Parallel Measurement of Ca2+ Binding and Fluorescence
Emission upon Ca2+ Titration of Recombinant Skeletal Muscle
Troponin C
MEASUREMENT OF SEQUENTIAL CALCIUM BINDING TO THE REGULATORY
SITES*
Fernando
Fortes de Valencia
,
Adriana Aparecida
Paulucci§,
Ronaldo Bento
Quaggio,
Ana
Cláudia
Rasera da Silva,
Chuck S.
Farah, and
Fernando
de Castro Reinach
From the Departamento de Bioquímica, Instituto de
Química, Universidade de São Paulo, CP 26 077, São Paulo SP CEP 05599-970, Brazil
Received for publication, September 27, 2002, and in revised form, January 13, 2003
 |
ABSTRACT |
Calcium binding to chicken recombinant
skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W),
5-hydroxytryptophan (F29HW), or 7-azatryptophan
(F29ZW) at position 29 was measured by flow dialysis and by
fluorescence. Comparative analysis of the results allowed us to
determine the influence of each amino acid on the calcium binding
properties of the N-terminal regulatory domain of the protein. Compared
with TnC, the Ca2+ affinity of N-terminal sites was:
1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW,
and 3) decreased slightly in F29HW. The Ca2+
titration of F29ZW monitored by fluorescence displayed a
bimodal curve related to sequential Ca2+ binding to the two
N-terminal Ca2+ binding sites. Single and double mutants of
TnC, F29W, F29HW, and F29ZW were constructed by
replacing aspartate by alanine at position 30 (site I) or 66 (site II)
or both. Ca2+ binding data showed that the Asp
Ala
mutation at position 30 impairs calcium binding to site I only, whereas
the Asp
Ala mutation at position 66 impairs calcium binding to both
sites I and II. Furthermore, the Asp
Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for
replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or
7-azatryptophan. We conclude that position 29 influences the affinity
of site I and that Ca2+ binding to site I is dependent on
the previous binding of metal to site II.
 |
INTRODUCTION |
One of the first events in skeletal muscle contraction is calcium
binding to the N-terminal sites of troponin C
(TnC).1 Structural
modifications resulting from this association are thought to be
transmitted to other components of the thin filament, which ultimately
allows the effective association of myosin to actin and force
generation (for review, see Refs. 1-4). TnC has a dumbbell-shaped
structure with the globular N-terminal and C-terminal domains separated
by a nine-turn
-helix (5, 6). Calcium binds with high affinity
(Kd
10
7 M) to the
two EF-hand Ca2+/Mg2+ binding sites located at
the TnC C-terminal domain (sites III and IV, the C-terminal sites) and
with low affinity (Kd
10
5
M) to the two EF-hand Ca2+-specific binding
sites located in the TnC N-terminal domain (sites I and II, the
N-terminal sites) (7). Because of the affinity and kinetic properties
of the C-terminal sites they are believed to be always occupied by
Ca2+ or Mg2+ in vivo and are
involved in the stable TnC binding to TnI (8). Ca2+ binding
to the two TnC N-terminal sites results in a greatly increased affinity
for the TnI C-terminal and inhibitory regions, resulting in their
dissociation from actin and disinhibition of the actomyosin interaction.
The affinity and cooperativity of calcium binding to TnC have been
studied extensively. These parameters are determined through direct
measurement of free calcium concentration in association systems
(direct techniques) (7, 9-11) or through spectroscopically monitored
titrations (indirect techniques) (12-31). These studies largely agree
on the value of the binding constants but disagree with regard to the
cooperativity parameter. Cooperativity between N-terminal sites has
been detected only by indirect techniques, raising the possibility that
potentially damaging conditions, the extensive time needed to prepare
samples, or the intrinsically high errors involved in direct techniques
disturb or conceal the N-terminal site cooperativity. In one of the
studies using indirect techniques (25) we constructed a spectral probe
mutant of recombinant skeletal chicken TnC, named TnCF29W (or simply
F29W), in which Phe-29 was replaced by Trp. This position is
immediately adjacent to Ca2+ binding site I (residues
30-41), and the substitution was expected to cause only minor
alterations in both apo and Ca2+-saturated forms of the
wild type N-terminal domain structure. Upon Ca2+ binding to
F29W N-terminal sites there is a 3-fold increase in the intensity of
fluorescence emission at 340 nm. This mutant binds Ca2+
with a Hill coefficient of 1.96, reflecting a high degree of cooperativity between sites I and II. High values of cooperativity for
N-terminal sites were also detected by far-UV CD measurements of wild
type chicken recombinant TnC (23, 26). Later reports, however,
indicated that the properties of the F29W N-terminal domain are
different from those of TnC (29, 32, 33), and NMR studies using the
isolated N-terminal domain of TnC indicated that sites I and II are
sequentially occupied (with a nearly 10-fold affinity difference) with
no cooperativity between them (32).
To resolve this discrepancy and to understand the influence of position
29 of chicken TnC on the properties of N-terminal sites, we introduced
the amino acid analogs 5-hydroxytryptophan (5-HW) and
7-azatryptophan (7-ZW) into position 29 of TnC (35-40). Ca2+ binding by these new mutants (F29HW and
F29ZW, respectively), as well as F29W and TnC was analyzed
using a direct flow dialysis calcium binding assay and indirectly by
fluorescence. We have also constructed fluorescent and nonfluorescent
mutants with Asp
Ala replacements at position X of sites I or II or
both sites to assert the dependence of N-domain calcium binding
properties on each site. These mutants were submitted to flow dialysis
and/or fluorescence assays. Our data indicate that: 1) tryptophan at position 29 increases the N-terminal site calcium affinity nearly 6-fold compared with TnC; 2) 5-HW at position 29 lowers the
N-terminal site calcium affinity to levels slightly below those of TnC;
3) 7-ZW at position 29 increases the N-terminal site
affinities to an intermediate level, between the F29W and TnC
N-terminal site affinities; 4) the flow dialysis data obtained with Asp
Ala mutants indicate that when Asp at the first position (X) of
site II is replaced by Ala, site I does not bind Ca2+.
Conversely, calcium binding to site II is maintained by Asp
Ala
mutation at position X of site I. 5) The fluorescence data indicate
lower values of cooperativity between F29W N-terminal sites than
reported previously and a still lower N-terminal site cooperativity
value for F29HW. 6) 7-ZW probe at position 29 senses the binding of calcium to the higher affinity N-terminal site (site II) by increasing fluorescence and to the lower (in the case of
F29ZW) affinity N-terminal site (site I) by decreasing the
fluorescence in a bimodal fluorescence change. This observation indicates stepwise calcium binding to the regulatory N-terminal sites.
In conclusion, the data obtained with isolated TnC and its mutants
support a model for stepwise Ca2+ binding to N-terminal
sites, suggesting that site I does not bind Ca2+ when site
II is unoccupied. Our results also indicate that mutations in position
29 alter the properties of the regulatory sites.
 |
MATERIALS AND METHODS |
Construction of TnC Mutants--
The D30A and D66A (8) and F29W
mutants (25) were used to obtain the mutants D30A/D66A, F29W/D30A,
F29W/D66A, and F29W/D30A/D66A using the overlap extension PCR method
(41). The primers to generate the Asp
Ala mutation at position 30 on the F29W gene were 5'-C ACC ACC GTC CGC AGC CCA CAT GTC
AAA-3' and 5'-TTT GAC ATG TGG GCT GCG GAC GGT GGT G-3'. The
Asp
Ala mutation at position 66 was generated with the primers 5'-C
GCT GCC ATC CTC GGC CAC CTC CTC GAT GA-3' and 5'-TC ATC GAG
GAG GTG GCC GAG GAT GGC AGC G-3'. The PCR products were
purified from low melting agarose gels and used in the overlap
extension reactions. These final products were digested with
BamHI and NdeI and inserted to pET3a (42).
Mutations were confirmed by sequencing.
Production of TnC and TnC Mutants--
The expression and
purification of TnC and the mutant F29W have been described (10, 25,
43). The same procedure was used to express and purify D/A and F29 D/A
mutants. To incorporate 5-HW or 7-ZW into TnC
mutants a derivative of W3110TrpA33 (44) was constructed by infecting
this strain with phage
DE3 (42) and transforming with plasmid pLysS
(45). The new strain, named W3110TrpA33(DE3)pLysS, carries the
auxotrophic marker for tryptophan from the mother strain, the T7 RNA
polymerase gene from phage
DE3 under control of the
isopropyl-1-thio-
-D-galactopyranoside-inducible promoter
lacUV5, and the lysozyme gene from plasmid pLysS. By transforming this strain with plasmid pET-F29W or pET-F29W D/A we could
strictly regulate the production of large amounts of the desired
mRNA. To incorporate the Trp analogs we grew transformed W3110TrpA33(DE3)pLysS cells in M9 minimal medium containing 200 µg/ml
ampicillin, 20 µg/ml chloramphenicol, 5 mg/liter thiamin, and 20 µg/ml L-tryptophan. When the culture reached an
A600 nm
1.0, cells were centrifuged at 5,000 rpm for 10 min at 4 °C and resuspended in M9 minimal medium plus 200 µg/ml ampicillin, 20 µg/ml chloramphenicol, 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside, and 100 µg/ml
5-HW or 7-ZW instead of
L-tryptophan, followed by 3 h of additional incubation
at 37 °C with vigorous shaking (37). The protein purification
process was the same of that of TnC (10), yielding 10-20 mg of
protein/liter of culture.
Estimate of Tryptophan Analog Incorporation into
F29HW and F29ZW--
Trp analog incorporation
was estimated based on a comparative analysis of the absorbance and
emission spectra of the proteins. The absorbance spectra of proteins
(18-24 µM) and free amino acids (100 µM)
were obtained in 5 M guanidine HCl. The F29W,
F29HW, and F29ZW absorbance spectra were
normalized by subtracting the TnC absorbance spectrum to discount the
contribution of the phenylalanines. The percentage of 5-HW
incorporation into mutant F29HW (H%) was estimated using
the equation
|
(Eq. 1)
|
where S
5HW,
S
SF29W, and S
F29HW are
the areas between 269 and 324 nm of the normalized absorbance spectra
of 5-HW, F29W, and F29HW, respectively. The
7-ZW incorporation into F29ZW is more
straightforward because the emission spectra is red shifted with a
maximum near 380 nm. We subtracted the two normalized F29ZW emission spectra obtained by excitation at 287 or 315 nm, and the
difference (seen as a small peak near 340 nm) was transformed back to
absolute values. This area was compared with that of a sample with a
known F29W concentration and assumed to represent the amount of F29W
contaminating the F29ZW sample.
Ca2+ Binding to TnC and TnC Mutants Determined by
Flow Dialysis--
Flow dialysis experiments were performed as
described previously (11) with some modifications. The buffers used in
dialysis to remove bound Ca2+ from proteins were: buffer A,
which contained 20 mM MOPS, 100 mM KCl, 1 mM DTT, 5 mM EDTA, pH 7.0; 2) buffer A with 2 mM EDTA; 3) buffer A but with no EDTA. The proteins were
dialyzed with each buffer for 12 h with two changes. We have found
that using Spectra/Por dialysis tubing in all steps gives the most
reproducible results. Protein concentration was determined as described
previously (46) using as standard a TnC sample whose concentration was determined through amino acid analysis (Met and Val content). Protein
concentration in the dialysis chamber was 13 µM and was adjusted with the same buffer used for the flow dialysis experiment. The Marquardt-Levenberg nonlinear regression algorithm of Jandel Scientific SigmaPlot 2.0 package was used for curve fitting. The number
of calcium sites of each protein was inferred from the saturation
plateau obtained with pCa values between 4.5 and 4.0. This
avoided the introduction of another parameter in the curve fitting
associated with nonspecific calcium binding at lower pCa values. The data from TnC or F29X (where X = W,
HW, or ZW) were fitted using Equation 2.
|
(Eq. 2)
|
The data from D30A and F29X/D30A were fitted using
Equation 3.
|
(Eq. 3)
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The data from D66A, D30A/D66A, F29X/D66A, and
F29X/D30A/D66A were fitted using Equation 4,
|
(Eq. 4)
|
where Kd1, n1,
Kd2, and n2 are the
apparent dissociation constant and Hill coefficients, respectively, for
the C-terminal sites and the N-terminal sites.
Fluorescence Measurements of Calcium Binding to F29X
and F29X/D30A--
The fluorescence experiments were
performed in a Hitachi F-4500 spectrofluorometer with 2.5 nm excitation
slit and 5 nm emission slit for F29W and 5 nm for excitation and
emission slits for all other mutants. The excitation wavelength used
was 287 nm for F29W and F29W/D30A and 315 nm for F29X and
F29X/D30A. The temperature of the cell was kept at
25 °C. We used the calcium-free protein batch used in the flow
dialysis experiments. The volume of the protein solution in the
fluorescence assays was 2.5 ml, and calcium was added to maintain its
concentration the same as in the flow dialysis experiments. In this
way, we could use the mean pCa values obtained in the flow
dialysis experiments to correlate to the data obtained in the
fluorescence experiments. Curve fitting for F29W, F29HW,
and F29ZW/D30A (negative variation for
F29ZW/D30A) was performed adjusting the experimental data
to Equation 5.
|
(Eq. 5)
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Curve fitting for F29W/D30A and F29HW/D30A was
performed adjusting the experimental data to Equation 6,
|
(Eq. 6)
|
where F is the relative fluorescence change,
Kd2 the apparent dissociation constant for
calcium binding to the N-terminal sites (or the microscopic
dissociation constant to the remaining N site for the F29/D30A mutants)
and n2 the Hill coefficient reflecting the
cooperativity between the two N sites. Curve fitting for
F29ZW was performed adjusting the experimental data to
Equation 7
|
(Eq. 7)
|
where F is the relative fluorescence change,
f21 is the relative fluorescence change
associated with the filling of the site II, Kd21
is the microscopic dissociation constant for site II,
f12 is the relative fluorescence change
associated with the filling of the site I, and
Kd12 is the microscopic dissociation constant
for site I.
 |
RESULTS |
Incorporation of Tryptophan Analogs into F29HW and
F29ZW Mutants--
Fig.
1A shows the normalized
absorbance spectra of the three isolated amino acids, Trp,
5-HW, and 7-ZW. Both 5-HW and
7-ZW have red shifted absorbance compared with tryptophan.
This characteristic is maintained in the absorbance spectra of the
corresponding recombinant TnC mutants, F29W, F29HW, and
F29ZW (Fig. 1B). Fig. 1, C-E,
demonstrates the similarity between the free amino acid and the
corresponding protein absorbance spectra.

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Fig. 1.
Absorbance spectra of Trp, 5-HW,
7-ZW, and F29W, F29HW, and F29ZW
mutants. A, normalized absorbance spectra of Trp ( ),
5-HW ( ), and 7-ZW ( ). B,
normalized absorbance spectra of F29W ( ), F29HW ( ),
and F29ZW ( ). C-E, comparison of the
normalized free and protein-incorporated amino acid analog absorbance
spectra. The symbols are the same as in A and B.
The buffer contained 20 mM MOPS, 100 mM KCl, 1 mM DTT, and 5 M guanidine HCl, pH 7.0.
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The normalized excitation and emission spectra of the three amino acids
and the three mutants are shown in Fig.
2. The fluorescence of F29W increases
nearly three times upon addition of Ca2+ (pCa
2.0), as reported previously (25). When excited at 315 nm, the
fluorescence of F29HW increases nearly seven times upon addition of Ca2+ (pCa 2.0) (this increase drops
to nearly three times when the excitation wavelength is 287 nm). The
fluorescence of F29ZW displays a bimodal response to
Ca2+, increasing nearly 40% from pCa 9.0 to
pCa 5.0 and decreasing nearly 20% from pCa 5.0 to pCa 3.0.

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Fig. 2.
Excitation and emission fluorescence spectra
of Trp, 5-HW, and 7-ZW, and F29W,
F29HW, and F29ZW mutants with and without
calcium. A, normalized excitation fluorescence spectra of
Trp ( ), 5-HW ( ), and 7-ZW ( ). Emission
was detected at 340 nm for Trp and 5-HW and at 380 nm for
7-ZW. B, normalized excitation fluorescence
spectra of F29W at pCa 9.0 ( ) and at pCa 2.0 ( ), F29HW at pCa 9.0 ( ) and at
pCa 2.0 ( ), and F29ZW at pCa 9.0 ( ), at pCa 5.0 ( ), and at pCa 3.0 ( ).
The emission wavelengths were 340 nm for F29W and F29HW and
380 nm for F29ZW. C, normalized emission
fluorescence spectra of Trp, 5-HW, and 7-ZW.
Excitation was at 287 nm ( ) and 315 nm for Trp ( , normalized
against 5-HW spectrum, and , normalized against
7-ZW spectrum) and 315 nm for 5-HW ( ) and
7-ZW ( ). D, normalized emission fluorescence
spectra of F29W excited at 287 nm, at pCa 9.0 ( ) and
pCa 2.0 ( ), F29HW excited at 315 nm, at
pCa 9.0 ( ) and pCa 2.0 ( ) and
F29ZW excited at 315 nm, at pCa 9.0 ( ),
pCa 5.0 ( ), and pCa 3.0 ( ). The buffer
contained 20 mM MOPS, 100 mM KCl, and 1 mM DTT.
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Quantitative analysis of the absorbance and emission spectra of the
recombinant TnC indicates 87-96% analog incorporation efficiency for
F29HW and nearly 96% for F29ZW (see
"Materials and Methods"). Because of this incorporation ratio the
results obtained with both analog mutants are not significantly
distorted by contamination with F29W. We used the same procedure in
producing all of the other F29HW D/A and F29ZW
D/A mutants.
Calcium Binding to TnC and Its Mutants--
Fig.
3A shows the calcium binding
curves to TnC and to the three fluorescent mutants, F29W,
F29HW, and F29ZW as determined by flow
dialysis. Calcium binding curves indicate that all four proteins bind
four Ca2+/molecule and that near saturation is achieved
between pCa 4.5 and 4.0.

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Fig. 3.
Calcium binding to TnC and to F29
mutants. Flow dialysis (white symbols) and/or
fluorescence measurements (black symbols) were used to
determine the characteristics of calcium binding to TnC ( in
A), F29W (squares in A, B,
and C), F29HW (triangles up in
A, B, and D), and F29ZW
(triangles down in A, B, and
E). Each set of points is the mean of three experiments.
Error bars representing the standard deviations are often
occluded by the symbols. In B, C,
D, and E the pCa values used with the
fluorescence data were obtained from the corresponding flow dialysis
experiments. A compares the Ca2+ binding to TnC
and to F29 mutants. B shows the fluorescence changes upon
Ca2+ binding of mutants F29W, F29HW, and
F29ZW. C, D, and E
correlate the fluorescence change of each mutant to the
Ca2+ saturation level. The buffer contained 20 mM MOPS, 100 mM KCl, and 1 mM
DTT.
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At pCa values less than 4, there is an increase in the
calcium binding ratio which is most possibly related to nonspecific calcium binding to TnC. Thus, pCa 4 was assumed to be the
maximum Ca2+ concentration to which we could relate any
dependent parameter. The TnC and F29HW Ca2+
binding curves are similar regarding the overall transition. Between
pCa 5.5 and pCa 4.0, the most evident difference
among the binding curves is the displacement of the F29W and
F29ZW curves toward higher pCa values compared
with TnC and F29HW. This reflects an increase in the
affinity of the low affinity N-terminal sites. The C-terminal sites
have affinities nearly 100 times higher than the N-terminal sites and
are represented by the lower part of curves (between pCa 7 and 5.5).
Fig. 3B compares the relative fluorescence changes of F29W,
F29HW, and F29ZW. The data were obtained under
the same conditions as the flow dialysis experiments, that is, in
absence of EGTA. The fluorescence experiments showed that the
half-transition point of F29HW occurs 0.7 pCa
unit after the F29W transition. This confirms the lower calcium
affinity of F29HW N-terminal sites with respect to the F29W
N-terminal sites. The F29ZW fluorescence change is bimodal,
with the maximum value at
pCa 5 and a final decrease of
nearly 20%, which plateaus at pCa 3.5.
In Fig. 3, C-E, we compare flow dialysis and fluorescence
data for each mutant to correlate the fluorescence changes with calcium
saturation level and to determine the point in the free calcium
titration which corresponds to the fluorescence change. In the case of
F29W and F29HW (Fig. 3, C and D), the
greater part of the fluorescence change occurs above the pCa
where there are two bound calcium ions/molecule. This limit is better
defined for F29HW than for F29W. The difference in affinity
between F29HW C-terminal and N-terminal sites is nearly 0.7 pCa unit higher than between C-terminal and N-terminal sites
of F29W. These Ca2+-dependent fluorescence
changes provide direct evidence that calcium binding to the C-terminal
sites does not alter the fluorescence of the tryptophan or
5-HW incorporated at position 29 of TnC and that these
probes selectively report calcium binding to the regulatory N-terminal
sites of TnC.
Fig. 3E shows fluorescence of F29ZW during
titration with Ca2+. The maximal fluorescence value occurs
at pCa 5.0 and is associated in the calcium binding curve
with three bound Ca2+/molecule. Therefore, in the case of
this mutant, the probe can be used to distinguish between the binding
of the third and the fourth Ca2+ ions.
Fig. 4 shows calcium binding measured by
flow dialysis to TnC and its D/A mutants (A), F29W and F29W
D/A mutants (B), F29HW and F29HW D/A
mutants (C), and F29ZW and F29ZW D/A
mutants (D). The data for TnC and its D/A mutants show that
the Asp
Ala mutations at position 30 or 66 or the combined
mutations affect predominantly the calcium binding at pCa
lower than 6, i.e. in a range associated with binding to
N-terminal sites. This is in agreement with the structural independence
of the C- and N-domains of TnC. Near pCa 6, the TnC and D30A
binding curves diverge from the D66A and D30A/D66A binding curves. The
D66A and D30A/D66A binding curves are similar and indicate that these
mutants bind only two Ca2+ ions. The TnC and D30A binding
curves diverge only at pCa lower than 5, where the D30A
binding curve reaches a plateau (indicating a maximum of three
Ca2+ bound/protein). The TnC binding curve continues to
four bound Ca2+/molecule at pCa 4.

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Fig. 4.
Calcium binding to the D/A mutants determined
by flow dialysis. A shows the calcium binding to TnC ( )
and mutants D30A ( ), D66A ( ), and D30A/D66A ( ). B,
C, and D show, respectively, the calcium binding
to the mutants F29W, F29HW, F29ZW, and to the
corresponding D/A double or triple mutants. The symbols for
each D/A mutant are the same as in A. Each data set is the
median of two experiments. The error bars, representing the
standard deviations, are often occluded by the symbols. The
buffer contained 20 mM MOPS, 100 mM KCl, and 1 mM DTT.
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This pattern is also found in the calcium binding curves of the
fluorescent D/A mutants (Fig. 4, B-D). The difference is
that the presence of the fluorescent probes affects the pCa
at which the curves for the non-D/A and the corresponding D30A mutant
diverge (pCa
6 for F29W and F29W/D30A,
pCa
4.6 for F29HW and
F29HW/D30A, and pCa
5.4 for
F29ZW and F29ZW/D30A). The corresponding
fluorescent and nonfluorescent D66A and D30A/D66A calcium binding
curves largely superimpose over the entire pCa scale (Fig.
4).
Fig. 5 shows the fluorescence change upon
calcium binding to the fluorescent D/A mutants, which bind only one or
zero Ca2+ ions in their N-terminal domains. Fig.
5A compares the fluorescence change of each mutant upon
calcium binding (relative to the apo form = 100%). For all D30A
mutants, the Ca2+-induced fluorescence increase is much
less than that observed in the original fluorescent mutants that
possess two functional N-terminal Ca2+ binding sites. In
the case of F29ZW/D30A the fluorescence intensity decreases
slightly upon Ca2+ binding. For all D66A and D30A/D66A
mutants, the fluorescence change was very small or nonexistent.
Therefore, these latter mutants were not used in the subsequent
fluorescence following calcium titrations.

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Fig. 5.
Fluorescence change upon calcium binding to
F29 mutants and calcium titration comparing the calcium saturation
level of each mutant with the fluorescence change. A shows
the fluorescence increase of F29 mutants upon calcium binding relative
to the calcium free value, considered as 100%. Only the D/A mutants
whose fluorescence intensity varied more than 5% upon calcium addition
(the F29 D30A mutants) were used for parallel flow
dialysis/fluorescence following calcium titrations. B,
C, and D correlate the fluorescence change
(black symbols) of mutants F29W/D30A,
F29HW/D30A, and F29ZW/D30A, respectively, with
the calcium saturation level (white symbols) obtained in the
correspondent flow dialysis experiments, as in Fig. 3, C-E,
for the F29 single mutants. The buffer contained 20 mM
MOPS, 100 mM KCl, and 1 mM DTT.
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Fig. 5, B-D, displays the fluorescence and flow dialysis
Ca2+ binding curves for the fluorescent D30A mutants:
F29W/D30A, F29HW/D30A, and F29ZW/D30A,
respectively. Approximately 60% of the total fluorescence change of
F29W/D30A, F29HW/D30A, and F29ZW/D30A occurs
after the calcium saturation level reaches two Ca2+
ions/molecule and is complete when the calcium saturation level reaches
three Ca2+/molecule. This indicates that Ca2+
binding to the single functioning N-terminal site in these proteins (site II) is the event associated with the fluorescence change.
Fig. 6 further highlights the similarity
between fluorescent and nonfluorescent forms of D30A and compares the
fluorescence changes during calcium titration of the fluorescent
mutants with and without the D30A substitution. These results are
summarized in Table II. Fig. 6A compares the calcium binding
curves obtained by flow dialysis for fluorescent and nonfluorescent
D30A mutants. The four binding curves are similar, indicating that the
effect of the three different probes at position 29 on Ca2+
binding to site II is very small in the D30A mutants. Fig.
6B compares the fluorescence changes during calcium
titration of the fluorescent D30A mutants. The three
pCa1/2 values are very similar: ~5.5 for
F29W/D30A and F29HW/D30A and ~5.3 for
F29ZW/D30A. Fig. 6, C-E, compares the calcium
titrations followed by fluorescence of the fluorescent mutants with and
without the D30A mutation. Both F29W and F29W/D30A have a
pCa1/2 close to 5.5, and the curves differ only
slightly at low free calcium concentrations (Fig. 6C).
Interestingly, when 5-HW instead of tryptophan is present at position 29, the D30A mutation has a much greater effect: the pCa1/2 of F29HW is ~4.8 whereas
the pCa1/2 of F29HW/D30A is ~5.5. The slopes of both curves are similar (predicted Hill coefficient of
1.11 for F29HW and fixed to 1 for F29HW/D30A).
Fig. 6E compares the F29ZW and
F29ZW/D30A curves. The bimodal shape of the
F29ZW curve is replaced by a monotonic decrease in
F29ZW/D30A, with pCa1/2
5.3 (see
Table II).

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Fig. 6.
Flow dialysis and fluorescence following
calcium binding to D30A and F29 D30A mutants, and comparison of
fluorescence following calcium titration among F29 and F29 D30A
mutants. A shows the calcium binding curves of D30A and
F29 D30A mutants. B compares the fluorescence change of F29
D30A mutants. C, D, and E show the fluorescence
change upon calcium binding of mutants F29W versus F29W/D30A
(C), F29HW versus
F29HW/D30A (D), and F29ZW
versus F29ZW/D30A (E). The buffer
contained 20 mM MOPS, 100 mM KCl, and 1 mM DTT.
|
|
It is important to highlight that the mid-transition points of Fig. 3,
B and E, and Fig. 6E (mutant
F29ZW) are only apparent because the absolute increase and
decrease are not known. Hence, the apparent mid-transition points are
not reflected directly by the Kd values. We
chose the Kd values from the best fitting, and
small variations on the assumed absolute increase/decrease led to
significant variation on Kd values. We therefore
emphasize that the main indication of sequential binding is the bimodal profile of the fluorescence curve and that the affinities of site II
and site I (when site II is filled) do not need to be very different to
be in accordance with sequential binding.
 |
DISCUSSION |
Recombinant skeletal chicken TnC is devoid of Tyr and Trp.
Pearlstone et al. (25) replaced Phe at position 29 with Trp, allowing Ca2+ binding to regulatory (or N-terminal sites)
to be followed using fluorescence techniques. Initial tests indicated
that the most important properties of TnC were maintained in F29W,
including the Ca2+ affinity of the N-terminal sites. In
addition, the fluorescence data revealed a high degree of cooperativity
(Hill coefficient of 1.96), consistent with an all-or-none mechanism of
Ca2+ binding to the two N-terminal sites. Chandra et
al. (29) found that the F29W mutant is deficient in its ability to
relieve TnI inhibition of actomyosin S1 ATPase in a
Ca2+-dependent manner. Using the same system, a
small increase in N-domain F29W calcium affinity was detected. Li
et al. (32) confirmed this affinity increase (0.5 pCa unit, determined by far-UV CD-Ca2+
titration) as well as an increased negative ellipticity of the Ca2+-loaded state at 221 nm. Yu et al. (33)
showed that the apo N-domain of F29W is conformationally less stable at
lower temperatures compared with the isolated TnC N-domain.
Here we used direct binding assays to show that the F29W N-terminal
site calcium affinity is at least 6-fold greater than that of TnC
N-terminal sites (Table I). The addition
of a hydroxyl at position 5 of Trp side chain (F29HW
mutant) reverts this affinity increase. When 7-ZW is
incorporated at position 29, the N-terminal site affinity increases
nearly 3-fold compared with TnC. Therefore, the N-domain calcium
binding properties of TnC are indeed quite sensitive to the nature of
the amino acid side chain at this position (Phe in the native protein;
Trp, 5-HW, or 7-ZW in the mutants).
View this table:
[in this window]
[in a new window]
|
Table I
Number of sites, apparent dissociation constants, and Hill coefficients
for TnC and its mutants measured by flow dialysis
|
|
This influence of position 29 on TnC N-domain Ca2+ affinity
is not surprising because in the crystal structure of chicken
2Ca2+-TnC (i.e. unloaded N-domain) an extensive
network of interactions involving Phe-29, Met-48, Glu-41, Thr-44, and
Val-45, can be observed. In both the x-ray and NMR-determined
calcium-saturated structures (34, 47, 48) the Phe-29 side chain is
adjacent to Phe-75, Ile-37 and Val-45. Phe-29 and the residues cited
above form a "hydrophobic patch" that is exposed upon calcium
binding to the N-terminal sites, as TnC goes from the "closed" to
the "open" form. It is therefore very likely that the structure of
TnC should be sensitive to the introduction, at position 29, of a
bulkier indole side chain with or without potential hydrogen bond
donors and acceptors in the six-membered ring. It is generally accepted that in the thin filament, this exposed hydrophobic patch interacts with TnI in the presence of Ca2+. In the absence of TnI,
calcium binding to TnC would force the hydrophobic patch to become
solvent-exposed. This explains the reduced calcium affinity of the
N-terminal sites in free TnC compared with that measured in the
TnC·TnI complex (7). Pearlstone et al. (25) and da Silva
et al. (11) reported TnC mutants with increased N-terminal
site Ca2+ affinity by replacing, one by one, the five
highly hydrophobic residues Val-45, Met-46, Met-48, Leu-49, and Met-82
from the hydrophobic patch by Thr, Gln, Ala, Thr and Gln, respectively.
These substitutions were expected to lower the energetic barrier
between the closed and open forms and, thus, increase the calcium
affinity of the N-terminal sites. The increased Ca2+
affinity observed for the F29W N-terminal sites could be the result of
a destabilization of the closed form (because of steric clashes
involving Trp-29) and/or a stabilization of the open form because of
the presence of the N1-H (indole) hydrogen bond donor while
maintaining at least some of the original interactions of the
six-membered ring in the open form. The observation that the Ca2+ affinity of the F29HW N-terminal sites was
very similar to that of native TnC (dissociation constants 2.4 × 10
5 M and 1.9 × 10
5
M, respectively, Table I) suggests that the stabilization
or destabilization of the N-terminal domain structure by
5-HW is similar for both the closed and open forms. The
mutant F29ZW displayed an intermediate N-terminal site
Ca2+ affinity, between that of F29W and TnC (nearly 2 times
smaller than that of F29W and 3 times greater than that of TnC, Table I). These data indicate that the decrease in hydrophobic character of
the residue at position 29, initially from Phe to Trp, should not be
extended as a general rule to explain the influence of that position on
N-terminal sites Ca2+ affinity, as highlighted by Tikunova
et al. (49) for a series of 27 F29W mutants with hydrophobic
residues replaced by glutamine. In the case of F29HW
another possibility is an interaction involving the hydroxyl group of
5-hydroxytryptophan residue and the carboxylic group of Glu-41. This
could hamper the reorientation of the Glu-41 side chain which is
necessary for calcium to bind to site I (48). This interaction could
mimic, to a lesser degree, the replacement of Glu at position 41 by
Ala, a mutation reported to raise the site I calcium dissociation
constant from 16 µM to 1.3 mM and impairs
opening of N-domain even with site I filling (50).
The fluorescence data obtained for F29W, F29HW, and
F29ZW (Table II) were all
compatible with those from flow dialysis experiments. The observation
of disparate fluorescence behavior for F29ZW was expected
because the photophysics of 7-ZW are the most complex of
the three chromophores used, with high quantum yield variation and
maximum emission sensitivity upon environment changes (51). Rosenfeld
and Taylor (52) reported a bimodal fluorescence change using skeletal
rabbit TnC labeled at Cys-98 with
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine which occurred at low ionic strength (0 M KCl) and was
absent at 0.1 M KCl. The present F29ZW
titrations were performed at 100 mM KCl, which argues that
the bimodal form of the Ca2+ titrations followed by
fluorescence is not the result of subtle variations in the ionic
strength which accompany the additions of Ca2+.
Furthermore, the presence of 2 mM Mg2+ does not
significantly alter the pCa1/2 of the transition
(data not shown). Although a bimodal curve poses statistical problems to the fitting procedure, the first and second phases of the emission curve may be related to the calcium binding to the N-terminal sites II
and I, respectively. The F29ZW fluorescence increase is not
caused by calcium binding to C-terminal sites because it occurs
principally at pCa values lower than 6.
View this table:
[in this window]
[in a new window]
|
Table II
Dissociation constants and Hill coefficients for F29W,
F29HW, F29ZW, and its D30A mutants measured by
fluorescence
For the F29ZW mutant Kd21 and
Kd12 are microscopic dissociation constants,
respectively, for sites II and I. For the D30A mutants,
Kd2 is the site II microscopic dissociation
constant.
|
|
D30A and F29X/D30A bind three Ca2+ ions, and
the Ca2+ binding curves reveal equal affinities among the
remaining N-domain site (site II of each mutant) (Table I). The
influence of the residue at position 29 on the Ca2+ binding
patterns is abolished when Ca2+ does not bind to site I;
that is, site II Ca2+ affinity is not reduced by any of the
four tested residues at position 29 in mutants with a nonfunctional
site I. Assuming that the transition from the closed to open state in
the N-domain is associated with Ca2+ binding to site I, as
suggested by Li et al. (50), the difference in affinity
among the TnC and Phe-29 N-terminal sites may be related to the opening
of the N-domain.
All mutants with alanine at position 66 (D66A and
F29X/D66A) or with alanines at position 66 and 30 (D30A/D66A and F29X/D30A/D66A) bind only two calciums and
have similar calcium binding curves, as determined in the flow dialysis
experiments (Fig. 4). In mutants with a modified site I the saturation
level of three Ca2+/molecule is reached at the same
pCa as TnC and F29X mutants. We conclude that
the sites I and II contribute differently to the Ca2+
binding properties of the N-domain. One possible explanation is that
the replacement of aspartate at position 66 by alanine disturbs both
sites, whereas the same replacement at position 30 is functionally
restricted to site I. Another explanation is that previous filling of
site II is essential for calcium binding to site I at physiological
calcium concentrations, as suggested by Gagné et al.
(53) and Strynadka et al. (48). According to this proposal,
Ca2+ binding to site II would "set the stage" for
calcium binding to site I. An unoccupied site I would thus exist in two
conformations: one previous to Ca2+ binding to site II and
other after Ca2+ binding to site II. The data presented
here indicate that site I is essentially defunct without previous site
II filling. Strong site I functional dependence on site II filling is
compatible with the experimentally determined stepwise filling and lack
of cooperativity of the N-terminal domain (32). The different
Ca2+ binding curves of TnC and fluorescent TnCs may be a
consequence of differences exclusively among the sites I or among the
stabilities of the open forms, which would be sensitive to residue at
position 29.
The F29W and F29W/D30A fluorescence changes during Ca2+
titration shown in Fig. 6 occur in the same pCa range, but
F29W transition displays a greater Hill coefficient (Table II). This
observation is compatible with the hypothesis that binding to site II
activates site I (obligatory sequential binding model) and that
Ca2+ binding to F29W site I causes the large fluorescence
increment. On the other hand, the F29HW fluorescence
transition occurs nearly 0.7 pCa unit after the
F29HW/D30A fluorescence transition. Therefore, site II in
F29HW has an affinity nearly five times lower than in
proteins with only site II functional. Furthermore, the site I affinity
(when site II is filled) is nearly 2-fold lower than site II affinity
in the F29HW mutant. Fig. 6E compares the
F29ZW and F29ZW/D30A fluorescence following
Ca2+ titration. The F29ZW/D30A fluorescence
change has a pCa1/2 of nearly 5.6, corresponding
to a dissociation constant nearly 5 × 10
6 M,
which is of the same order of magnitude as that observed for the
F29W/D30A and F29HW/D30A mutants. The bimodal shape of the F29ZW fluorescence change may be related to an increase in
fluorescence caused by Ca2+ binding to site II and a
fluorescence decrease associated with Ca2+ binding to site
I (that is, the fluorescence of the saturated form is lower than the
fluorescence of the form with only site II filled). The affinity of the
site I (when site II is filled) would be close to that of site II
(Table II).
Previous results related to cooperativity between TnC sites I and II or
III and IV are contradictory. Earlier reports (7, 12, 13) excluded the
cooperativity parameter from the fitting process. Some later studies
(15) found no cooperativity between the sites in each domain, even
though data were allowed to fit to the Hill equation. Nevertheless,
some reports detected cooperativity between C-terminal sites but no
cooperativity between N-terminal sites (9, 16, 54, 56). All but one of
these studies (9) utilized EGTA buffers to control the free calcium
concentration. Pearlstone et al. (25) and Tikunova et
al. (49) have reported the highest value for cooperativity between
N-terminal sites. However, Pearlstone et al. (57)
highlighted the difficulty in conferring biological significance to
cooperativity inferred from Ca2+ titrations using indirect
spectroscopic methods. This is mainly because of Hill coefficients
greater than unity which were predicted for processes known to be
related to the filling of only one site (57). Although our fluorescence
data led in some cases to predicted Hill coefficients different from
unity (Table II), we consider that there is no secure reason to assume
all those values as real indications of positive or negative
cooperativity. Also, direct Ca2+ binding data from flow
dialysis experiments involve such variability (reflected in Hill
coefficients from 0.75 to 1.51, Table I) that we do not consider them
to confirm the existence or nonexistence of cooperativity between sites
I and II or III and IV. Furthermore, the obligatory sequential binding
model does not maintain the correspondence between Hill coefficients
and cooperativity.
The near-UV CD spectra changes associated with calcium binding (data
not shown) indicate that the F29W D/A and F29HW D/A mutants do not suffer large decreases in CD bands at wavelengths above 260 nm
upon calcium binding. Pearlstone et al. (57) suggested that
this decrease is associated with opening of the N-domain, based on the
characteristics of cardiac TnC (cTnC) and the E41A mutant of TnC. These
proteins show neither the CD band decrease nor the opening of N-domain
upon calcium binding (53, 55). In our study, the data suggest that the
replacement of Asp by Ala at position 30 impairs opening of the
N-domain of mutants F29W/D30A and F29HW/D30A. The
calcium-associated fluorescence increase of these mutants may be
related to the effect of metal binding to site II on the environment of
the probe at position 29 and reinforces the opening of N-domain as the
cause of the large fluorescence increase of F29W and F29HW
mutants upon calcium binding. Because there is no large increase in
fluorescence of F29W/D30A and F29HW/D30A mutants even at
pCa values near 2 (data not shown), calcium binding to the
mutated sites I which would eventually occur at these high calcium
concentrations does not lead to N-domain opening (as in E41A mutant and
in cardiac TnC).
Our results indicate that position 29 strongly influences the
Ca2+ affinity of TnC N-terminal sites. F29W has N-terminal
sites with higher affinity than TnC, and the addition of
5-HW reverts this increase. Incorporation of
7-ZW results in intermediate N-terminal site affinities
between those of F29W and of TnC. The direct binding data of D/A
mutants suggest that site I is essentially defunct without previous
Ca2+ binding to site II and that the differences among TnC
and F29X N-domain site affinities are the consequence of
differences in the site I affinities only (when site II is filled). The
fluorescence data of F29W/D30A and F29HW/D30A mutants may
be explained assuming obligatory sequential Ca2+ binding
(first to site II) with the Ca2+ binding to site I as the
cause of the fluorescence increase and N-domain opening. The bimodal
F29ZW fluorescence change is the result of a fluorescence
increase caused by Ca2+ binding to site II and a subsequent
fluorescence decrease associated with Ca2+ binding to site I.
 |
FOOTNOTES |
*
This work was supported in part by the Fundação
de Amparo à Pesquisa do Estado de São Paulo and the Howard
Hughes Medical Institute.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Graduate fellow from Fundação Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior and
supported by Fundação Universidade de Brasília. To
whom correspondence should be addressed: Dept. de Biologia Celular,
Instituto de Ciências Biológicas, Universidade de
Brasília, ICC SUL, Campus Universitário, Asa Norte,
Brasília DF CEP 70919-970, Brazil. Tel.: 55-61-307-2192; Fax: 55-61-273-4608; E-mail: ffvalenc@unb.br.
§
Graduate fellow from the Fundação de Amparo à
Pesquisa do Estado de São Paulo.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M209943200
 |
ABBREVIATIONS |
The abbreviations used are:
TnC, recombinant
chicken troponin C;
TnI, troponin I;
DTT, dithiothreitol;
MOPS, 4-morpholinepropanesulfonic acid;
5-HW, 5-hydroxytryptophan;
7-ZW, 7-azatryptophan;
F29, Phe-29,
used to indicate broad spectrum of Phe-29 mutations;
F29W, F29HW, and F29ZW, recombinant TnCs with
phenylalanine at position 29 replaced by tryptophan,
5-hydroxytryptophan, or 7-azatryptophan, respectively;
F29X (where X = W, HW,
or ZW), any single mutant with phenylalanine at position 29 replaced by tryptophan, 5-hydroxytryptophan, or 7-azatryptophan;
D/A, any protein with phenylalanine at position 29 and at least one
replacement of aspartate by alanine;
F29X D/A, any double
or triple mutant with tryptophan or an analog at position 29 and at
least one replacement of aspartate by alanine;
N-domain, N-terminal
domain sites I and II of TnC or its mutants;
C-domain, C-terminal
domain sites III and IV of TnC or its mutants;
pCa1/2, value of
-log[Ca2+]free corresponding to 50% of the
maximal associated transition.
 |
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