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

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

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

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

. 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).

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

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

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

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

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

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).

)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

2.6 · 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

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-

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

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

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

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

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

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

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

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

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

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].

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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