Tài liệu Báo cáo khoa học: An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding docx

An engineered disulfide bridge mimics the effect of
calcium 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 (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 (DDG
#
¼ 33.5 kJÆmol
)1
at 25 °C) between
calcium saturation (at 100 mm CaCl
2
) 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 25 °C and 5 mm 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 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.
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1523
mutational 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 stabiliza-
tion of this unfolding region results in global stabiliza-
tion 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 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 92.5 °C from <0.3 min to 35.9 min and increased
the temperature, after which half of the initial activity
is lost within 30 min (T
50
), by 16.7 °C [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 100 °C [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 using crystal soaking 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.
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1524 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS
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 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 concentration 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 mM
Tris ⁄ HCl buffer, pH 7.5, containing 5 mM 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.
P. Du
¨
rrschmidt et al. Engineered disulfide bridge mimics calcium effect
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1525
bands 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 (k
obs
) 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 lgmL
)1
(e), 20 lgmL
)1
(,),
5 lgmL
)1
(h) and 1 lgmL
)1
(n)] was incubated in 50 mM
Tris ⁄ HCl, pH 7.5, containing 5 mM CaCl
2
and 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 mM Tris ⁄ HCl buffer,
pH 7.5, containing 5 m
M CaCl
2
and GdnHCl. After 5 min (s),
30 min (h)and3h(n), nondegraded protein was quantified as
described in the Experimental procedures. Each value is the aver-
age from three independent experiments.
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1526 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS
at 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
)9
s
)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
)7
s
)1
. Following
Eyring’s equation:
k ¼
k
B
Á T
h
Á e
À
DG
#
RT
ð1Þ

with k
B
, 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
5mm CaCl
2
and 25 ° C) 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

CaCl
2
, 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 CaCl
2
, even G8C ⁄ N60C shows a non-
linear correlation between ln k
obs
and the GdnHCl
concentration, whereas at 100 mm CaCl
2
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 CaCl
2
, 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 CaCl
2
,
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 mM CaCl
2
and
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 k
obs
values fitted from the measuring data according to a first-order
reaction.
P. Du
¨
rrschmidt et al. Engineered disulfide bridge mimics calcium effect
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1527
merely 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 CaCl
2
(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 mM Tris ⁄ HCl, pH 7.5,
containing 2 m
M (s), 5 mM (h) or 100 mM (n) CaCl
2
and the indica-
ted concentration of guanidine hydrochloride (GdnHCl). k
obs
values

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 mM 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.
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1528 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS
absence of GdnHCl, the rate constant of unfolding of
pWT (k
0Ca2+
) was 3.01 ± 0.42 · 10
)3
Æs
)1
. In the pres-
ence of 100 mm CaCl

2
, where complete saturation of
all four calcium-binding sites is assumed, the rate
constant of unfolding (k
4Ca2+
) cannot be determined
directly because unfolding in the absence of GdnHCl
is too slow to be detectable. However, the k
4Ca2+
value could be obtained from the chevron plot in the
presence of 100 mm CaCl
2
(Fig. 6), by using linear
extrapolation [27], to zero GdnHCl and amounts to
4.12 ± 1.16 · 10
)9
Æs
)1
. The comparison of the rate
constants in the presence of 100 mm EDTA and
100 mm CaCl
2
reveals a deceleration of the unfolding
process by almost six orders of magnitude after bind-
ing of all four calcium ions, corresponding to an
increase in Gibbs free energy of activation of unfold-
ing, at 25 °C, 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 CaCl
2
(Fig. 6) amounts to 1.76 · 10
)13
s
)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 [IC
50
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 spectro-
scopic methods. In the presence of 2 and 5 mm CaCl
2
,

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
CaCl
2
, 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 mM 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.
P. Du
¨
rrschmidt et al. Engineered disulfide bridge mimics calcium effect
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1529
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 % 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 CaCl
2
, 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–
5mm CaCl
2
, by the addition of isopropanol leads to a
reduction of the observed rate constants of unfolding.
The k
obs
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 CaCl
2
(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 CaCl
2
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 % 5mm CaCl
2
and is saturated at 100 mm
CaCl
2
(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 mutations 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
5mm CaCl
2
reflects the high sensitivity of the cross-
linked region against unfolding. From the data of ther-
moinactivation measurements [13], where the half life
at 92.5 °C was determined to 35.9 min for G8C ⁄ N60C
and 0.3 min for pWT, the Gibbs free energy of activa-
tion at 92.5 °C 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 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 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
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1530 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS
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 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 (£ 5mm) 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 CaCl
2
(saturation concentra-
tion) or (b) by the introduction of the disulfide bridge

G8C ⁄ N60C (for CaCl
2
‡ 5mm).
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
CaCl
2
, incubated at 65 °C 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 CaCl
2
and 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
37 °C, as previously described [39,40]. The activity towards
N
fragments
D
Ca
2+
local
unfolding
global
unfolding
N
*
N

Ca
2+
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
Ca
2+
) unfolds globally to the com-
pletely unfolded state D. The introduced disulfide bridge,
G8C ⁄ N60C, restricts unfolding.
P. Du
¨
rrschmidt et al. Engineered disulfide bridge mimics calcium effect
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1531
Abz-AGLA-Nba was measured at 25 ° C 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 CaCl
2
and incubated for
10 min. Inactivation owing to the remaining GdnHCl was
insignificant.
Fluorescence and CD measurements
Fluorescence spectroscopy was carried out at 25 °C 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
k
obs
.
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
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1532 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS
proteases reflected in the thermal inactivation kinetics.
Biotechnol Appl Biochem 32, 189–195.
12 Mansfeld J, Vriend G, Van den Burg B, Eijsink VG &
Ulbrich-Hofmann R (1999) Probing the unfolding
region in a thermolysin-like protease by site-specific
immobilization. Biochemistry 38, 8240–8245.
13 Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van
den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink
VG (1997) Extreme stabilization of a thermolysin-like
protease by an engineered disulfide bond. J Biol Chem
272, 11152–11156.
14 Eijsink VG, Dijkstra BW, Vriend G, van der Zee JR,
Veltman OR, van der Vinne B, van den Burg B, Kempe
S & Venema G (1992) The effect of cavity-filling muta-
tions on the thermostability of Bacillus stearothermophi-
lus neutral protease. Protein Eng 5, 421–426.
15 Durrschmidt P, Mansfeld J & Ulbrich-Hofmann R
(2001) Differentiation between conformational and
autoproteolytic stability of the neutral protease from
Bacillus stearothermophilus containing an engineered dis-
ulfide bond. Eur J Biochem 268, 3612–3618.
16 Van den Burg B, Vriend G, Veltman OR, Venema G &
Eijsink VG (1998) Engineering an enzyme to resist boil-

ing [see comments]. Proc Natl Acad Sci USA 95, 2056–
2060.
17 Colman PM, Jansonius JN & Matthews BW (1972) The
structure of thermolysin: an electron density map at 2–3
A
˚
resolution. J Mol Biol 70, 701–724.
18 Matthews BW, Jansonius JN, Colman PM, Schoenborn
BP & Dupourque D (1972) Three-dimensional structure
of thermolysin. Nature 238, 37–41.
19 Matthews BW, Colman PM, Jansonius JN, Titani K,
Walsh KA & Neurath H (1972) Structure of thermo-
lysin. Nature 238, 41–43.
20 Tajima M, Urabe I, Yutani K & Okada H (1976) Role
of calcium ions in the thermostability of thermolysin
and Bacillus subtilis var. amylosacchariticus neutral pro-
tease. Eur J Biochem 64, 243–247.
21 Voordouw G & Roche RS (1974) The cooperative bind-
ing of two calcium ions to the double site of apothermo-
lysin. Biochemistry 13, 5017–5021.
22 Weaver LH, Kester WR, Ten Eyck LF & Matthews
BW (1976) Enzymes and proteins from thermophilic
microorganisms. In Symposium on Enzymes and Proteins
from Thermophilic Microorganisms (Zuber H, ed.), pp.
31. Birkha
¨
user-Verlag, Basel.
23 Veltman OR, Vriend G, van den Burg B, Hardy F,
Venema G & Eijsink VG (1997) Engineering thermo-
lysin-like proteases whose stability is largely indepen-

dent of calcium. FEBS Lett 405, 241–244.
24 Veltman OR, Vriend G, Berendsen HJ, Van den Burg
B, Venema G & Eijsink VG (1998) A single calcium
binding site is crucial for the calcium-dependent thermal
stability of thermolysin-like proteases. Biochemistry 37,
5312–5319.
25 Eijsink VG, Vriend G, Van Den Burg B, Venema G &
Stulp BK (1990) Contribution of the C-terminal amino
acid to the stability of Bacillus subtilis neutral protease.
Protein Eng 4, 99–104.
26 Nakamura S, Tanaka T, Yada RY & Nakai S (1997)
Improving the thermostability of Bacillus stearothermo-
philus neutral protease by introducing proline into the
active site helix. Protein Eng 10, 1263–1269.
27 Tanford C (1970) Protein denaturation. C. Theoretical
models for the mechanism of denaturation. Adv Protein
Chem 24, 1–95.
28 Muta Y & Inouye K (2002) Inhibitory effects of alco-
hols on thermolysin activity as examined using a fluor-
escent substrate. J Biochem (Tokyo) 132, 945–951.
29 Bhuyan AK & Udgaonkar JB (1998) Multiple kinetic
intermediates accumulate during the unfolding of horse
cytochrome c in the oxidized state. Biochemistry 37,
9147–9155.
30 Sauder JM, MacKenzie NE & Roder H (1996) Kinetic
mechanism of folding and unfolding of Rhodobacter
capsulatus cytochrome c2. Biochemistry 35, 16852–
16862.
31 Zaidi FN, Nath U & Udgaonkar JB (1997) Multiple
intermediates and transition states during protein

unfolding. Nat Struct Biol 4, 1016–1024.
32 Kim PS & Baldwin RL (1990) Intermediates in the fold-
ing reactions of small proteins. Annu Rev Biochem 59,
631–660.
33 Panchal SC, Bhavesh NS & Hosur RV (2001) Real time
NMR monitoring of local unfolding of HIV-1 protease
tethered dimer driven by autolysis. FEBS Lett 497, 59–64.
34 D’Amico S, Marx JC, Gerday C & Feller G (2003)
Activity-stability relationships in extremophilic enzymes.
J Biol Chem 278, 7891–7896.
35 Pace CN, Grimsley GR, Thomson JA & Barnett BJ
(1988) Conformational stability and activity of ribonu-
clease T1 with zero, one, and two intact disulfide bonds.
J Biol Chem 263, 11820–11825.
36 Yamaguchi S, Takeuchi K, Mase T, Oikawa K,
McMullen T, Derewenda U, McElhaney RN, Kay CM
& Derewenda ZS (1996) The consequences of engineer-
ing an extra disulfide bond in the Penicillium camember-
tii mono- and diglyceride specific lipase. Protein Eng 9,
789–795.
37 Ikegaya K, Sugio S, Murakami K & Yamanouchi K
(2003) Kinetic analysis of enhanced thermal stability of
an alkaline protease with engineered twin disulfide
bridges and calcium-dependent stability. Biotechnol
Bioeng 81, 187–192.
38 Almog O, Gallagher DT, Ladner JE, Strausberg S,
Alexander P, Bryan P & Gilliland GL (2002) Structural
basis of thermostability. Analysis of stabilizing muta-
tions in subtilisin BPN¢. J Biol Chem 277, 27553–27558.
39 Kunitz M (1947) Crystalline soybean trypsin inhibitor.

II. General properties. J Gen Physiol 30, 291–299.
P. Du
¨
rrschmidt et al. Engineered disulfide bridge mimics calcium effect
FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS 1533
40 Fujii M, Takagi M, Imanaka T & Aiba S (1983) Mole-
cular cloning of a thermostable neutral protease gene
from Bacillus stearothermophilus in a vector plasmid and
its expression in Bacillus stearothermophilus and Bacillus
subtilis. J Bacteriol 154, 831–837.
41 Nishino N & Powers JC (1980) Pseudomonas aeruginosa
elastase. Development of a new substrate, inhibitors,
and an affinity ligand. J Biol Chem 255, 3482–3486.
42 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
43 Wharton SA, Martin SR, Ruigrok RW, Skehel JJ &
Wiley DC (1988) Membrane fusion by peptide analo-
gues of influenza virus haemagglutinin. J Gen Virol 69,
1847–1857.
Engineered disulfide bridge mimics calcium effect P. Du
¨
rrschmidt et al.
1534 FEBS Journal 272 (2005) 1523–1534 ª 2005 FEBS