ion concen-tration increases (> 5 mm), does the disulfide-contain-ing 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
Fig 5 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
incu-bated in 50 m M Tris ⁄ HCl buffer, pH 7.5, containing 5 m M CaCl2and
guanidine hydrochloride (GdnHCl) A protein concentration of
100 lgÆmL)1 was used, except for the fluorescence experiments
at ‡ 7 M GdnHCl, where the protein concentration was 5 lgÆmL)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.
Trang 6merely enhance the calcium affinity, but has an
addi-tional stabilizing effect
The difference in the observed rate constants of
unfolding between pWT and G8C⁄ N60C at calcium
ion concentrations of > 5 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
(Fig 1) To assign the contribution of bound calcium ions at calcium binding-site III, the variant W55F, car-rying a mutation in the respective binding site, was included in the measurements The specific activity of W55F and the content of secondary structure meas-ured 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 concentra-tions of < 5 mm and > 50 mm, but differ at concen-trations between these values (Fig 7) This means that
a higher calcium concentration is needed for occupa-tion 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 bind-ing sites
From the change of the unfolding rate constant under extreme conditions of calcium availability, we were able to estimate the contribution of the incorpor-ation of calcium ions into pWT to the Gibbs free energy of activation of unfolding under native condi-tions When 100 mm EDTA was added to pWT for complete removal of all bound calcium ions in the
Fig 6 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 CaCl 2 The
enzymes (5 lgÆmL)1) were incubated in 50 m M Tris ⁄ HCl, pH 7.5,
containing 2 m M (s), 5 m M (h) or 100 m M (n) CaCl 2 and the
indica-ted concentration of guanidine hydrochloride (GdnHCl) kobsvalues
of unfolding were determined by fluorescence measurements, as
described in the Experimental procedures.
Fig 7 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 pres-ence of 7.25 M guanidine hydrochloride (GdnHCl) The enzymes (5 lgÆmL)1) were incubated in 50 m M Tris ⁄ HCl, pH 7.5, containing CaCl 2 , as indicated, and 7.25 M GdnHCl k obs values of unfolding were determined by fluorescence measurements, as described in the Experimental procedures.
Trang 7absence of GdnHCl, the rate constant of unfolding of
pWT (k0Ca2+) was 3.01 ± 0.42· 10)3Æs)1 In the
pres-ence 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)9Æs)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
bind-ing of all four calcium ions, correspondbind-ing to an
increase in Gibbs free energy of activation of
unfold-ing, at 25C, of 33.5 ± 1.0 kJÆmol)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)13s)1, which
corres-ponds 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
intro-duction 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
auto-proteolysis, 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
iso-propanol [IC50¼ 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
spectro-scopic methods In the presence of 2 and 5 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
unfold-ing behavior of pWT and G8C⁄ N60C is the result of
autoproteolysis
Discussion
Autoproteolysis indicates an unfolding intermediate
Regarding unfolding and autoproteolysis at the com-monly used calcium ion concentration (5 mm), the
Fig 8 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 lgÆmL)1) were incubated in 50 m M Tris ⁄ HCl, pH 7.5, containing 20% (v ⁄ v) isopropanol, 2 or 5 m M CaCl 2 and guanidine hydrochloride (GdnHCl) of the indicated concentration k obs values
of unfolding were determined by fluorescence measurements, as described in the Experimental procedures.
Trang 8results 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 5 m GnHCl (Figs 4 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
concen-tration, the rate of autoproteolysis depends on the
protein concentration (Fig 4) For G8C⁄ N60C in the
presence of 5 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
con-ditions, 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 4 and 5), which
indicates that unfolding occurs in more than one step
[29–31]
Reduction of autoproteolysis, in the presence of 2–
5 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
con-centration of 100 mm CaCl2(Fig 7), their contribution
to the Gibbs free energy of activation of unfolding is
33.5 kJÆmol)1 This difference in kinetic stability cor-responds 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 com-parison 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 occu-pied at 5 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 muta-tions in calcium binding-site III, whereas mutamuta-tions in binding site IV have much smaller effects
At low calcium concentrations (2 mm in Fig 7), cal-cium 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 chev-ron plot (Fig 5), except at low calcium ion concentra-tions (< 5 mm) (Fig 6) The stabilization energy of 13.2 kJÆmol)1 by the introduced disulfide bridge at
5 mm CaCl2 reflects the high sensitivity of the cross-linked region against unfolding From the data of ther-moinactivation measurements [13], where the half life
at 92.5C was determined to 35.9 min for G8C ⁄ N60C and 0.3 min for pWT, the Gibbs free energy of activa-tion at 92.5C can be calculated for pWT and G8C⁄ N60C as 100.1 kJÆmol)1 and 114.6 kJÆmol)1, respectively Correspondingly, the disulfide bridge yields an increase of the kinetic stability at this tem-perature by 14.5 kJÆmol)1, which is similar to the value obtained here at 25C
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 unfold-ing 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
Trang 9can 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
sta-bilized by the disulfide bridge connecting residues 8
and 60 Engineered disulfide bridges producing a
sim-ilar effect on protein stability as calcium ions have
been also reported for subtilisin BPN¢ or alkaline
pro-tease [37,38] However, under calcium saturation
(Figs 5 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 9
Following this scheme, calcium binding-site III
deter-mines 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
con-centrations (£ 5 mm) and unfolding proceeds via the
(locally unfolded) intermediate state that is visualized
by the subsequently faster autoproteolysis (for < 5 m
GdnHCl) After reduction in the rate of
autoproteo-lysis 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
indica-ted 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
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 concentra-tion) or (b) by the introduction of the disulfide bridge G8C⁄ N60C (for CaCl2‡ 5 mm)
Experimental procedures
Chemicals
Casein was purchased from Merck (Darmstadt, Germany), Abz-AGLA-Nba from Bachem (Heidelberg, Germany), GdnHCl from ICN Biomedicals GmbH (Eschwege, Ger-many), isopropanol from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany), and tris(hydroxymethyl)amino-methane (Tris) from Amersham Biosciences (Uppsala, Sweden) All other reagents were the purest ones avail-able
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
muta-genesis 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 stearo-thermophilus, cloned into pET-28b(+) (J Mansfeld, unpub-lished data) As checked by the progam what if [5], the mutation W55F should not dramatically disturb the pro-tein geometry The sequence of the mutated plasmid was verified by dideoxy sequencing before using the SalI frag-ment 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 5 mm CaCl2, incubated at 65C for 8 min 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 buf-fer, pH 7.5, containing 5 mm CaCl2and 20% (v⁄ v) isopro-panol as eluent The protein concentration was determined
by using the bicinchoninic acid protein assay (Pierce, Ger-many) with BSA as standard
Activity assay
The activity towards casein as substrate was determined at
37C, as previously described [39,40] The activity towards
N
fragments D
Ca2+
local unfolding
global
unfolding
N*
NCa2+
Fig 9 Unfolding scheme The native state with unoccupied
cal-cium binding-site III (N) unfolds via an intermediate state (N*),
which is susceptible to autoproteolysis Under calcium-saturation
conditions the native state (N Ca2+) unfolds globally to the
com-pletely unfolded state D The introduced disulfide bridge,
G8C ⁄ N60C, restricts unfolding.
Trang 10Abz-AGLA-Nba was measured at 25C at a substrate
con-centration of 20 lm The increase in fluorescence emission
at 415 nm after excitation at 340 nm was recorded over
10 min [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 lgÆmL)1 To
con-centrate the protein and to remove GdnHCl, the samples
were treated with sodium deoxycholate [15] The
precipi-tates 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,
Ger-many) The amount of intact protein was calculated from
the intensity of the protein band
Autoproteolysis in sample solutions containing <20
lgÆmL)1 protein was followed by determining the residual
activity towards casein or the synthetic fluorogenic
sub-strate, Abz-AGLA-Nba Before assaying, the sample
solu-tion was diluted at least 20-fold into 50 mm Tris⁄ HCl
buffer, pH 7.5, containing 5 mm CaCl2 and incubated for
10 min Inactivation owing to the remaining GdnHCl was
insignificant
Fluorescence and CD measurements
Fluorescence spectroscopy was carried out at 25C with a
FluoroMax-2 spectrofluorometer (Yvon-Spex, Grasbrunn,
Germany) with excitation at 295 nm The slit width was
5 nm for both excitation and emission The ratio of the
emission at 334 nm and 354 nm was used for determining
the kinetic constants [43]
CD measurements in the far-UV region were performed
by using a 62-A DS CD spectrophotometer (Aviv,
Lake-wood, NJ, USA) in a quartz cell of 1 mm path length The
signal at 222 nm was used for the determination of kinetic
constants
As the fluorescence and CD spectra of the unfolded and
the degraded protein are nearly identical [15], the progress
curves in the presence of GndHCl reflect the decrease of
native molecules, and autoproteolysis does not disturb the
measurements
Kinetic analysis
Kinetic measurements of both unfolding and
autoproteoly-sis were started by the addition of protein to the
GdnHCl-containing solution The progress curves were fitted to a
single exponential function, yielding the rate constants
kobs
Acknowledgements
The authors thank G Vriend for preparation of Fig 1, V G Eijsink for plasmid pGE501 encoding the gene of the wild-type sequence of the neutral pro-tease from B stearothermophilus, and R Golbik for stimulating discussions The financial support of the Kultusministerium des Landes Sachsen-Anhalt and the Deutsche Forschungsgemeinschaft is gratefully acknowledged
References
1 Rawlings ND & Barrett AJ (1995) Evolutionary families
of metallopeptidases Methods Enzymol 248, 183–228
2 Holmes MA & Matthews BW (1982) Structure of ther-molysin refined at 1.6 A resolution J Mol Biol 160, 623–639
3 Pauptit RA, Karlsson R, Picot D, Jenkins JA, Niklaus-Reimer AS & Jansonius JN (1988) Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A˚ resolution and comparison with the homologous but more thermostable enzyme thermolysin J Mol Biol 199, 525–537
4 Takagi M, Imanaka T & Aiba S (1985) Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus J Bacteriol 163, 824–831
5 Vriend G & Eijsink V (1993) Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases J Comput Aided Mol Des 7, 367–396
6 Eijsink VG, Veltman OR, Aukema W, Vriend G & Venema G (1995) Structural determinants of the stabi-lity of thermolysin-like proteinases Nat Struct Biol 2, 374–379
7 Veltman OR, Vriend G, Middelhoven PJ, van den Burg
B, Venema G & Eijsink VG (1996) Analysis of struc-tural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis Protein Eng 9, 1181–1189
8 Veltman OR, Vriend G, Hardy F, Mansfeld J, van den Burg B, Venema G & Eijsink VG (1997) Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases Eur J Biochem
248, 433–440
9 Ulbrich-Hofmann R, Arnold U & Mansfeld J (1999) The concept of the unfolding region of approaching the mechanism of enzyme stabilization J Mol Catalysis B: Enzymatic 7, 125–131
10 Schellenberger A & Ulbrich R (1989) Protein stabiliza-tion by blocking the native unfolding nucleus Biomed Biochim Acta 48, 63–67
11 Mansfeld J & Ulbrich-Hofmann R (2000) Site-specific and random immobilization of thermolysin-like