Báo cáo khoa học: Tertiary structure in 7.9 M guanidinium chloride ) the role of Glu53 and Asp287 in Pyrococcus furiosus endo-b-1,3-glucanase pot

Tertiary structure in 7.9 M guanidinium chloride
)
the role
of Glu53 and Asp287 in Pyrococcus furiosus
endo-b-1,3-glucanase
Roberta Chiaraluce
1
, Rita Florio
1
, Sebastiana Angelaccio
1
, Giulio Gianese
2
,
Johan F. T. van Lieshout
3
, John van der Oost
3
and Valerio Consalvi
1
1 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’ Sapienza Universita
`
di Roma, Italy
2 Ylichron Srl c ⁄ o ENEA Casaccia Research Center, S. Maria di Galeria, Italy
3 Laboratory of Microbiology, Wageningen University, the Netherlands
Endo-b-1,3-glucanase (EC 3.2.1.39) from the hyper-
thermophilic archaeon Pyrococcus furiosus (pfLamA) is
a laminarinase that displays considerable residual ter-
tiary structure in 7.9 m guanidinium chloride (GdmCl)
[1]. A high DG
H

2
O
value of 61.5 kJÆmol
)1
is associated
with the partial unfolding of pfLamA which, in
7.9 GdmCl, maintains the ability to bind calcium with
substantial recovery of native tertiary structure, a
unique property of this enzyme [2].
pfLamA belongs to family 16 glycoside hydrolases
[3], a family composed by 748 enzymes (http://www.
cazy.org/). According to their substrate specificity, the
enzymes of this family can be assigned to different
subgroups [4] ( ⁄ )
Keywords
double mutant; glycoside hydrolases;
laminarinase; protein stability;
thermodynamic stability
Correspondence
V. Consalvi, Dipartimento di Scienze
Biochimiche ‘A. Rossi Fanelli’ Universita
`
‘La Sapienza’, P.le A. Moro 5, 00185 Rome,
Italy
Fax: +39 06 4440062
Tel: +39 06 49910939
E-mail:
(Received 26 July 2007, revised 8 October
2007, accepted 10 October 2007)
doi:10.1111/j.1742-4658.2007.06137.x

The thermodynamic stability of family 16 endo-b-1,3-glucanase
(EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is
decreased upon single (D287A, E53A) and double (E53A ⁄ D287A) muta-
tion of Asp287 and Glu53. In accordance with the homology model predic-
tion, both carboxylic acids are involved in the composition of a calcium
binding site, as shown by titration of the wild-type and the variant proteins
with a chromophoric chelator. The present study shows that, in P. furiosus,
endo-b-1,3-glucanase residues Glu53 and Asp287 also make up a calcium
binding site in 7.9 m guanidinium chloride. The persistence of tertiary
structure in 7.9 m guanidinium chloride, a feature of the wild-type enzyme,
is observed also for the three variant proteins. The DG
H
2
O
values relative to
the guanidinium chloride-induced equilibrium unfolding of the three vari-
ants are approximatelty 50% lower than that of the wild-type. The
destabilizing effect of the combined mutations of the double mutant is
non-additive, with an energy of interaction of 24.2 kJÆmol
)1
, suggesting a
communication between the two mutated residues. The decrease in the
thermodynamic stability of D287A, E53A and E53A ⁄ D287A is contained
almost exclusively in the m-values, a parameter which reflects the solvent-
exposed surface area upon unfolding. The decrease in m-value suggests that
the substitution with alanine of two evenly charged repulsive side chains
induces a stabilization of the non-native state in 7.9 m guanidinium chlo-
ride comparable to that induced by the presence of calcium on the wild-
type. These results suggest that the stabilization of a compact non-native
state may be a strategy for P. furiosus endo-b-1,3-glucanase to thrive under

adverse environmental conditions.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; BAPTA, 5,5¢-Br
2
-1,2-bis(O-aminophenoxy)ethan-N,N,N¢,N¢-tetraacetic acid; GdmCl, guanidinium
chloride; pfLamA, endo-b-1,3-glucanase from Pyrococcus furiosus; SVD, singular value decomposition.
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6167
which share the same b-jelly roll fold but display nota-
ble differences in their primary structure. Twenty-three
crystal structures of members of this family have been
solved ( however, the crystal
structure of the laminarinase subfamily is still missing
[4,5]. Seventeen out of the 23 available crystal struc-
tures demonstrate the presence of at least one metal
binding site ( />uni-stuttgart.de ⁄ ) [4,5]. Sequence alignments of
pfLamA from different sources suggested that Asp287
[2,6], a residue conserved in most family 16 glycoside
hydrolases, may be part of a calcium binding site
( ⁄ ). According to this
hypothesis, a 3D homology model of pfLamA has pre-
dicted the presence of one or two potential binding
sites for metals and two pairs of negatively charged
amino acid residues have been assumed to be involved
in calcium binding: Glu53 and Asp287, and Glu239
and Glu246 [2].
In the present work, and on the basis of pfLamA
homology modeling prediction [2], residues Glu53 and
Asp287 were replaced with alanine residues by site-
directed mutagenesis, either individually (E53A,
D287A) or simultaneously (E53A ⁄ D287A), in order to

demonstrate their involvement in calcium binding in
native conditions and in the presence of 7.9 m GdmCl.
The thermodynamic stability of pfLamA variant pro-
teins has been studied by GdmCl-induced unfolding
equilibrium experiments. The thermodynamic charac-
terization of the double mutant provided more informa-
tion than a study of single mutants, especially with
respect to the direct or indirect involvement of residues
Glu53 and Asp287 either in electrostatic interactions
with other protein residues or in metal binding [7].
Glu53 and Asp287 are negatively charged at neutral pH
and contribute to the optimization of electrostatic
charges balance of pfLamA in the native state, indepen-
dently of their interaction with calcium. The role of
electrostatic interactions in protein stability has been
widely investigated and the stabilizing effect of salt
bridge networks on the native state of hyperthermophil-
ic proteins has been proposed on the basis of several
computational and experimental studies [8–12]. Studies
of proteins from hyperthermophiles have provided an
array of hypotheses on the structural determinants
responsible for their resistance to denaturation [13,14];
however, a unifying description remains elusive [15].
Investigations on protein stability are also necessary to
advance our skills in designing new catalysts resistant to
temperature and extreme solvent conditions.
In addition to their role in the stabilization of
pfLamA native state, Glu53 and Asp287 could also
contribute to the persistence of residual tertiary struc-
ture in the non-native state in 7.9 m GdmCl [1]. The

study of the properties of non-native states of proteins
has received considerable attention in the last 10 years
because the residual structure within the unfolded state
may play an important role in a protein’s energetics
and function [16–19]. Changes in the denatured states
induced by mutations can therefore affect protein sta-
bility [20,21] and, in thermophilic proteins, the persis-
tence of residual structure in non-native states may
contribute toward avoiding irreversible denaturation
under extreme environmental conditions [22]. Under
denaturing conditions, the spectroscopic characteriza-
tion of the residual structure in proteins is very diffi-
cult, although it has been reported in several studies
[18,23–25].
A measure of the residual structure in the unfolded
form of a protein is the thermodynamic parameter m,
as obtained in equilibrium unfolding studies [26–28].
This parameter represents the rate of change of the
free energy of unfolding as a function of denaturant
concentration and is proportional to the amount of
additional surface area exposed upon unfolding [26].
The m-value may provide information about the resid-
ual structure present in the denatured states of mutants
in comparison to that of the wild-type protein [26,29].
Mutations affecting m-values are more likely to change
the accessible surface area of the unfolded form rather
than that of the native state; thus, a change in m-value
is generally considered to reflect a change in the com-
pactness of the denatured state [26]. The present study
reports on the thermodynamic stability of pfLamA

single mutants E53A, D287A and double mutant
E53A ⁄ D287A, as well as the binding of calcium to the
mutant proteins in native conditions. The single and
combined mutations dramatically decrease the thermo-
dynamic stability of the proteins with a significant
decrease of m-values relative to the GdmCl-induced
unfolding equilibrium. A double-mutant thermody-
namic cycle reveals a non-additive effect of the muta-
tions on the thermodynamic parameters [30,31]. The
effect of the mutations indicates a key role for Glu53
and Asp287 in the interactions responsible for the
residual structure in the non-native state, as well as for
calcium binding of pfLamA in the native state and in
7.9 m GdmCl.
Results
Spectroscopic characterization of the mutants in
native conditions and in 7.9
M GdmCl
In native conditions, the near-UV CD spectra of the
three mutants E53A, D287A and E53A ⁄ D287A are
Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R. Chiaraluce et al.
6168 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS
very similar to those of the wild-type enzyme, except
for minor differences in the ellipticity signals all cen-
tered around the same main aromatic bands of the
native wild-type (Fig. 1A). The fluorescence emission
spectra of native wild-type and mutant enzymes are all
centered at the same maximum emission wavelength of
342 nm and have similar emission fluorescence intensi-
ties (Fig. 1B). Analogously, the far-UV CD spectra are

virtually superimposable (data not shown). These
results indicate that the mutations had no effect on the
secondary and tertiary structure arrangements of the
protein and suggest that, in the native state, the effect
of the mutations are directed and localized to the
mutated residue.
In the presence of 7.9 m GdmCl, and analogous to
that observed with the wild-type pfLamA [1,2], the
near-UV CD spectra of the three mutants indicate
the presence of substantial residual tertiary structure
(Fig. 1C). Minor differences in the near-UV CD spec-
tra of the mutants, in comparison to that of the wild-
type, are evident in the 275–290 nm region, where the
positive ellipticity is progressively reduced in the three
mutants from the D287A to the double mutant, and in
the 260–270 nm region where the negative ellipticity of
the E53A ⁄ D287A mutant is somewhat decreased
(Fig. 1C). The changes observed in the near-UV CD
spectra of the three mutants indicate that, in 7.9 m
GdmCl, the environment of the aromatic residues is
slightly perturbed. In particular, for the double
mutant, the decrease in the dichroic activity around
260 nm suggests that Phe residues are locked differ-
ently in their tertiary contacts compared to the wild-
type (Fig. 1C).
In 7.9 m GdmCl, the maximum fluorescence emis-
sion wavelength of the three mutants is shifted to
357 nm, similar to the wild-type in the same condi-
Fig. 1. Spectral properties of pfLamA wild-type and mutants: effect of GdmCl in the presence and absence of CaCl
2

. Near-UV CD (A, C, E)
and fluorescence (B, D, F) spectra of pfLamA wild-type (– ÆÆ–), D287A (—–), E53A ( ) and E53A ⁄ D287A (– ) –) were recorded at 20 °C
after 20 h of incubation of the protein in native conditions (20 m
M Tris ⁄ HCl, pH 7.4) (A, B) and in 7.9 M GdmCl, pH 7.4, in the absence (C, D)
and presence (E, F) of 40 m
M CaCl
2
. The spectral properties of all the proteins under native conditions are unchanged upon addition of
40 m
M CaCl
2
(data not shown). Near-UV CD spectra (A, C, E) were recorded in a 1-cm quartz cuvette at 0.6 mgÆmL
)1
protein concentration.
Fluorescence spectra (B, D, F) were recorded at 40 lgÆmL
)1
protein concentration (290 nm excitation wavelength).
R. Chiaraluce et al. Role of Glu53 and Asp287 in the stability in 7.9
M GdmCl
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6169
tions, but the relative fluorescence intensities are
increased to a different extent (Fig. 1D). The increase
in relative fluorescence intensity emission at 342 nm is
approximately two-fold for the wild-type and 2.5, 2.6
and 2.8-fold for the D287A, E53A and the double
mutant, respectively (Fig. 1D). Noteworthy, similar to
that reported for the wild-type enzyme [1,2], the far-
UV CD spectra of the three mutants are not affected
by equilibrium incubation at increasing concentrations
of GdmCl up to 7.9 m (data not shown).

Equilibrium transition studies in GdmCl
The effect of increasing GdmCl concentrations (0–8 m)
on the structure of the three mutants was analyzed in
comparison to the effect exerted on the wild-type
pfLamA in 20 mm Tris ⁄ HCl, pH 7.4, containing
100 lm dithiothreitol and 100 lm EDTA. The intrinsic
fluorescence emission intensity of the three mutants
increases after 20 h of incubation at increasing GdmCl
concentrations (Fig. 2) and, in 7.9 m GdmCl, the max-
imal fluorescence emission wavelength shifts to 357 nm
(Fig. 1D). The changes in relative intrinsic fluorescence
emission intensity of the mutants show a sigmoidal
dependence on GdmCl concentration and follow a
two-state denaturation process without any detectable
intermediate, similar to that reported for the wild-type
pfLamA (Fig. 2) [2]. The changes are fully reversible
upon dilution of the denaturant, and the transition
midpoints are at 6.2 ± 0.15 m for D287A and the
double mutant, and at 6.0 ± 0.12 m for E53A
(Fig. 2), with the values being slightly lower than that
of the wild-type pfLamA, which is at 6.7 ± 0.13 m
GdmCl (Fig. 2) [2]. Table 1 shows the thermodynamic
parameters values obtained for wild-type and mutant
forms of pfLamA. The m
g
value of 9.2 kJÆmol
)1
ÆM
)1
of the wild-type is approximately 30% lower than the

value predicted from the number of the aminoacid res-
idues (approximately 13 kJÆmol
)1
ÆM
)1
for 263 amino
acid residues) [28], in accordance with the persistence
of residual structure in 7.9 m GdmCl. The mutants are
thermodynamically less stable than the wild-type, with
a significant decrease of DG
H
2
O
and m
g
values, suggest-
ing that the mutations considerably affect the stability
of pfLamA. The double mutant shows a slightly smal-
ler stability than either the single mutants and the
similarity between the DDG
H
2
O
values of the variant
proteins (Table 1) indicates that the double mutant
E53A ⁄ D287A is more stable than expected from the
sum of the stability change from single mutants E53A
and D287A, and hence the effect of the mutations is
non-additive. Calculation of the energy of interaction
between two mutated residues, DD G

int
, according to
Eqn (5), yields a value of. 24.2 ± 1.87 kJÆmol
)1
.
Effect of calcium on pfLamA mutants in 7.9
M
GdmCl and in native conditions
The addition of 40 mm CaCl
2
to calcium-depleted
samples of D287A and E53A in 7.9 m GdmCl causes
changes in their tertiary structure, but these are much
Fig. 2. GdmCl-induced fluorescence changes of pfLamA wild-type
(r, e), D287A (m, n), E53A (d, s) and E53A ⁄ D287A (j, h). Con-
tinuous lines are the nonlinear regression to Eqn (3) of the fluores-
cence data at varying denaturant concentrations, as described in
the Experimental Procedures. The reversibility points (empty sym-
bols) were not included in the nonlinear regression analysis. All
spectra were recorded at 20 °C after 20 h of incubation at the indi-
cated GdmCl concentrations.
Table 1. Thermodynamic parameters for GdmCl-induced unfolding
equilibrium of pfLamA wild-type and mutants. All data were
obtained at 20 °Cin20m
M Tris ⁄ HCl, pH 7.4, containing 100 lM
dithiothreitol and 100 lM EDTA. DG
H
2
O
and m

g
values were
obtained from Eqn (3); [GdmCl]
0.5
was calculated from Eqn (4).
Data are reported as the mean ± SE of the fit. DDG
H
2
O
¼ DG
H
2
O
mutant ) DG
H
2
O
wild-type. The SE value relative to DDG
H
2
O
was
calculated according to: [SE(DDG
H
2
O)
]
2
¼ [SE(DG
H

2
O
wild-type)]
2
+
[SE(DG
H
2
O
mutant)]
2
.
Protein
[GdmCl]
0.5
(M)
DG
H
2
O
(kJÆmol
)1
)
m
g
(kJÆmol
)1
ÆM
)1
)

DDG
H
2
O
(kJÆmol
)1
)
Wild-type 6.7 ± 0.13 61.5 ± 1.23 9.2 ± 0.18 0
D287A 6.2 ± 0.15 36.5 ± 0.91 5.9 ± 0.15 )25.0 ± 1.53
E53A 6.0 ± 0.12 33.9 ± 0.68 5.6 ± 0.11 )27.6 ± 1.40
E53A ⁄ D287A 6.2 ± 0.15 33.1 ± 0.83 5.3 ± 0.13 )28.4 ± 1.48
Role of Glu53 and Asp287 in the stability in 7.9
M GdmCl R. Chiaraluce et al.
6170 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS
less pronounced than those observed for the wild-type,
indicating the involvement of Glu53 and Asp287 in the
interaction with the cation (Fig. 1E,F). The regain of
aromatic chirality at 295 nm and in the 260–270 nm
region for D287A is similar to that observed for the
wild-type enzyme, whereas it is much less evident for
E53A (Fig. 1E). With the double mutant, the near-UV
CD spectrum in 7.9 m GdmCl shows very minor
changes upon addition of CaCl
2
(Fig. 1E). The intrin-
sic fluorescence emission spectra of the mutants in
7.9 m GdmCl are affected by the presence of 40 mm
CaCl
2
to different extents (Fig. 1F). With D287A, the

intrinsic fluorescence emission intensity at 342 nm is
1.6-fold decreased, similar to the wild-type enzyme,
and the maximum emission wavelength is shifted to
347 nm, 4 nm higher than with the wild-type [2]
(Fig. 1F). For E53A and the double mutant, the
changes of intrinsic emission fluorescence are less evi-
dent: the relative intensities are decreased 1.3-fold and
1.2-fold and the maximum emission wavelengths are
shifted to 350 nm with E53A and to 356 nm with the
double mutant, 7 nm and 13 nm more red-shifted than
that observed with the wild-type [2] (Fig. 1F). The far-
UV CD spectra of the three mutants in 7.9 m GdmCl,
which are the same as those measured in the absence
of denaturant, are not affected by the addition of
CaCl
2
(data not shown).
Changes in both near-UV CD and fluorescence spec-
tra occur by titration in 7.9 m GdmCl with CaCl
2
,
from 0.2 nm to 150 mm unchelated Ca
2+
, although
with remarkably different amplitudes, depending on
the protein form (Fig. 3). The amplitude of the elliptic-
ity changes at 295 nm decreases from the wild-type to
D287A and E53A and, above 200 lm Ca
2+
, no further

changes are observed (Fig. 3A). For the double
mutant, only minor changes in the dichroic activity at
295 nm are detected over the whole range of the cation
concentration (Fig. 3A). Nonlinear regression analysis
of the [Q]
295
data for the wild-type pfLamA was used
to define two limiting slopes, intersecting at a value
which suggests that 2 mol of Ca
2+
per mol of enzyme
are required to reach an apparent saturation effect
(Fig. 3A) [2]. The changes in the fluorescence proper-
ties in 7.9 m GdmCl induced by CaCl
2
, represented by
a blue-shift of the maximum emission wavelength and
by a quenching of the fluorescence intensities (Fig. 1F),
are reported in Fig. 3B as the intensity-averaged emis-
sion wavelength (

k) calculated according to Eqn (1)
and follow a hyperbolic dependence on CaCl
2,
similar
to that observed for [Q]
295
.
In native conditions, the addition of 40 mm CaCl
2

to the mutants did not affect the near-UV, the far-UV
CD and the fluorescence properties (data not shown),
similar to that reported for the pfLamA wild-type [2].
The interaction of the mutants with calcium, in native
conditions, was studied by titration with CaCl
2
in the
Fig. 3. Interaction of calcium with pfLamA wild-type and mutant
forms in 7.9
M GdmCl. (A) Left axis: [Q]
295
of wild-type (e) and
D287A (n); right axis: [Q]
295
of E53A (s) and E53A ⁄ D287A (h)
measured from near UV-CD spectra at 22 l
M protein concentration.
(B) Intensity-averaged emission wavelength

k of wild-type (e),
D287A (n), E53A (s) and E53A ⁄ D287A (h) measured at 1.2 l
M
protein concentration from fluorescence spectra (290 nm excitation
wavelength). All spectra were recorded at 20 °C, 5 min after each
Ca
2+
addition. [Q]
295
is reported after removal of the high-frequency
noise and the low-frequency random error by the singular value

decomposition algorithm (SVD) [2] in the spectral region 250–
310 nm.

k was calculated according to Eqn (1). The two limiting
slopes, calculated by nonlinear regression analysis to the [Q]
295
and
to

k data, intersect at a point corresponding to [Ca
2+
unchelat-
ed] ⁄ [protein] ¼ 2. The reported unchelated Ca
2+
concentrations
intervals, calculated according to [49], are 0.2 n
M to 140 mM and
0.2 n
M to 7.6 mM for [Q]
295
and fluorescence changes, respec-
tively.
R. Chiaraluce et al. Role of Glu53 and Asp287 in the stability in 7.9
M GdmCl
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6171
presence of the chromophoric chelator 5,5¢-Br
2
-1,2-
bis(O-aminophenoxy)ethan-N,N,N¢,N¢-tetraacetic acid
(BAPTA) and compared with the results obtained with

the wild-type enzyme [2]. The fitting of the titration
data by caligator software [32] allows a quantitative
determination of the corresponding binding constants,
as reported in Table 2. All the mutants bind calcium
with a significantly lower affinity compared to the
wild-type. Similar to that observed for the wild-type,
the v
2
obtained by fitting the data of E53A and
D287A titration to a two calcium binding sites model
are both lower than that obtained by fitting the same
data to a one calcium binding site model (Table 2). In
the case of the double mutant, the v
2
value relative to
the fitting to a one calcium binding site model is lower
than that relative to the fitting to a two calcium bind-
ing sites model. Furthermore, the higher value of the
second binding constant suggests that only one of the
two binding sites may be functional in E53A ⁄ D287A
(Table 2).
Effect of calcium on the equilibrium transitions
in GdmCl
Incubation of pfLamA mutants at increasing GdmCl
concentrations (0–8 m)in20mm Tris ⁄ HCl, pH 7.4,
containing 100 lm dithiothreitol, 100 lm EDTA and
40 mm CaCl
2
for 20 h at 20 °C results in changes in
the intrinsic fluorescence emission (Fig. 4). The

GdmCl-induced unfolding process in the presence of
40 mm CaCl
2
is reversible and, by contrast to the wild-
type (Fig. 4A), does not follow a simple two-state
mechanism, as suggested by the lack of coincidence of
the changes in relative fluorescence intensity and in

k
and by the hysteresis of the reversibility process
(Fig. 4). At the end of the transition, the intrinsic fluo-
rescence emission intensity at 342 nm is increased
1.7-fold for D287A, 2.1-fold for E53A and 2.4-fold for
the double mutant and the fluorescence maximum
emission wavelength is shifted to 347 nm for D287A,
350 nm for E53A and to 356 nm for the double
mutant (Fig. 4, insets). Notably, in 7.9 m GdmCl and
40 mm CaCl
2
, the maximum fluorescence emission
wavelength of the wild-type was still centred at 342 nm
[2]. The fluorescence emission spectra of the three vari-
ants measured after incubation in 7.9 m GdmCl and
40 mm CaCl
2
are comparable with those resulting from
the progressive addition of CaCl
2
to the proteins in
7.9 m GdmCl (Fig. 1F, Fig. 4, insets).

8-Anilinonaphthalene-1-sulfonic acid ammonium
salt (ANS) fluorescence and acrylamide
quenching
The amphiphilic dye ANS has affinity for hydrophobic
clusters present in tertiary structure elements, which
are not tightly packed within a fully folded structure.
The accessibility of hydrophobic residues of pfLamA
wild-type and variant proteins in 7.9 m GdmCl was
compared with that in native conditions, by analysis
with the fluorescent probe ANS. The fluorescence
emission spectrum of ANS in the presence of all the
variant proteins in 7.9 m GdmCl shows a modest, two-
fold increase in intensity compared to that in native
conditions, without any change in the maximum fluo-
rescence emission wavelength (results not shown). This
suggests that, in 7.9 m GdmCl, the hydrophobic sur-
face area of the mutants is not significantly exposed,
similar to that observed for the wild-type. The
uncharged fluorescence quencher acrylamide was used
to probe the accessibility of the hydrophobic core and
the dynamic properties of the three mutants in com-
parison to the wild-type in native conditions and in
7.9 m GdmCl. Effective acrylamide quenching con-
stants from the modified Stern–Vollmer plots for the
proteins in the native state were 7.9 m
)1
, 8.6 m
)1
,
8.4 m

)1
and 9.4 m
)1
for the wild-type and D287A,
E53A and the double mutant, respectively. In 7.9 m
GdmCl, the acrylamide quenching constants
were 13.0 m
)1
, 9.9 m
)1
, 11.5 m
)1
and 10.3 m
)1
for the
wild-type, D287A, E53A and the double mutant,
respectively. A quantitative analysis of the data is not
Table 2. Calcium binding constants for pfLamA wild-type and mutants determined in the presence of the chromophoric chelator BAPTA.
v
2
represents the best fit of the absorbance data. Replicate determinations indicate a standard deviation for the calcium binding constants
K
1
and K
2
less than 5%.
Protein
2Ca
2+
binding sites 1 Ca

2+
binding site
K
1
(M
)1
) K
2
(M
)1
) v
2
K
1
(M
)1
) v
2
Wild-type 5.0 · 10
7
2.6 · 10
5
9.8 · 10
)5
3.3 · 10
7
2.9 · 10
)4
D287A 2.7 · 10
5

2.6 · 10
4
2.5 · 10
)4
3.4 · 10
5
3.1 · 10
)4
E53A 1.4 · 10
6
1.5 · 10
5
9.6 · 10
)4
1.1 · 10
6
1.7 · 10
)3
E53A ⁄ D287A 4.6 · 10
5
2.5 · 10
1
3.0 · 10
)3
4.4 · 10
5
3.1 · 10
)4
Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R. Chiaraluce et al.
6172 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS

possible because pfLamA wild-type and variant pro-
teins are heterogeneously emitting systems; however,
the results indicate that the fluorophores accessibility
of the protein variants to the uncharged quencher, in
comparison with the wild-type, decreases in 7.9 m
GdmCl and increases in native conditions.
Discussion
The results obtained with pfLamA mutant forms indi-
cate that residues Glu53 and Asp287 are involved in
calcium binding, in accordance to the homology mod-
elling. The pfLamA single (D287A and E53A) and
double (E53A ⁄ D287A) mutants in 7.9 m GdmCl show
a residual tertiary structure comparable to that of the
wild-type; however, the integrity of the calcium bind-
ing site formed by Asp287 and Glu53 is essential for
interaction with the cation in 7.9 m GdmCl.
An interesting finding of the present study is the sig-
nificant decrease in the thermodynamic stability of the
three pfLamA mutants in comparison to the wild-type,
which shows a high DG
H
2
O
value of 61.5 kJÆmol
)1
asso-
ciated with its partial unfolding [2]. The DG
H
2
O

associ-
ated with the reversible fluorescence changes at
increasing GdmCl concentration is decreased from 1.7-,
1.8- to 1.9-fold with respect to the wild-type pfLamA,
going from D287A, to E53A and to the double mutant,
respectively. Notably, the transition midpoints for the
fluorescence changes of the three mutants are not sig-
nificantly changed with respect to pfLamA wild-type;
hence, the decrease of DG
H
2
O
values is mainly due to a
decrease in m
g
values. The decrease in m
g
value
observed in all the pfLamA variant proteins is signifi-
cant (1.6-fold) and not unprecedented for other single
[22,33,34] and double-mutant proteins [7]. The mecha-
nism responsible for m
–
mutant proteins, which display
a m
g
value lower than that of the wild-type, is usually
referred to a decrease in the solvent-exposed surface
area upon unfolding. This is more frequently ascribed
to an increase in the compactness of the residual struc-

ture in the non-native state ensemble, rather than to an
Fig. 4. GdmCl-induced fluorescence changes of pfLamA mutant
forms in the presence of CaCl
2.
(A) D287A, (B) E53A and (C)
E53A ⁄ D287A. Fluorescence changes are reported as relative fluores-
cence intensity at 342 nm (left axis: j, h) and as intensity-averaged
emission wavelength

k (right axis: d, s) calculated according to
Eqn (1). The wild-type reversible transition in the presence of 40 m
M
CaCl
2
monitored by relative fluorescence intensity at 342 nm (left
axis: e, r) is also shown in (A) for comparison [2]. The solid lines
through the mutants unfolding data points (filled symbols) are
intended to guide the eye of the reader and do not represent the fit-
ting of the data. Reversibility points are indicated by empty symbols.
All spectra were recorded at 20 °C after 20 h of incubation at the
indicated GdmCl concentrations at 40 lgÆmL
)1
protein concentration.
Insets show intrinsic fluorescence emission spectra of the mutants
measured after 20 h of incubation in 7.9
M GdmCl and 40 mM CaCl
2
(continuous), the spectra resulting from the addition of CaCl
2
to the

mutants after 20 h of incubation in 7.9
M GdmCl (dotted) and the
spectra of the native mutants in 20 m
M Tris ⁄ HCl pH 7.4 (dashed). All
spectra were recorded at 20 °C (290 nm excitation wavelength).
R. Chiaraluce et al. Role of Glu53 and Asp287 in the stability in 7.9
M GdmCl
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6173
increase of the accessible surface area of the native state
[26,27,29]. Similar to that reported for most m
–
mutants
[16], the spectral properties of the pfLamA variants in
7.9 m GdmCl do not indicate to a significant increase
in the structure of the non-native state to support the
significant decrease in m
g
. Consistent with these results
and similar to that observed for the wild-type, the ANS
binding experiments indicate that, in 7.9 m GdmCl, the
hydrophobic surface area of pfLamA mutants is not
significantly exposed. An increase in compactness of
the non-native state ensemble of the variants is sug-
gested by the decreased fluorophores accessibility to the
uncharged quencher acrylamide in 7.9 m GdmCl com-
pared to that of the wild-type. In native conditions, the
spectral properties of the three variants point to tertiary
structures almost identical to that of the wild-type;
however, the higher fluorophores accessibility to the
uncharged quencher suggests a less compact native

state for the three mutant proteins. A decrease in m
g
value upon single mutation has been also referred, in
some cases, to the population of a third intermediate
state during chemical unfolding [35]; however, in our
experimental conditions, the presence of an intermedi-
ate state was not observed for any of the pfLamA vari-
ants. Thus, the decrease of m
g
value may be ascribed to
an increase in the compactness of the non-native state
in 7.9 m GdmCl.
The effect of the double mutation on the thermody-
namic parameters was non-additive, being lower than
the sum of the effects of the two single mutations.
Non-additivity is generally observed when two mutated
residues communicate directly or indirectly through
electrostatic interactions or structural perturbation, so
that they do not behave independently [7,36]. There-
fore, the comparison of the thermodymamic parame-
ters of the pfLamA double mutant with those of single
mutants may provide information about any direct or
indirect interconnection between the two mutated resi-
dues. The non-additivity of the stability change can be
expressed by the free energy coupling DDG
int
, a param-
eter calculated from a double-mutant cycle (Scheme 1)
that reflects the interaction energy between the two
mutated residues, Asp287 and Glu53. The positive

value of the interaction free energy between the two
mutated residues in pfLamA indicates that the double
mutant is more stable than predicted, on the assump-
tion that the effects of the two single mutants would
be additive. A significant DDG
int
(above 20 kJÆmol
)1
)
has been related either to a direct communication
between two residues or a short range steric interaction
involving a mediating residue or a ligand [37]. The
large interaction free energy between the two pfLamA
mutated residues (DDG
int
¼ 24.2 ± 1.87 kJÆmol
)1
)is
in accordance with the calcium binding results
described in the present study. The analysis of electro-
static interactions in pfLamA model, including histi-
dine residues and considering a distance threshold of
6A
˚
, reveals that Glu53 may be involved in a large salt
bridge network of seven ion pairs, three of which are
strong ion pairs (distance threshold of 4 A
˚
) whereas
the Asp287 is involved in three ion pairs, only one of

which is a strong ion pair (Fig. 5). The putative cal-
cium binding site is localized in proximity of the well
conserved Asp287 residue in family 16 [2,6]. The
homology model suggests ionic interactions between
Fig. 5. Model structure of the calcium binding site of wild-type
pfLamA. The pfLamA model is represented in teal blue cartoons.
The two acidic residues binding calcium and those with which they
form ion pair interactions are shown as stick models with superim-
posed violet CPK space-filling models. Carbon, oxygen and nitrogen
atoms are displayed with green, red and blue colors, respectively.
Calcium ion is represented as an Nb sphere model with yellow
color and a superimposed violet CPK space-filling model. Orange
dashes indicate ion pair interactions. This figure was rendered using
PYMOL [50].
Scheme 1
Role of Glu53 and Asp287 in the stability in 7.9
M GdmCl R. Chiaraluce et al.
6174 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS
the cation and the carboxylate moiety of both Glu53
and Asp287 (Fig. 5). In native conditions, the spectro-
scopic studies of pfLamA variant proteins with the
chromophoric chelator BAPTA show the involvement
of Glu53 and Asp287 in the interaction with calcium
and indicate that a second binding site might be pres-
ent, as predicted by the homology model [2]. In 7.9 m
GdmCl, the capability to interact with calcium with
a consistent recovery of tertiary structure is still
observed, to a lesser extent than the wild-type, for
D287A and E53A, but not for D287A ⁄ E53A. Hence,
the integrity of the calcium binding site formed by

Asp287 and Glu53 is essential for interaction with
Ca
2+
in 7.9 m GdmCl. The second calcium binding
site revealed in native conditions by the chromophoric
chelator BAPTA and located by modeling between
Glu239 and Glu246 [2], either does not bind calcium
in 7.9 m GdmCl or it interacts with the cation without
affecting the enzyme tertiary structure.
In the presence of calcium, the GdmCl equilibrium
transitions are complex and hysteretic for all the three
mutants, indicating that the cation may stabilize some
refolding intermediate(s) and ⁄ or increase the popula-
tion of some states that are not evident under calcium
depletion. The comparison with the simple two state
GdmCl transition of the wild-type in the presence of
calcium [2] suggests that the integrity of the cation
binding site formed by Glu53 and Asp287 prevents the
population of folding intermediates.
The single or double substitution of Glu53 and
Asp287 by alanine decreases the capability of pfLamA
to interact with calcium as well as its thermodynamic
stability but not its intrinsic resistance to denaturation,
as indicated by the minor differences in the transition
midpoints. The destabilizing effect appears to be
mainly realized through a stabilization of the non-
native state in 7.9 m GdmCl, rather than a destabiliza-
tion of the native state, as suggested by the decrease
of m
g

and of fluorophores accessibility to acrylamide
for all the variants compared to the wild-type. The
replacement by Ala of any of the negatively charged
residues shown to be involved in the composition of a
calcium binding site induces a stabilization of the non-
native state in 7.9 m GdmCl comparable, but not iden-
tical, to that exerted by calcium on the wild-type [2].
Ion pair networks play an important role in protein
stability [8], and their involvement in interactions with
cations offers new perspective in the analysis of the
contribution of ions as cofactors in protein folding [38]
and in the design of variants of proteins with enhanced
stability [14]. Changes in the denatured states induced
by mutation affect protein stability [20,21] and, for
thermophilic proteins, the persistence of residual
structure in non-native states may contribute to avoid
irreversible denaturation under extreme environmental
conditions [22]. The stabilization of a compact non-
native state may represent a strategy for P. furiosus
endo-b-1,3-glucanase to thrive under the most adverse
environmental conditions.
Experimental procedures
Site-directed mutagenesis
The pfLamA D287A mutant was prepared by overlap exten-
sion PCR [39], using the wild-type construct pET9d::LamA
as template [6], with the primers 5¢-GCAAAG
ATGGTGGTGG
CATATGTAAGGGTTTAC-3¢ (sense)
and 5¢-GTAAACCCTTACATA
TGCCACCACCATCT

TTGC-3¢ (antisense). The E53A mutant and the double
mutant (E53A ⁄ D287A) of pfLamA were produced using as
primers 5¢-GCACGATG
CGTTTGAAGG-3¢, and its com-
plementary oligonucleotide. The mutated bases are under-
lined. The mutant forms of pfLamA E53A and
E53A ⁄ D287A were produced using as template the wild-type
construct pET9d::LamA [6] and the mutant construct
pET24d::LamA D287A, respectively, using the Quik-
Change
TM
site-directed mutagenesis kit from Stratagene
(La Jolla, CA, USA). The kit employs double-stranded
DNA as template, two complementary oligonucleotide prim-
ers containing the desired mutation, and DpnI endonuclease
to digest the parental DNA template. Oligonucleotides were
synthesized by MWG-Biotech AG (Anzinger, Germany).
Escherichia coli strain DH5a cells were transformed.
The coding regions of the mutated pfLamA gene were
sequenced to confirm the mutations and then E. coli
strain HMS174 (DE3) cells were transformed and used for
expression.
Enzyme preparation and assay
pf LamA wild-type was functionally produced in E. coli
BL21(DE3) strain, and the three mutant forms were func-
tionally produced in E. coli HMS174(DE3) strain and puri-
fied according to Kaper et al. [40]. The conditions used for
expression and purification of the mutant proteins in E. coli
were as described for the wild-type enzyme. The protein
concentration was determined for wild-type and mutant

forms at 280 nm using e
280
¼ 83070 m
)1
· cm
)1
calculated
according to Gill and von Hippel [41]. The enzyme activity
was determined by measuring the amount of reducing sug-
ars released upon incubation in 0.1 m sodium phosphate
buffer, pH 6.5, containing 5 mgÆmL
)1
of laminarin, at 60
or 80 °C for 10 min, as described previously [40]. Calcium-
depleted protein was obtained by extensive dialysis with
100 lm EDTA and 100 lm EGTA in 10 mm Tris ⁄ HCl,
pH 7.4. All the precautions required to prevent Ca
2+
R. Chiaraluce et al. Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6175
contamination were followed during the preparation and
storage of protein and buffer solutions [32]. Calcium-loaded
protein refers to the protein in the presence of 40 mm CaCl
2
.
Chemicals and buffers
ANS, dithiothreitol, EDTA, GdmCl, and laminarin were
from Fluka (Buchs, Switzerland). 3¢,5¢-dinitrosalicylic acid
was purchased from Sigma (St Louis, MO, USA). BAPTA
was from Molecular Probes Europe BV (Leiden, the Neth-

erlands). Buffer solutions were filtered (0.22 lm) and care-
fully degassed. All buffers and solutions were prepared
with ultra-high quality water (ELGA UHQ, High Wy-
combe, UK). Buffers for calcium titrations were prepared
as previously described [32].
Spectroscopic techniques
Intrinsic fluorescence emission and 90° light scattering mea-
surements were carried out with a LS50B Perkin Elmer
spectrofluorimeter (Perkin Elmer, Waltham, MA, USA)
using a 1.0-cm pathlength quartz cuvette. Fluorescence
emission spectra were recorded at 300–450 nm (1 nm sam-
pling interval) at 20 °C with the excitation wavelength set
at 290 nm. 90° light scattering was measured at 20 °C with
both excitation and emission wavelength set at 480 nm to
check for the presence of aggregated particles.
Far-UV (185–250 nm) and near-UV (250–320 nm) CD
measurements were performed at 20 °C in a 0.1–0.2-cm and
1.0-cm pathlength quartz cuvette, respectively. CD spectra
were recorded on a Jasco J-720 spectropolarimeter (Jasco
Inc., Easton, MD, USA). The results are expressed as the
mean residue ellipticity [Q] assuming a mean residue weight
of 110 per amino acid residue. In all the spectroscopic mea-
surements, 100–250 lm EDTA was always present unless
otherwise stated.
Experiments with the fluorescent dye ANS were per-
formed at 20 °C by incubating each protein sample, wild-
type and variant proteins, with ANS at 1 : 20 molar ratio.
After 5 min, fluorescence emission spectra were recorded at
400–600 nm with the excitation wavelength set at 390 nm.
The maximum fluorescence emission wavelength and

the intensity of the hydrophobic probe ANS depend on the
environmental polarity (e.g. on the hydrophobicity of the
accessible surface of the protein) [42].
Fluorescence quenching was carried out by adding
increasing amounts of acrylamide (0–104 mm) to solutions
containing wild-type or variant proteins (40 lgÆmL
)1
)in
20 mm Tris ⁄ HCl, pH 7.4, 100 lm dithiothreitol and
100 lm EDTA. Emission spectra (300–400 nm) were
recorded at 20 °C 5 min after each acrylamide addition
with the excitation wavelength set at 290 nm. The effective
quenching constants were obtained from modified Stern–
Vollmer plots by analyzing F
0
⁄ DF versus 1 ⁄ [acrylamide]
(23 data points) [43].
GdmCl-induced unfolding and refolding
For equilibrium transition studies, protein samples (final
concentration 40–50 lgÆmL
)1
) were incubated at 20 °Cat
increasing concentrations of GdmCl (0–8 m)in20mm
Tris ⁄ HCl, pH 7.4, containing 100 lm dithiothreitol and
100 lm EDTA and, when indicated, 40 mm CaCl
2
. After
20 h, the equilibrium was reached and intrinsic fluores-
cence emission and far-UV CD spectra (0.2-cm cuvette)
were recorded in parallel at 20 °C. To test the reversibil-

ity of the unfolding, protein samples were unfolded at
20 °C in 7.8 m GdmCl at 0.8 mgÆmL
)1
protein concentra-
tion in 25 mm Tris ⁄ HCl, pH 7.4, containing 100 lm
dithiothreitol and 100 lm EDTA, in the presence and
absence of 40 mm CaCl
2
. After 20 h, refolding was
started by 20-fold dilution of the unfolding mixture, at
20 °C, into solutions of the same buffer used for unfold-
ing containing decreasing GdmCl concentrations. The
final protein concentration was 40 lgÆmL
)1
. After 24 h,
which had been established as sufficient to reach equilib-
rium, intrinsic fluorescence emission and far-UV CD spec-
tra were recorded at 20 °C.
Data analysis
The changes in intrinsic fluorescence emission spectra at
increasing GdmCl concentrations were quantified as the
intensity-averaged emission wavelength,

k [44] calculated
according to

k ¼ RðI
i
k
i

Þ=RðI
i
Þð1Þ
where k
i
and I
i
are the emission wavelength and its corre-
sponding fluorescence intensity at that wavelength, respec-
tively. This quantity is an integral measurement, negligibly
influenced by the noise, which reflects changes in the shape
and position of the emission spectrum. Far-UV CD and
near-UV CD spectra from GdmCl and Ca
2+
titrations were
analyzed by the singular value decomposition (SVD) algo-
rithm [1,45] using the software matlab (MathWorks, South
Natick, MA, USA).
SVD is useful to find the number of independent com-
ponents in a set of spectra and to remove the high-fre-
quency noise and the low-frequency random error. CD
spectra in the 210–250 nm region or in the 250–310 region
(0.2 nm sampling interval) were placed in a rectangular
matrix A of n columns, one column for each spectrum
collected in the titration. The A matrix is decomposed by
SVD into the product of three matrices: A ¼ U*S*V
T
where U and V are orthogonal matrices and S is a diago-
nal matrix. The columns of U matrix contain the basis
spectra and the columns of the V matrix contain the

denaturant or the Ca
2+
dependence of each basis spec-
trum. Both U and V columns are arranged in terms of
their decreasing order of the relative weight of informa-
Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl R. Chiaraluce et al.
6176 FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS
tion, as indicated by the magnitude of the singular values
in S. The diagonal S matrix contains the singular values
that quantify the relative importance of each vector in U
and V. The signal-to-noise ratio is very high in the earliest
columns of U and V and the random noise is mainly
accumulated in the latest U and V columns. The wave-
length averaged spectral changes induced by increasing
denaturant or Ca
2+
concentrations are represented by the
columns of matrix V; hence, the plot of the columns of V
versus the denaturant or Ca
2+
concentrations provides
information about the observed transition.
GdmCl-induced equilibrium unfolding was analyzed by
fitting baseline and transition region data to a two-state lin-
ear extrapolation model [46] according to:
DG
unfolding
¼ DG
H
2

O
þ m
g
½GdmCl¼ÀRT ln K
unfolding
ð2Þ
where DG
unfolding
is the free energy change for unfolding
for a given denaturant concentration, DG
H
2
O
is the free
energy change for unfolding in the absence of denaturant
and m
g
is a slope term that quantitates the change in
DG
unfolding
per unit concentration of denaturant, R is
the gas constant, T is the temperature and K
unfolding
is the
equilibrium constant for unfolding. The model expresses
the signal as a function of denaturant concentration:
y
i
¼
y

N
þ m
N
½X
i
þðy
D
þ m
D
½X
i
Þ
Ã
exp½ðÀDG
H
2
O
À m
g
½X
i
Þ=RT
1 þ exp½ðÀDG
H
2
O
À m
g
½X
i

Þ=RT
ð3Þ
where y
i
is the observed signal, y
N
and y
D
are the native
and denatured baseline intercepts, m
N
and m
D
are the
native and denatured baseline slopes, [X]
i
is the denaturant
concentration after the ith addition, DG
H
2
O
is the extrapo-
lated free energy of unfolding in the absence of denaturant,
m
g
is the slope of a G unfolding versus [X] plot, R is the
gas constant and T is the temperature. [GdmCl]
0.5
is the
denaturant concentration at the midpoint of the transition

and, according to Eqn (2), is calculated as:
½GdmCl
0:5
¼ DG
H
2
O
=m
g
ð4Þ
The free energy coupling parameter DDG
int
, which reflects
the interaction energy between the two mutated residues
Asp287 and Glu53, is calculated from a double mutant
cycle (Scheme 1) [47] where the changes in the free energy
relative to the GdmCl unfolding are denoted by
DG
D287AÀWT
¼ DG
WT
À DG
D287A
; DG
E53A=D287AÀE53A
¼ DG
E53A
À DG
E53A=D287A
; DG

E53AÀWT
¼ DG
WT
À DG
E53A
; DG
E53A=D287AÀD287A
¼ DG
D287A
À DG
E53A=D287A
where DG is the free energy change between the denatured
and the native state for pfLamA wild-type (WT) and the
protein variants E53A, D287A and E53A ⁄ D287A. Hence,
the free energy coupling parameter DDG
int
is calculated by:
DDG
int
¼ DG
E53AÀWT
À DG
E53A=D287AÀD287A
¼ DG
D287AÀWT
À DG
E53A=D287AÀE53A
ð5Þ
The standard error (SE) for determination of DDG
int

was
determined by:
ðSE
DDGint
Þ
2
¼ðSE
DGE53A=D287A
Þ
2
þðSE
DGE53A
Þ
2
þðSE
DGD287A
Þ
2
þðSE
DGWT
Þ
2
ð6Þ
Calcium titrations and determination of binding
constants
Calcium-depleted wild-type and mutant forms of pfLamA
(9–16 lm) were titrated with CaCl
2
in the presence of
24 lm of the chromophoric chelator BAPTA [48].

BAPTA concentration was determined by measuring the
absorbance at 263 nm using e
239.5
¼ 1.6 · 10
4
m
)1
Æcm
)1
[32]. Titrations were performed at 20 °Cin10mm
Tris ⁄ HCl pH 7.5 by the addition of 1–2 lL of CaCl
2
solutions ranging from 0.015 mm to 20.0 mm to a 1 mL
protein solution containing 24 lm BAPTA. Absorbance
spectra were monitored between 200–450 nm after each
CaCl
2
addition. To determine protein binding constants
and number of binding sites, the variation of BAPTA
absorbance as a function of calcium addition was fitted
by nonlinear analysis using the caligator software [32].
The v
2
value as calculated by the program was used as
the measure of the goodness-of-fit. All the precautions
required to prevent Ca
2+
contamination were followed
during the preparation and storage of the proteins and
buffer solutions [32].

Calcium titration of calcium-depleted wild-type and
mutant forms of pfLamA in 7.9 m GdmCl (in 25 mm
Tris ⁄ HCl, pH 7.4 containing 200 lm dithiothreitol and
250 lm EDTA) was performed by addition of increasing
CaCl
2
concentrations (0–40 mm) under continuous stir-
ring. Five minutes after each CaCl
2
addition, near-UV
CD (240–320 nm, 22 lm protein concentration) and
fluorescence (300–450 nm, 1.2 l m protein concentration)
spectra were recorded at 20 °C. The spectral changes
observed after each CaCl
2
addition were not affected by
a longer incubation time. The concentration of unchelated
Ca
2+
was calculated using the program WINMAXC, version
2.40 [49] ( />Acknowledgements
This work was supported by a Grant from ‘Progetti
strategici MIUR Legge 499⁄ 97’, Project Genefun and
from FIRB 2003 RBNE034XSW. We thank Dr
Roberto Contestabile for helpful discussion.
R. Chiaraluce et al. Role of Glu53 and Asp287 in the stability in 7.9 M GdmCl
FEBS Journal 274 (2007) 6167–6179 ª 2007 The Authors Journal compilation ª 2007 FEBS 6177
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