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INTRODUCTION |
The EF-hand is the most common Ca2+ binding motif
found in nature (1). EF-hand proteins and their functions are numerous and diverse (for review see Ref. 2). In general, EF-hand domains can be
classified functionally into two groups, those that regulate cellular
activities through a reversible change in structure upon Ca2+ binding and release (such as the N-terminal regulatory
domain of troponin C (TnC)1
and both domains of calmodulin (CaM)) and those that simply
buffer/transport Ca2+ (such as parvalbumin and calbindin
D9K) or anchor protein complexes (such as the C-terminal
domain of TnC). Often the nonregulatory EF-hand domains bind
Mg2+ competitively and display higher Ca2+
affinity than do the regulatory domains. However, it is becoming clear
that regulatory EF-hand proteins, such as the N-terminal of CaM can
also bind Mg2+ with a physiologically relevant affinity (3,
4). Thus, it is important to elucidate the mechanisms behind EF-hand
Mg2+ binding.
The canonical EF-hand consists of 29 consecutive residues, with two
helices flanking a 12-residue loop. The chelating loop residues in
positions 1(+x), 3(+y), 5(+z), 7 (
y), 9(x), and 12 (z) ligate
Ca2+ through seven oxygen atoms arranged three
dimensionally on the axes of a pentagonal bipyramid (for review see
Refs. 5 and 6). Factors that control Ca2+ affinity are
complex and involve residues within and outside of the
Ca2+-binding loop (7-11). The mechanisms utilized for
EF-hand Mg2+ binding are less understood. The smaller
Mg2+ cation is typically complexed by some of the same loop
residues used for Ca2+ binding, although through six oxygen
atoms arranged in an octahedral geometry (for review see Refs. 11 and
12). Interestingly, synthetic EF-hand peptides were observed to bind
Mg2+ when their Ca2+-binding loops contained a
Z acid pair, chelating residues at loop positions +z and
z (13, 14).
The metal binding properties of TnC have been studied extensively using
various biophysical and biochemical techniques (15-19). These studies
have demonstrated that the two C-domain EF-hands have ~10-fold
higher Ca2+ affinity and greater than 100-fold slower
Ca2+ exchange rates than the two N-domain EF-hands (18,
20). In addition to Ca2+, the C-domain sites also
competitively bind Mg2+ with a physiologically relevant
affinity. Because of their high Ca2+ and Mg2+
affinities and slow exchange rates, the C-domain sites are thought to
play a structural role in muscle function by anchoring TnC into the Tn Complex.
The N-domain sites of TnC are considered to be
Ca2+-specific under physiological Mg2+
concentrations, and generally accepted to be directly involved in the
Ca2+-dependent regulation of muscle contraction
(for review see Refs. 21 and 22). However, addition of Mg2+
has been shown to decrease the Ca2+ sensitivity of the
regulatory domain of fluorescent TnCs in isolation (19, 23-27), in the
Tn complex (23, 28), and in reconstituted muscle fibers (26, 29).
Furthermore, several groups have demonstrated that increased
[Mg2+] caused a decrease in Ca2+ sensitivity
of Tn-regulated actomyosin ATPase and force development (26, 27, 30,
31). The question whether Mg2+ competes with
Ca2+ for the N-domain sites remains unresolved and
controversial. Some research groups have suggested the presence of
auxiliary Mg2+ binding sites in TnC (15, 23), whereas
others hypothesized Mg2+ as a direct Ca2+
competitor for the N-terminal regulatory sites (19, 27, 32).
Because TnC regulates muscle contraction as a part of the troponin
complex and not in isolation, it is important to understand the
interactions of TnC with TnI. The binding of TnI to TnC increases the
Ca2+ sensitivity of the N- and C-domains of TnC ~10-fold
(15, 33). TnC and TnI interact in an antiparallel orientation (34, 35) such that the Ca2+-dependent binding of the
N-terminal regulatory domain of TnC to the C-domain of TnI is an early
step in the generation of force in skeletal muscle (21). Previous
studies demonstrated that region 96-116 of TnI interacts with actin
and was largely responsible for the ability of TnI to inhibit the
activity of the actomyosin ATPase that could be reversed upon
TnC-Ca2+ binding (36-38). Recent studies implicated
additional residues within 116-148 of TnI as being important for the
complete inhibitory activity and regulatory interactions with actin and
TnC (39-42). Thus, the Ca2+-dependent binding
of the regulatory domain of TnC to TnI-(96-148) may be a good
model system to study the Ca2+-dependent
interactions between TnI and TnC.
The F29W mutation in chicken skeletal TnC (TnCF29W) has
been frequently used to study metal and ligand interactions with the
regulatory N-domain sites of TnC (9, 20, 43-46). TnCF29W
is a physiologically active protein that produced maximal isometric tension with a Ca2+ sensitivity indistinguishable from that
of recombinant TnC, when reconstituted into skinned skeletal fibers
(47). Interestingly, whereas Ca2+ causes a large increase
in TnCF29W fluorescence, Mg2+ does not alter
the fluorescence properties of Trp29 (44, 45, 48).
Previously, we have demonstrated that an endogenous Z acid pair was
required for high affinity Mg2+ binding to the first
EF-hand of fluorescent CaMF19W (3). We wanted to test if
Mg2+ binding could be engineered into the first EF-hand of
TnCF29W by substituting Gly in position 34 with Asp, thus
introducing a Z acid pair (Fig. 1). We
also wanted to determine how competitive Mg2+ binding to
the first EF-hand of TnC would affect the physiological properties of
G34DTnCF29W reconstituted into skinned skeletal muscle
fibers.
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Fig. 1.
Predicted structures of the first
Ca2+-binding loops of TnCF29W and
G34DTnCF29W. Loop residues 29 through 41 are shown in
a ribbon/stick configuration for TnCF29W and
G34DTnCF29W as rendered by RASMOL (72). The F29W and G34D
mutations were predicted by the computer software program WHAT IF (73)
and constructed from the coordinates of the N-terminal domain crystal
structure of chicken skeletal TnC (Protein Data Bank number
1AVS) (74). These cartoons are not meant to represent the actual
structures but are used simply to demonstrate the potential effect of
the G34D mutation and to demonstrate the location of the F29W mutation
adjacent to the first Ca2+-binding loop. The spherical
Ca2+ ion is shown to be coordinated by residues at
positions 30 (+x), 32 (+y), 34 (+z),
36 (y), 38 (x), and 41 (z) that
form the base and apexes of the pentagonal bipyramid geometry.
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EXPERIMENTAL PROCEDURES |
Materials--
Phenyl-Sepharose CL-4B, EDTA, and EGTA were
purchased from Sigma. Quin-2 was purchased from Calbiochem (La Jolla,
CA). All other chemicals were of analytical grade. The TnI-(96-148)
peptide was synthesized and purified by the Alberta Peptide Institute (Edmonton Alberta, Canada).
Protein Mutagenesis and Purification--
The construction and
expression of intact chicken skeletal TnCF29W and isolated
N-domain residues 1-90, TnC
, both in pET3a, have been described (44, 49-50). Chicken skeletal fast
TnI was prepared as described for the rabbit protein (51). The
G34DTnCF29W mutant was constructed from the
TnCF29W plasmid by primer-based site-directed mutagenesis
using the QuikChange site-directed mutagenesis kit from Stratagene (La
Jolla, CA). The mutation was confirmed by DNA sequence analysis. The
plasmids for TnCF29W and G34DTnCF29W were
transformed into Escherichia coli BL21(DE3)pLysS cells
(Novagen) and purified as described previously (9).
Determination of Ca2+ and Mg2+
Affinities--
All steady-state fluorescence measurements were
performed using a PerkinElmer Life Sciences LS5 spectrofluorimeter at
15 °C. Trp fluorescence was excited at 275 nm and monitored at 345 nm as microliter amounts of CaCl2 or MgCl2 were
added to 1 ml of each TnCF29W mutant (0.3 µM)
in 200 mM MOPS (to prevent pH changes upon addition of
metal), 90 mM KCl, 2 mM EGTA, 1 mM
DTT, pH 7.0, at 15 °C. The [Ca2+]free was
calculated using the computer program EGCA02 developed by Robertson and
Potter (52). The Ca2+ and Mg2+ affinities are
reported as dissociation constants
Kd(Ca) and
Kd(Mg), respectively. Each
Kd represents a mean of 3-5 titrations fit with a
logistic sigmoid function mathematically equivalent to the Hill
equation, as previously described (8, 9).
Determination of Apparent TnI-(96-148) Peptide
Affinities--
Trp fluorescence was monitored as described in the
previous paragraph. Microliter amounts of TnI-(96-148) were added to 1 ml of each TnCF29W mutant (0.6 µM) in 200 mM MOPS, 90 mM KCl, 2 mM EGTA, 1 mM [Ca2+]free, 1 mM
DTT, pH 7.0, at 15 °C. Each apparent peptide affinity represents a
mean of 3 titrations fit with a logistic sigmoid function.
Determination of Ca2+ and Mg2+
Association and Dissociation Rates--
Ca2+ and
Mg2+ association (kon(Ca) and
kon(Mg)) and dissociation rates
(koff(Ca) and koff(Mg))
were measured using an Applied Photophysics Ltd. (Leatherhead, UK)
model SX.18 MV stopped-flow instrument with a dead time of 1.4 ms at
15 °C. The samples were excited using a 150-watt xenon arc source.
Ca2+ and Mg2+ binding kinetics of the
N-terminal domain within intact TnCF29W and
G34DTnCF29W were obtained utilizing Trp fluorescence
changes excited at 275 nm with emission monitored through a UV
transmitting black glass filter (UG1 from Oriel, Stratford, CT).
Ca2+ dissociation rates in the absence of
Mg2+ were also measured using the fluorescent
Ca2+ chelator Quin-2. Whereas the fluorescence of
Trp29 was selective for N-terminal Ca2+
dissociation, Quin-2 fluorescence reported Ca2+
dissociation from both the N- and C-domains within TnCF29W.
However, the Ca2+ dissociation rate from the N-terminal
domain within TnCF29W was easily distinguished from the
rate of Ca2+ dissociation from the C-terminal domain
because the latter rate was >95-fold slower in the absence
and presence of TnI-(96-148) or intact TnI. Quin-2 fluorescence was
excited at 330 nm, with its emission monitored through a 510-nm broad
band-pass interference filter (Oriel). Each data set represents an
average of 10-15 traces, fit with a single exponential (variance
<2 × 10
4). The curve fitting program (by P. J. King, Applied Photophysics Ltd.) uses the nonlinear
Levenberg-Marquardt algorithm. The buffer used in all the
Ca2+ stopped-flow experiments was 10 mM MOPS,
90 mM KCl, 1 mM DTT, pH 7.0.
Calculation of Ca2+ and Mg2+ Association
Rates--
The Ca2+ and Mg2+ association rates
were calculated using the equation kon = koff/Kd, where
koff represents the concerted release of two
Ca2+ or single Mg2+ ions and
Kd represents the binding event of two
Ca2+ or one Mg2+ to the N-domain of TnC, as
previously described (3, 8).
Muscle Fiber Experiments--
Single fibers were isolated the
day of use from bundles of rabbit psoas muscle that had been stored in
a glycerinating solution at 20 °C no longer than 1 month.
Solutions and the mechanical setup utilized for force measurements were
as previously described (53). Briefly, a single fiber was soaked in
relaxing solution containing 1% (v/v) Triton X-100 for 5 min to remove
any residual sarcolemma and sarcoplasmic reticulum. The fiber was then
tied down with pens in troughs attached to a servo-controlled DC torque motor (Cambridge Technologies, Watertown, MA) and an isometric force
transducer (model 403A, Cambridge Technologies) as previously described
(54). Fiber sarcomere length, width, and depth were measured with a
video camera (Sony model XC-ST70) and an image analysis system (Simple
PCI, Compix Inc., Cranberry Township, PA). Resting sarcomere length was
set between 2.50 and 2.60 µm. The fiber was then activated in a
pCa (-log[Ca2+]) 4.0 solution and rapidly
slackened after isometric force reached plateau. The analogue output of
the force transducer was digitized using a DaqBoard/2000 and Daqview
software (Iotech Inc., Cleveland, OH). The total force was measured
between the plateau and baseline levels. The same procedure was
utilized to obtain the resting force level of the fiber in a
pCa 9.0 solution. The active force generated by the fiber in
the various pCa solutions was calculated as the total force
minus the resting force. Three active force measurements were performed
in pCa 4.0 with the final activation taken as the maximal
force generated by the native fiber (i.e. prior to
extraction of TnCendogenous), which led to an average force
per cross-sectional area of 89 ± 4 kilo newton/m2.
Force versus pCa was then established or the
fiber was then soaked for 2 min in a TnC extraction solution containing
5 mM EDTA, 10 mM HEPES, and 0.5 mM
trifluoperazine dihydrochloride at pH 7.0 (55). The average
force in pCa 4.0 generated after TnCendogenous
extraction was 5 ± 1% of the maximal force generated by the
native fibers. The fiber was then washed three times in pCa
9.0 solution to remove any residual trifluoperazine dihydrochloride and
then soaked for 2 min in a pCa 9.0 solution containing 16.7 µM recombinant TnCF29W or
G34DTnCF29W. The maximal force generated by the recombinant
TnCs averaged 93 ± 1 and 62 ± 5% for TnCF29W
and G34DTnCF29W, respectively. Because additional fibers
reconstituted with 33.4 µM G34DTnCF29W or
TnCF29W did not improve the maximal amount of force
recovery or affect the force versus pCa
relationship, these data were then combined with the 16.7 µM measurements. All of the reconstituted fibers were
then exposed to a series of pCa solutions varying from
pCa 9.0 to 3.0 and the active force versus
pCa was measured. Every fourth activation was performed at
pCa 4.0 to which each adjacent and randomized pCa
was normalized. Because fibers reconstituted with recombinant TnC did
not completely recover maximal force, force versus
[Ca2+] curves were also performed on fibers in which TnC
was partially extracted. Four fibers were soaked in TnC extraction
solution for variable times to partially extract
TnCendogenous.
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RESULTS |
Effect of Ca2+ and Mg2+ on the Fluorescence
Spectra of the N-terminal Domains within TnCF29W and
G34DTnCF29W--
TnCF29W undergoes an
~2.4-fold increase in Trp fluorescence upon Ca2+ binding
to its N-domain sites at 15 °C (Fig.
2A). No change in Trp
fluorescence was observed upon addition of 50 mM
Mg2+ to TnCF29W (Fig. 2A). We
previously demonstrated that high Mg2+ binding to the first
EF-hand of CaM required the presence of an endogenous Z acid pair (3).
When a Z acid pair was introduced into the first EF-hand of
TnCF29W (i.e. G34DTnCF29W), addition
of Ca2+ led to an ~2.2-fold increase in Trp fluorescence
(Fig. 2B). Furthermore, G34DTnCF29W also
underwent an ~1.4-fold increase in its Trp fluorescence upon addition
of Mg2+ (Fig. 2B). These results demonstrate
that substitution of Gly-34 with Asp in the first EF-hand of TnC led to
Mg2+ binding to this EF-hand.
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Fig. 2.
Effect of Ca2+ and
Mg2+ on the fluorescence spectra of TnCF29W and
G34DTnCF29W. Fluorescence emission spectra of
TnCF29W (panel A) or G34DTnCF29W
(panel B) in the apo, Ca2+ saturated, or
Mg2+ saturated states. The spectra were recorded with an
excitation wavelength of 275 nm. Protein concentration was 1 µM in 1 ml of 200 mM MOPS, 90 mM
KCl, 2 mM EGTA, 1 mM DTT, pH 7.0, at 15 °C.
Trp fluorescence spectra were recorded before the addition of metals
(Apo), after the addition of either 50 mM
Mg2+ (+Mg2+) or 1 mM
[Ca2+]free (+Ca2+).
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Measurement of the Mg2+ Binding Affinity for
TnC
and the N-terminal
Domains within TnCF29W and
G34DTnCF29W--
Fig.
3A shows the
Mg2+-dependent increase in Trp fluorescence of
G34DTnCF29W (
). G34DTnCF29W exhibited a
half-maximal increase in Trp fluorescence at 295 ± 10 µM. However, addition of Mg2+ (up to 50 mM) caused no change in the Trp fluorescence signal of
TnCF29W (Fig. 3A, 
). Thus, as expected, the
G34D mutation in TnCF29W incorporates physiological
Mg2+ binding to the first EF-hand of
TnCF29W.
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Fig. 3.
Measurement of Mg2+ binding and
dissociation from G34DTnCF29W and TnCF29W.
Panel A shows the Mg2+-dependent
change in Trp fluorescence as a function of -log[Mg2+]
for G34DTnCF29W () or TnCF29W (). For
panels A and B, the protein, buffer, and experimental
conditions were as described in the legend to Fig. 2. Trp fluorescence
was monitored at 345 nm, with excitation at 275 nm. Each data point
represents an average ± S.E. of 3-5 titrations. Panel
B shows the Mg2+-dependent decrease in Trp
fluorescence of Ca2+ saturated G34DTnCF29W
(), TnCF29W (), or
TnC ( ) as a function of
-log[Mg2+]. Initially each protein was subjected to 10 µM [Ca2+]free (pCa
5.0) and then titrated with increasing concentrations of
Mg2+. 100% Trp fluorescence corresponds to the
Ca2+ bound state, whereas 0% fluorescence corresponds to
the Mg2+ saturated state in the presence of 10 µM Ca2+. The Trp fluorescence decreased 71, 88, and 98% of the total fluorescence change between the
Ca2+ bound and apo states for G34DTnCF29W,
TnCF29W, and TnC,
respectively. The Ca2+ affinity of
TnC was shown to be nearly
identical to that of TnCF29W (44). Panel C shows
Mg2+ dissociation from G34DTnCF29W and
TnCF29W. The time course of the change in Trp fluorescence is shown as EDTA
dissociates Mg2+ from the N-terminal domains of
G34DTnCF29W and TnCF29W. Each protein (2 µM) in the same buffer as Fig. 2 plus 10 mM
Mg2+ was rapidly mixed with an equal volume of 20 mM EDTA in the same buffer at 15 °C. Trp fluorescence
was monitored through a UV-transmitting black glass filter (UG1 from
Oriel) with excitation at 275 nm. The traces have been staggered for
clarity. Each trace is an average of at least 15 traces, and the data
were fit with a single exponential (excluding TnCF29W,
which was flat, variance <2 × 104). The kinetic
traces were triggered at time 0, the first ~2 ms of premixing time is
shown (the apparent lag phase), and the average trace was fit after
mixing was complete. Control experiments in which each protein (2 µM) in the same buffer as Fig. 2 plus 10 mM
Mg2+ was rapidly mixed with an equal volume of the same
buffer were flat lines.
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Fig. 3B shows that in the presence of 10 µM
[Ca2+]free the Trp fluorescence signal of
TnC (), TnCF29W
(), and G34DTnCF29W () was greater than 90%
saturated, which Mg2+ subsequently decreased in a
concentration-dependent manner. Mg2+
half-maximally decreased the Trp fluorescence of
TnC, TnCF29W and
G34DTnCF29W at 7.3 ± 0.8, 9 ± 1, and 1.9 ± 0.2 mM, respectively. Thus, the Ca2+-dependent fluorescence of
TnC, TnCF29W, and G34DTnCF29W was reversed by
Mg2+ binding, presumably because of the closing of the
N-terminal hydrophobic pocket of TnC and not because of any type of
interference from Mg2+ binding to the C-terminal domain.
Knowing the Kd(Ca) for each N-terminal
domain protein (determined as described later) and assuming competitive
Mg2+ binding, the Kd(Mg) of
TnC, TnCF29W,
and G34DTnCF29W was calculated to be ~1.8
mM, 2.2 mM, and 303 µM,
respectively. Thus, the calculated Mg2+ affinity for
G34DTnCF29W was identical to that measured directly as
described above. Furthermore, even though TnCF29W does not
undergo a change in fluorescence upon Mg2+ binding (Fig.
3A, ) its N-terminal domain appears to bind
Mg2+ and displace Ca2+. Table
I summarizes the data for
Mg2+ binding to TnCF29W and
G34DTnCF29W.
Measurement of the Mg2+ Dissociation Rate from the
N-terminal Domain within G34DTnCF29W--
Fluorescence
stopped-flow measurements were done to determine the rate of
Mg2+ dissociation from G34DTnCF29W. Fig.
3C shows the time course of the EDTA-induced decrease in Trp
fluorescence for G34DTnCF29W. At 15 °C, EDTA dissociated
Mg2+ from G34DTnCF29W at 570 ± 15 s1. There was no change in Trp fluorescence when the same
experiment was performed with TnCF29W because
Mg2+ binding to the N-terminal sites of TnCF29W
does not lead to a change in the Trp fluorescence.
Measurements of Ca2+ Binding Affinities for the
N-terminal Domains within TnCF29W and
G34DTnCF29W in the Absence and Presence of 3 mM
Mg2+--
The Ca2+ binding affinities of
TnCF29W and G34DTnCF29W were measured following
the Ca2+ induced increases in Trp fluorescence in the
absence and presence of 3 mM Mg2+ at 15 °C.
Fig. 4A shows
that in the absence of Mg2+, TnCF29W (
) and
G34DTnCF29W (
) exhibited half-maximal
Ca2+-dependent increases in Trp fluorescence at
3.3 ± 0.1 and 1.9 ± 0.1 µM Ca2+,
respectively. Therefore, in the absence of Mg2+,
G34DTnCF29W exhibited an ~1.7-fold increase in its
Ca2+ affinity, relative to that of TnCF29W.
Fig. 4A also shows that in the presence of 3 mM
Mg2+, TnCF29W () and G34DTnCF29W
() exhibited half-maximal increases in Trp fluorescence at 5.9 ± 0.4 and 11.6 ± 0.9 µM Ca2+,
respectively. Therefore, in the presence of 3 mM
Mg2+, G34DTnCF29W exhibited an ~2-fold
decrease in its Ca2+ sensitivity, relative to that of
TnCF29W. Furthermore, 3 mM Mg2+
shifts the Ca2+ sensitivity of TnCF29W and
G34DTnCF29W ~1.8- and 6-fold, respectively. Again,
assuming competitive Mg2+ binding, the
Kd(Mg) of TnCF29W and
G34DTnCF29W was ~3.8 mM and 590 µM, respectively. These values are in good agreement with
the direct and competitive Mg2+ binding studies described
above (see Table I). Thus, in the presence of 3 mM
Mg2+ both proteins are capable of opening their N-terminal
hydrophobic pockets in a Ca2+-dependent manner,
albeit with decreased Ca2+ sensitivity. Table
II summarizes the data for
Ca2+ binding to TnCF29W and
G34DTnCF29W.
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Fig. 4.
Measurement of Ca2+ binding to
TnCF29W, G34DTnCF29W, the
TnCF29W·TnI-(96-148) complex, or the
G34DTnCF29W·TnI-(96-148) complex in the presence and
absence of 3 mM Mg2+. Panel A shows
the Ca2+-dependent increase in Trp fluorescence
as a function of -log[Ca2+] for TnCF29W
(), G34DTnCF29W (), TnCF29W + 3 mM Mg2+ (), or G34DTnCF29W + 3 mM Mg2+ (). One-hundred percent fluorescence
corresponds to a 2.4-, 2.2-, 2.4-, and 1.6-fold fluorescence increase
for TnCF29W, G34DTnCF29W, TnCF29W + 3 mM Mg2+, or G34DTnCF29W + 3 mM Mg2+, respectively. Experimental conditions
were the same as described in the legend to Fig. 3, panel A. Panel B shows the TnI-(96-148)-dependent
decrease in Trp fluorescence as a function of -log[TnI-(96-148)] for
Ca2+-saturated TnCF29W () or
G34DTnCF29W (). 100% Trp fluorescence corresponds to
the Ca2+-saturated state, whereas 0% Trp fluorescence
corresponds to the Ca2+-TnI-(96-148)-saturated state of
each TnCF29W. The Trp fluorescence decreased 55 and 49% of
the total fluorescence change between the Ca2+-saturated
and apo states for TnCF29W and G34DTnCF29W,
respectively. Panel C shows the Ca2+-dependent increase in Trp fluorescence as a function of
-log[Ca2+] for the TnCF29W·TnI-(96-148)
complex (), the G34DTnCF29W·TnI-(96-148) complex
(), the TnCF29W·TnI-(96-148) complex + 3 mM Mg2+ (), or the
G34DTnCF29W·TnI-(96-148) complex + 3 mM
Mg2+ () under the same buffer conditions as panel
A. The TnC protein concentration was 0.3 µM, whereas
the TnI-(96-148) peptide concentration was 3 µM.
One-hundred percent fluorescence corresponds to a 1.4-, 1.4-, 1.4-, and
1.1-fold fluorescence increase for the
TnCF29W·TnI-(96-148) complex, the
G34DTnCF29W·TnI-(96-148) complex, the
TnCF29W·TnI-(96-148) complex + 3 mM
Mg2+, or the G34DTnCF29W·TnI-(96-148)
complex + 3 mM Mg2+, respectively.
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Table II
Summary of Ca2+ binding and exchange with TnCF29W and
G43D TnCF29W following the change in Trp fluorescence
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Measurement of Ca2+ Binding Affinities for the
N-terminal Domains within
TnCF29W·TnI-(96-148) and
G34DTnCF29W·TnI- (96-148) Complexes in
the Absence and Presence of 3 mM Mg2+--
A
partial decrease in Ca2+-saturated TnCF29W and
G34DTnCF29W fluorescence occurring upon binding of
TnI-(96-148) was utilized to determine the apparent peptide binding
affinity (39). Fig. 4B demonstrates that the apparent
TnI-(96-148) affinities for Ca2+ saturated
TnCF29W and G34DTnCF29W were nearly identical
at 457 ± 16 and 508 ± 9 nM, respectively. Thus,
any differences in Ca2+ sensitivity observed between the
two proteins in the presence of the peptide could not be because of
differences in peptide affinities. The Ca2+ binding
affinities for the TnCF29W·TnI-(96-148) and
G34DTnCF29W·TnI-(96-148) complexes were measured
following the Ca2+-induced increases in Trp fluorescence in
the absence and presence of 3 mM Mg2+ at
15 °C. Fig. 4C shows that in the absence of
Mg2+, the TnCF29W·TnI-(96-148) () and
G34DTnCF29W·TnI-(96-148) () complexes exhibited
half-maximal Ca2+-dependent increases in Trp
fluorescence at 267 ± 3 and 147 ± 2 nM
Ca2+, respectively. Therefore, in the absence of
Mg2+, the G34DTnCF29W·TnI-(96-148) complex
exhibited an ~1.7-fold increase in its Ca2+ affinity,
relative to that of the TnCF29W·TnI-(96-148) complex.
Consistent with intact TnI binding to TnC, TnI-(96-148) enhanced the
Ca2+ sensitivity of the regulatory domain of
TnCF29W ~12-fold (14, 32) and to G34DTnCF29W
~13-fold (see Table II). Fig. 4C also shows that in the
presence of 3 mM Mg2+, the
TnCF29W-TnI-(96-148) () and
G34DTnCF29W·TnI-(96-148) () complexes exhibited
half-maximal increases in Trp fluorescence at 397 ± 13 and
765 ± 41 nM Ca2+, respectively.
Therefore, in the presence of 3 mM Mg2+, the
G34DTnCF29W·TnI-(96-148) complex exhibited ~1.9-fold
decrease in Ca2+ sensitivity, relative to that of the
TnCF29W·TnI-(96-148) complex. Furthermore, 3 mM Mg2+ shifts the Ca2+ sensitivity
of the TnCF29W·TnI-(96-148) and
G34DTnCF29W·TnI-(96-148) complexes ~1.5- and 5.2-fold,
respectively. Again, assuming competitive Mg2+ binding, the
Kd(Mg) of TnCF29W and
G34DTnCF29W in complex with TnI-(96-148) was ~6.2
mM and 714 µM, respectively. Thus, unlike
Ca2+ binding to the regulatory sites of TnCF29W
and G34DTnCF29W, Mg2+ binding is not enhanced
by TnI-(96-148) binding and may actually be slightly decreased (see
Table I). Furthermore, in the presence of 3 mM
Mg2+, TnI-(96-148) increased the Ca2+
sensitivity to the regulatory domains of both TnCF29W and
G34DTnCF29W ~15-fold. Thus, Mg2+ did not
affect the enhancement of the Ca2+ affinity to the
regulatory domain of the TnCs caused by TnI-(96-148) binding,
consistent with Mg2+ acting only as a competitor for
Ca2+ binding.
Measurement of Ca2+ Dissociation Rates from the
N-terminal Domains within TnCF29W and
G34DTnCF29W Induced by EGTA, Quin-2, or
Mg2+--
Fluorescence stopped-flow measurements,
utilizing the EGTA-induced decreases in Trp fluorescence, were
conducted to determine the rates of Ca2+ dissociation from
the N-terminal sites of TnCF29W and
G34DTnCF29W. Fig.
5A shows the time
course of the EGTA-induced decreases in Trp fluorescence as
Ca2+ was dissociated from TnCF29W and
G34DTnCF29W (Trp EGTA traces). At 15 °C,
excess EGTA removed Ca2+ from TnCF29W and
G34DTnCF29W at 342 ± 5 and 403 ± 8 s1, respectively. Under saturating Ca2+
conditions, the presence of 3 mM Mg2+ did not
affect the rates of Ca2+ dissociation from
TnCF29W or G34DTnCF29W (data not shown, Table
II). These results were consistent with the Ca2+
dissociation rate from the regulatory sites of the fluorescent TnCDanz not being affected by Mg2+ (56).
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Fig. 5.
Measurement of Ca2+ dissociation
from TnCF29W or G34DTnCF29W in the presence or
absence of either TnI-(96-148) or intact TnI using EGTA, Quin-2, or
Mg2+. Panel A shows the rate of Ca2+
dissociation from TnCF29W and G34DTnCF29W
following the decreases in Trp fluorescence or the increase in Quin-2
fluorescence. The time course of the decrease in Trp fluorescence is
shown as EGTA dissociates Ca2+ from the N-terminal domains
of G34DTnCF29W and TnCF29W (Trp EGTA
traces). Each protein (2 µM) in 10 mM
MOPS, 90 mM KCl, 1 mM DTT, pH 7.0, plus 100 µM Ca2+ was rapidly mixed with an equal
volume of the same buffer plus 5 mM EGTA at 15 °C. Trp
fluorescence was monitored as described in the legend to Fig. 3,
panel C. Panel A also shows the time course of
the increase in Quin-2 fluorescence as Quin-2 dissociates
Ca2+ from the N-terminal domains of G34DTnCF29W
and TnCF29W (Quin-2 traces). Each protein (6 µM) in 10 mM MOPS, 90 mM KCl, 1 mM DTT, pH 7.0, plus 30 µM Ca2+
was rapidly mixed with an equal volume of the same buffer plus 150 µM Quin-2 at 15 °C. Quin-2 fluorescence was monitored
through a 510-nm broad band-pass interference filter with excitation at
330 nm. All the traces have been normalized and displaced vertically
for clarity. Panel B shows the time course of decrease in
Trp fluorescence as Mg2+ displaced Ca2+ from
the N-terminal domains of TnCF29W or
G34DTnCF29W (Trp Mg2+ traces). Each protein (4 µM) in the same buffer as panel A plus 15 µM Ca2+ was rapidly mixed with an equal
volume of the same buffer plus 150 mM Mg2+. Trp
fluorescence was monitored as described in the legend to Fig. 3,
panel C. The traces have been normalized and staggered for
visual clarity. Panel C shows the rate of Ca2+
dissociation from TnCF29W and G34DTnCF29W
following the decreases in Trp fluorescence in the presence of
TnI-(96-148) or the increase in Quin-2 fluorescence in the presence of
intact TnI. The time course of the decrease in Trp fluorescence is
shown as EGTA-dissociated Ca2+ from the regulatory
Ca2+ binding sites of the
G34DTnCF29W·TnI-(96-148) and the
TnCF29W·TnI-(96-148) complexes (Trp
TnI96-148 EGTA traces). Each TnC protein (0.6 µM) plus TnI-(96-148) peptide (6 µM) in 10 mM MOPS, 90 mM KCl, 1 mM DTT, pH
7.0, plus 100 µM Ca2+ was rapidly mixed with
an equal volume of the same buffer plus 10 mM EGTA at
15 °C. Trp fluorescence was monitored as described in the legend to
Fig. 3, panel C. Panel C also shows the time
course of the increase in Quin-2 fluorescence as Quin-2 dissociates
Ca2+ from the regulatory Ca2+ binding sites of
the G34DTnCF29W·TnI and the TnCF29W·TnI
complexes (Quin-2 intact TnI traces). Each TnC (3 µM) plus intact TnI (30 µM) in 10 mM MOPS, 90 mM KCl, 1 mM DTT, pH
7.0, plus 10 µM Ca2+ was rapidly mixed with
an equal volume of the same buffer plus 150 µM Quin-2 at
15 °C. Quin-2 fluorescence was monitored as described in panel
A. All the traces have been normalized and displaced vertically
for clarity. All the traces in Panels A-C are an average of
at least 15 traces fit with a single exponential as described in the
legend to Fig. 3, panel C (variance <2 × 104).
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To verify that the Trp signal changes were accurately reporting the
true Ca2+ dissociation rates and not a slower or faster
structural change, Ca2+ dissociation rates in the absence
of Mg2+ were also measured with the fluorescent
Ca2+ chelator Quin-2. Fig. 5A also shows the
time course of the Quin-2-induced increases in fluorescence as
Ca2+ was dissociated from TnCF29W and
G34DTnCF29W (Quin-2 traces). Nearly identical
Ca2+ dissociation rates were measured using Quin-2
fluorescence for TnCF29W and G34DTnCF29W at
346 ± 3 and 397 ± 2 s1, respectively, as were
measured by the EGTA-induced Trp changes. Therefore,
G34DTnCF29W exhibited an ~1.2-fold faster N-terminal
Ca2+ dissociation rate, relative to that of
TnCF29W. Similarly, the C-terminal Ca2+
dissociation rates from TnCF29W and G34DTnCF29W
were nearly identical at 0.48 ± 0.01 and 0.56 ± 0.01 s1, respectively (data not shown).
Fig. 3B demonstrated that Mg2+ could reverse the
Ca2+-induced increase in Trp fluorescence for both
TnCF29W and G34DTnCF29W. If Mg2+
binding to the N-terminal domains of TnCF29W and
G34DTnCF29W was truly competitive with Ca2+
binding, then it would be expected that Mg2+ could not bind
these Ca2+ saturated sites until Ca2+
dissociates, as has been demonstrated for the Ca2+- and
Mg2+-binding protein parvalbumin (57, 58). Fig.
5B shows the time course of Mg2+ binding to
Ca2+-saturated TnCF29W and
G34DTnCF29W (Trp
Mg2+ traces) following the decreases
in Trp fluorescence in a stopped-flow apparatus at 15 °C.
Mg2+ was able to displace Ca2+ from
TnCF29W and G34DTnCF29W at 348 ± 2 and
399 ± 6 s1, respectively. Because these values are
virtually identical to the Ca2+ dissociation rates in the
absence of Mg2+, it is clear that Mg2+ binding
to Ca2+-saturated TnCF29W and
G34DTnCF29W was limited by their respective
Ca2+ dissociation rates, supporting the hypothesis that
Mg2+ binds competitively with Ca2+.
Measurement of Ca2+ Dissociation Rates from the
N-terminal Domains within TnCF29W and
G34DTnCF29W Induced by EGTA or Quin-2 in the Presence of
TnI-(96-148) or Intact TnI--
The rates of Ca2+
dissociation from TnCF29W and G34DTnCF29W
induced by EGTA were also measured in the presence of TnI-(96-148)
(Fig. 5C, Trp TnI-(96-148) EGTA traces). At
15 °C, EGTA removed Ca2+ from the
TnCF29W·TnI-(96-148) and
G34DTnCF29W·TnI-(96-148) complexes at 10.6 ± 0.2 and 6.0 ± 0.3 s1, respectively. Nearly identical
rates were measured using Quin-2 to remove Ca2+ from the
TnCF29W·TnI-(96-148) and
G34DTnCF29W·TnI-(96-148) complexes at 10.6 ± 0.2 and 6.3 ± 0.1 s1, respectively (data not shown).
Thus, the N-terminal Trp signal change for both proteins in the absence
and presence of TnI-(96-148) accurately reports Ca2+
binding and dissociation.
To verify that TnI-(96-148) is a satisfactory model system for the
regulatory domain binding of TnC to TnI, stopped-flow studies were also
conducted with intact chicken skeletal TnI. Fig. 5C also
shows the time course of the Quin-2-induced increases in fluorescence
as Ca2+ was dissociated from the TnCF29W·TnI
complex at 9.1 ± 0.8 s1 and the
G34DTnCF29W·TnI complex at 5.4 ± 0.5 s1 (Quin-2 intact TnI traces). Thus, the
Ca2+ dissociation rates from the TnCF29W·TnI
and G34DTnCF29W·TnI complexes were similar to the
Ca2+ dissociation rates when complexed with TnI-(96-148).
Furthermore, G34DTnCF29W complexed with either TnI or
TnI-(96-148) exhibited an ~1.7-fold slower Ca2+
dissociation rate, relative to that of the TnCF29W
complexes. Thus, the binding of TnI to the regulatory domain of
G34DTnCF29W slows Ca2+ dissociation ~73-fold,
whereas this effect on TnCF29W was slowed only ~37-fold.
However, both TnI-(96-148) and intact TnI similarly slowed the
Ca2+ dissociation rate from the C-terminal domains within
TnCF29W and G34DTnCF29W only ~4-fold (data
not shown).
Following the Trp fluorescence signal, in the presence of
TnI-(96-148), 3 mM Mg2+ did not affect the
rate of Ca2+ dissociation from TnCF29W but
increased the Ca2+ dissociation rate of the
G34DTnCF29W·TnI-(96-148) complex to that of the
TnCF29W·TnI-(96-148) complex (data not shown, Table II).
The reason for this Mg2+ effect on the
G34DTnCF29W·TnI-(96-148) complex is currently unknown.
One possibility is that the increased negative charge of Asp-34
decreased the rate of Ca2+ dissociation from the regulatory
domain of G34DTnCF29W in the presence of TnI-(96-148),
which becomes screened by the positive Mg2+ ions.
Calculated Ca2+ Association Rates to the N-terminal
Domains within TnCF29W and
G34DTnCF29W--
Knowing the affinity of Ca2+
(Kd(Ca)) from the Ca2+
dependence of the increase in TnCF29W and
G34DTnCF29W Trp fluorescence and the rate of
Ca2+ dissociation (koff(Ca)) from
these same sites, we could calculate the rate of Ca2+
association (kon(Ca)) from the equation:
kon = koff/Kd. The calculated
values of kon(Ca) for TnCF29W and
G34DTnCF29W were 1.0 × 108 and 2.1 × 108 M1 s1,
respectively at 15 °C. Thus, G34DTnCF29W possesses a
~2.1-fold faster calculated Ca2+ association rate than
does TnCF29W. Furthermore, the
kon(Mg) calculated for G34DTnCF29W
was ~1.9 × 106 M1
s1, consistent with the slower dehydration rate of
Mg2+ compared with Ca2+ that tends to limit the
rate of Mg2+ binding to proteins (12).
Measurement of Ca2+ Association Rates to the N-terminal
Domains within TnCF29W and G34DTnCF29W in the
Absence and Presence of 3 mM Mg2+--
To
verify these rapid, nearly diffusion controlled Ca2+
association rates, the Ca2+-induced increase in Trp
fluorescence was measured in a stopped-flow apparatus. Fig.
6A shows the time
course of the rapid Ca2+-induced increase in Trp
fluorescence of G34DTnCF29W as the [Ca2+] was
increased from 0 to 5 µM. As the [Ca2+]
increases, the observed rate of Ca2+ binding increases as
expected for a second order reaction. At 1, 2.5, and 5 µM
Ca2+, the reaction occurred at 903 ± 37, 1103 ± 56, and 1660 ± 126 s1, respectively. A plot of the
observed rate versus [Ca2+] was fit by a
linear regression (r2 = 0.985, Fig.
6C, ) from which the Ca2+ association rate to
G34DTnCF29W was estimated at 1.9 ± 0.2 × 108 M1 s1. Similar
studies with TnCF29W yielded a Ca2+ association
rate of 1.57 ± 0.06 × 108
M1 s1
(r2 = 0.995, Fig. 6C, ). Thus, the
measured Ca2+ association rates to the N-terminal domains
of TnCF29W and G34DTnCF29W were extremely rapid
and essentially the same as the calculated Ca2+
association rates.
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Fig. 6.
Measurement of the Ca2+
association rates to TnCF29W and G34DTnCF29W in
the absence and presence of 3 mM Mg2+.
Panel A shows the time course of the increase in Trp
fluorescence of G34DTnCF29W as increasing concentrations of
Ca2+ were rapidly mixed with the protein. 4 µM G34DTnCF29W in the same buffer as
described in the legend to Fig. 5 was rapidly mixed with the same
buffer in the presence of increasing [Ca2+] at 15 °C.
The indicated [Ca2+] represents the amount of
Ca2+ that was presented to the apo-TnC immediately after
mixing was complete. Trp fluorescence was monitored as described in the
legend to Fig. 3, panel C. Each trace is an average of at
least 10-15 traces fit with a single exponential as described in Fig.
3, panel C (except No Ca2+, which was
flat, variance <2 × 104). Panel B shows
the time course of the increases in Trp fluorescence of
G34DTnCF29W as increasing concentrations of
Ca2+ were rapidly mixed with the protein in the presence of
3 mM Mg2+. The experimental conditions and
analysis were identical to the reactions as demonstrated in panel
A, except 3 mM Mg2+ was added to the
buffer and greater concentrations of Ca2+ were required to
achieve similar fluorescence values. Panel C shows plots of
the observed Ca2+ association rate versus the
[Ca2+] in the mixing chamber immediately after mixing was
complete for TnCF29W (), G34DTnCF29W (),
TnCF29W plus 3 mM Mg2+ (), or
G34DTnCF29W plus 3 mM Mg2+ ().
Each point represents the average ± S.E. of at least three determinants. The data were
fit with a linear regression where the slope represents the calculated
Ca2+ association rate, except for G34DTnCF29W
in the presence of 3 mM Mg2+, which
demonstrated no Ca2+ dependence of its association rate as,
described under "Results."
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At 3 mM Mg2+, G34DTnCF29W was
greater than 90% saturated with Mg2+ (Fig. 3A,
). Fig. 6B shows the time course of the
Ca2+-induced increase in Trp fluorescence of
G34DTnCF29W in the presence of 3 mM
Mg2+ as the [Ca2+] was increased from 0 to 25 µM. As the [Ca2+] increased, the rate of
the reaction did not increase as expected for a second order reaction
but remained fairly static at 440 ± 24 s1 (Fig. 6,
B and C, ). These static observed
Ca2+ association rates, close to the Mg2+
dissociation rate from G34DTnCF29W (see Table I), were
consistent with the interpretation of competitive Ca2+ and
Mg2+ binding to the N-terminal domain of
G34DTnCF29W. Thus, in the presence of saturating
[Mg2+], Ca2+ cannot bind to the regulatory
domain of G34DTnCF29W until Mg2+ dissociates.
Similar studies with TnCF29W yielded a Ca2+
association rate in the presence of 3 mM Mg2+
of 0.45 ± 0.03 × 108
M1 s1
(r2 = 0.999, Fig. 6C, ). Thus, at
these [Mg2+] and [Ca2+] the
Mg2+ dissociation rate from the regulatory domain of
TnCF29W was not rate-limiting for Ca2+ binding,
although it was slower than in the absence of Mg2+.
Mathematical modeling of the Ca2+ association rate
experiments with TnCF29W in the presence of 3 mM Mg2+ predicted the Mg2+
dissociation rate to be ~6000 s1 assuming a
kon(Mg) of 2 × 106
M1 s1 leading to a
Kd(Mg) of 3 mM (data not
shown). Thus, 3 mM Mg2+ slows the
Ca2+ association rate to TnCF29W only
~3.5-fold but drastically slows the Ca2+ association rate
to G34DTnCF29W by ~4.3 × 105-fold.
Table II also compares the Ca2+ binding properties of
TnCF29W and G34DTnCF29W in the absence or
presence of 3 mM Mg2+, with or without
TnI-(96-148).
Over longer times (0-5 s), in the absence of Mg2+, as the
[Ca2+] was increased from 0 to 5 µM slow
decreases in the Trp fluorescence signal were observed (~1-2
s1) for both TnCF29W and
G34DTnCF29W (less than 5% of the total Trp change, data
not shown). The amplitudes of these slow decreases in Trp fluorescence
decreased with increasing [Ca2+] and were absent when the
[Ca2+] exceeded 5 µM. Computer modeling of
these reactions predicted that these decreases in Trp fluorescence were
associated with Ca2+ removal from the N-domain sites of the
TnCs by the high affinity C-domain sites that possess an ~100-fold
slower Ca2+ association rate and ~10-fold higher
Ca2+ affinity (20). Furthermore, the rapid increase in Trp
fluorescence for both TnCF29W and G34DTnCF29W
became too fast to observe as the [Ca2+] exceeded 5 µM. Unexpectedly, as the [Ca2+] was
increased from 5 µM up to 1 mM another slow
second order rate constant was observed for TnCF29W and
G34DTnCF29W at ~1.0 × 106 and 2.0 × 106 M1 s1,
respectively (less than 10% of the total Trp signal change, data not
shown). This second Ca2+ association rate process was
abolished in the presence of 3 mM Mg2+ for both
TnCs and was not observed with
TnC, which was missing the
C-terminal domain Ca2+ binding sites (data not shown).
Thus, these slow Ca2+ association rates were associated
with the C-terminal domain of the TnCs and were consistent with
previously calculated Ca2+ association rates to the
C-terminal Ca2+/Mg2+ sites of TnC (18, 20).
Thus, the Trp29 fluorescence was marginally influenced by
C-terminal Ca2+ binding as has been reported for
TnCDanz, TnCF52W, and TnCF78W (17,
24, 59).
Functional Properties of TnCF29W and
G34DTnCF29W--
To investigate the effects of increased
competitive binding of Mg2+ to the regulatory domain of
G34DTnCF29W compared with TnCF29W,
both proteins were reconstituted into skinned rabbit psoas muscle fibers. Fig. 7A
shows the Ca2+-dependent increase in skinned
psoas muscle force with TnCendogenous (
),
TnCF29W (), or G34DTnCF29W () in the
presence of physiological 1 mM
[Mg2+]free (60). Half-maximal force occurred
at 331 ± 7, 531 ± 62, and 1830 ± 460 nM
Ca2+ for TnCendogenous, TnCF29W,
and G34DTnCF29W, respectively. Thus, 1 mM
[Mg2+]free shifted the Ca2+
sensitivity of force development with G34DTnCF29W
~3.4-fold compared with TnCF29W, consistent with
physiological competitive Mg2+ binding to
G34DTnCF29W.
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Fig. 7.
Ca2+ sensitivity of force
development with unextracted and reconstituted psoas muscle fibers with
TnCF29W and G34DTnCF29W in the presence of 1 or
3 mM [Mg2+]free. Panel
A shows the Ca2+ dependence of isometric force
development in unextracted (TnCendogenous, ) and
reconstituted psoas muscle fibers with TnCF29W () or
G34DTnCF29W () in the presence of 1 mM
[Mg2+]free. Each trace represents the
mean ± S.E. of at least three separate fibers fit with a logistic
sigmoid curve mathematically equivalent to the Hill equation. The
conditions for the experiments are as described under "Experimental
Procedures." Panel B shows the Ca2+ dependence
of isometric force development in unextracted
(TnCendogenous,  ) and reconstituted psoas muscle fibers
with TnCF29W () or G34DTnCF29W () in the
presence of 3 mM [Mg2+]free.
Analysis and experimental conditions were the same as described in
panel A, except for the increased
[Mg2+]free. Panel C shows the
-fold decreases in the [Ca2+] that produced half-maximal
force ([Ca2+]50) for reconstituted fibers
with TnCF29W (in 1 mM
[Mg2+]free () or 3 mM
[Mg2+]free () or G34DTnCF29W
(in 1 mM [Mg2+]free () or 3 mM [Mg2+]free ())
versus percent of maximal force recovery compared with the
mean [Ca2+]50 of the unextracted fibers ([Ca2+]50 endogenous). Each
point represents the mean ± S.E. for all the individual
reconstituted fibers. Panel C also shows the -fold decrease
in [Ca2+]50 for fibers in which
TnCendogenous was partially extracted versus
percent of maximal force recovery compared with
[Ca2+]50 endogenous
(TnCendogenous Extraction, ). The data
from four fibers were fit with a linear regression to determine the
relationship between incomplete force recovery and decreased
Ca2+ sensitivity as described under "Results."
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Because the Mg2+ affinity for the regulatory domain of
G34DTnCF29W is higher than that of TnCF29W, as
the [Mg2+] in the fiber increases there should be a
larger shift in the Ca2+ sensitivity of force for muscle
fibers containing G34DTnCF29W, compared with those
containing TnCF29W. Fig. 7B shows the
Ca2+-dependent increase in skinned psoas muscle
force with TnCendogenous (), TnCF29W (),
or G34DTnCF29W () in the presence of 3 mM
[Mg2+]free. Half-maximal force occurred at
540 ± 25, 770 ± 18, and 5770 ± 800 nM
Ca2+ for TnCendogenous, TnCF29W,
and G34DTnCF29W, respectively. Thus, in the presence of 3 mM [Mg2+]free the
Ca2+ sensitivity of force development was ~7.5-fold lower
for G34DTnCF29W compared with TnCF29W.
Furthermore, as the [Mg2+]free was increased
from 1 to 3 mM, the Ca2+ sensitivity of force
development with G34DTnCF29W was decreased ~3.2-fold.
However, under the same conditions the Ca2+ sensitivity of
force development with TnCendogenous or TnCF29W
was decreased only ~1.5-fold. These results are consistent with competitive Mg2+ binding to the regulatory domains of all
the TnC proteins, with the largest effect occurring with
G34DTnCF29W because of its higher Mg2+
affinity. Table III summarizes the
skinned muscle results.
After TnC was extracted, force was decreased to 5 ± 1% of the
maximal force generated with TnCendogenous (data not
shown). TnCF29W was capable of recovering maximal force to
93 ± 1%, whereas G34DTnCF29W recovered only 62 ± 5% of the maximal force. It has been demonstrated that partial
extraction of TnC can reduce maximal force recovery and decrease the
Ca2+ sensitivity of force development (61-64). We were
concerned that the decreased Ca2+ sensitivity of force
development generated by G34DTnCF29W could be explained by
its incomplete force recovery and not by its increased Mg2+
affinity. Fig. 7C shows that upon partial extraction of
TnCendogenous () to levels of 87, 69, 44, and 37%,
maximal force recovery shifted the Ca2+ sensitivity of
force development 2.1-, 2.5-, 4.4-, and 5.4-fold, respectively. This
decrease in Ca2+ sensitivity of force development caused by
partial extraction of TnCendogenous was nearly identical to
that determined by Brandt et al. (62). A linear regression
fit to our data (r2 = 0.934) predicted that at
the average force recovery observed with G34DTnCF29W of
62%, the Ca2+ sensitivity of force development would be
decreased ~3.3-fold due only to incomplete force recovery. Fig.
7C also shows that the average decrease in Ca2+
sensitivity of force development for G34DTnCF29W
compared with TnCendogenous at 1 () and 3 mM () [Mg2+]free was 5.5- and
10.7-fold, respectively. However, after taking into account incomplete
force recovery, Mg2+ actually decreased the
Ca2+ sensitivity of force generated by
G34DTnCF29W at 1 and 3 mM ~1.7- and 3.2-fold,
respectively, compared with TnCendogenous. Treating the
data similarly for TnCF29W predicted that the shifts in
Ca2+ sensitivity of force development with
TnCF29W compared with TnCendogenous could
be entirely explained by incomplete force recovery. Therefore, in the
presence of 3 mM [Mg2+]free, the
~3.2-fold shift in Ca2+ sensitivity of force development
with G34DTnCF29W was similar to the ~2.0-fold shift in
Ca2+ affinity of G34DTnCF29W compared with
TnCF29W either in isolation or in the presence of
TnI-(96-148).
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DISCUSSION |
Whether Mg2+ competes with Ca2+ for the
N-terminal regulatory domain of TnC is unresolved (15, 19, 23, 27, 32).
Several groups demonstrated that as the [Mg2+] was
increased, the Ca2+ sensitivity of TnC, Tn-activated
actomyosin ATPase, and force development decreased (19, 23-31). These
results suggested that Mg2+ binding to the regulatory
domain of TnC, competitive or otherwise, affected its biochemical and
physiological properties. We studied Mg2+ binding and
exchange with the regulatory domain of TnC and its mutant, utilizing
the Trp fluorescence of TnCF29W, which possesses a Phe 
Trp mutation immediately preceding the first Ca2+-binding loop.
We have previously demonstrated that an endogenous Z acid pair in the
first EF-hand of CaMF19W was required for physiologically
relevant Mg2+ binding to this EF-hand (3). Because the
first EF-hand of TnC has a Gly at the +z position, we
hypothesized that substitution of Gly with Asp should enable the first
EF-hand of TnC to bind Mg2+. Indeed, when Gly-34 at the
+z position was substituted with Asp, the Trp fluorescence
of G34DTnCF29W increased ~1.4-fold upon addition of
Mg2+, with a Kd(Mg) ~ 295 µM. This same mutation in the absence of Mg2+
led to an ~1.8-fold increase in Ca2+ affinity of the
regulatory domain. However, in the presence of 3 mM
Mg2+, the Ca2+ sensitivity of
G34DTnCF29W decreased ~6-fold. From the
Kd(Ca) obtained in the absence and
presence of Mg2+, we calculated the
Kd(Mg) to be ~588 µM for
G34DTnCF29W. Thus, the measured
Kd(Mg) was in good agreement with the
calculated Kd(Mg). These experiments
demonstrate that introduction of an Asp residue at the +z
position in the first EF-hand of TnCF29W enabled this
EF-hand to competitively bind Mg2+ with a physiologically
relevant affinity. Consistent with our results, introduction of a Z
acid pair into the CD site of oncomodulin led to an ~12- and 52-fold
increased Ca2+ and Mg2+ affinity to this site,
respectively (65). Similarly, in CaMF19W, replacement of
Asp with Asn at the +z position of the first EF-hand led to
an ~5- and 58-fold decreased Ca2+ and Mg2+
affinity to the N-terminal sites, respectively (3, 8). Thus, it would
appear that modification of the +z position in some EF-hand
proteins modulates the Mg2+ affinity more so than the
Ca2+ affinity.
Interestingly, the fluorescence of TnCF29W did not change
upon binding Mg2+ but increased upon binding
Ca2+ to the regulatory sites (43, 44, 48). However, we have demonstrated that Mg2+ can competitively bind and displace
Ca2+ from TnCF29W at the Ca2+
dissociation rate of ~350 s1, with a
Kd(Mg) in the range of 2.2-3.8
mM at 15 °C. This low Mg2+ affinity of
TnCF29W was consistent with previously calculated values of
0.8-5 mM (averaging to 3.7 ± 0.6 mM)
using other fluorescent TnCs in which competitive Mg2+
binding was assumed (23-26, 28, 29, 56). We propose that Mg2+ binds to the second regulatory Ca2+
binding site of TnCF29W because the Trp residue adjacent to
the first Ca2+ binding site was unperturbed by the direct
Mg2+ binding studies. Recent structures of the
Ca2+/Mg2+-binding proteins calbindin
D9K and CaM have demonstrated that Mg2+ binding
primarily affects only the local structural environment of the
cation-binding loop with the effects in CaM primarily occurring in the
N-terminal portion of the loop (4, 66,
,
, and
Physiology and Cell Biology,
** Molecular and Cellular Biochemistry, and ¶ Oral
Biology, The Ohio State University, Columbus, Ohio 43210 and the
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ABSTRACT
INTRODUCTION
