Rotational and Translational Motion of Troponin C*
Martin C.
Moncrieffe
,
Steven
Eaton§,
eljko
Bajzer
,
Christopher
Haydock
,
James D.
Potter¶,
Thomas M.
Laue§, and
Franklyn G.
Prendergast
From the
Department of Biochemistry and Molecular
Biology, Mayo Foundation, Rochester, Minnesota 55905, ¶ Department
of Molecular and Cellular Pharmacology, University of Miami School of
Medicine, Miami, Florida 33101, and § Department of
Biochemistry, University of New Hampshire,
Durham, New Hampshire 03824
 |
ABSTRACT |
Time resolved fluorescence anisotropy and
sedimentation velocity has been used to study the rotational and
translational hydrodynamic behavior of two mutants of chicken skeletal
troponin C bearing a single tryptophan residue at position 78 or 154 in
the metal-free-, metal-bound-, and troponin I peptide (residues 96-116
of troponin I)-ligated states. The fluorescence anisotropy data of both
mutants were adequately described by two rotational correlation times, and these are compared with the theoretically expected values based on
the rotational diffusion of an idealized dumbbell. These data imply
that the motion of the N- and C-terminal domains of troponin C are
independent. They also suggest that in the metal-free, calcium-saturated and calcium-saturated troponin I peptide-bound states, troponin C is elongated, having an axial ratio of 4-5. Calcium
or magnesium binding to the high affinity sites alone reduces the axial
ratio to approximately 3. However, with calcium bound to sites III and
IV and in the presence of a 1:1 molar ratio of the troponin I peptide,
troponin C is approximately spherical. The metal ion and troponin I
peptide-induced length changes in troponin C may play a role in the
mechanism by which the regulatory function of troponin C is effected.
 |
INTRODUCTION |
The three-dimensional structures of troponin C
(TnC)1 (1, 2), and the
homologous protein calmodulin (CAM) (3, 4) revealed molecular
architectures composed of globular N- and C-terminal domains joined by
an extended
-helical linker. The globular domains contain a pair of
EF-hand motifs (5), which are designed to coordinate metal ions. On the
basis of the crystal structure of TnC, with calcium bound to the
C-terminal domain sites (1, 2), Herzberg et al. (6) propose
that the major conformational change that occurs when the regulatory
N-terminal domain sites bind calcium involves the exposure of a
hydrophobic patch resulting from the movement of the B/C helix away
from the N/A/D helices. Subsequent structural data, for example, the
high resolution NMR (7) and x-ray crystal structures of
calcium-saturated (8) TnC and the x-ray crystal structure of the
calcium-saturated N-terminal domain of TnC (9), have confirmed the
validity of this model. However, TnC is but one component of a
three-component complex, and the issue of whether the structural
changes that occur in "isolated" TnC directly reflect its behavior
in the functioning troponin complex remains unresolved. Additionally,
it has been proposed that the hydrophobic patch that is exposed in the
calcium-replete N- and C-terminal domains are binding sites for
troponin I (10), and the structure and dynamics that TnC displays under
such conditions is not known.
We have recently completed the steady-state optical spectroscopic
characterization of two mutants of chicken skeletal TnC bearing a
single Trp residue at positions 78 (F78W) and 154 (F154W) in the N- and
C-terminal domains,
respectively.2 The introduced
Trp residue in both mutants is located at position
z + 1 of the EF-hand, which is immediately after the last metal ion
coordinating residue in site II (F78W) and site IV (F154W), respectively. Both mutants were iso-functional with wild-type TnC in
the restoration of contractile activity to TnC-depleted-skinned muscle
fibers.3 The spectroscopic
properties of Trp-154 are sensitive to calcium and magnesium binding at
sites III and IV (11), while Trp-78 responds to calcium binding at both
the N- and C-terminal domain sites. On the basis of the spectroscopic
properties of the Trp residue, we surmised that the indole moiety is in
a rigid molecular environment and that this feature should render these
mutants useful in studying the overall dynamic behavior of TnC. The
presence of a single Trp residue in either domain may also provide
information about the local dynamics of the N- and C-terminal domains
of TnC.
This paper reports a study of the rotational and translational motion
of F78W and F154W. We have first used minimum perturbation mapping (12,
13) to explore possible conformation of the Trp residue in both F78W
and F154W starting from the x-ray crystal structure of 2-calcium TnC
(2). Additionally, time-resolved anisotropy decays of the Trp
fluorescence and sedimentation velocity experiments have been used to
obtain information regarding the rotational motion and the shape that
TnC adopts in the metal-free-, metal-bound (calcium and magnesium)-,
and calcium-troponin I peptide (residues 96-116 of troponin I)-ligated
states, respectively. Our results suggest that the dynamics of
metal-free, 2·Ca2+, and 2·Mg2+-TnC are consistent
with that of flexible dumbbell and is similar to what has been found
for the metal-free state of CAM (14). However, in the calcium-saturated
state (with or without TnIp) and the
2·Ca2+TnIp state, the apparent domain motions are
no longer detected, which is suggestive of a more rigid conformation.
The sedimentation velocity data suggests that in the apo-,
calcium-saturated- and calcium-saturated TnIp states, TnC
is elongated with an axial ratio of 4-5. Calcium or magnesium binding
to the high affinity C-terminal domain sites results in a contraction
of the axial ratio to approximately 3, which is consistent with that
found in the 2-calcium crystal structure of TnC (2), which has
dimensions of 75 Å × 25 Å. However, in the presence of TnIp and
calcium bound to the high affinity sites, TnC becomes approximately spherical.
 |
MATERIALS AND METHODS |
Minimum Perturbation Mapping--
The initial structures used to
obtain minimum perturbation maps were generated from the 2-calcium
x-ray crystallographic coordinates of TnC (2), with bad contacts
removed by 5 steps of conjugate gradient minimization. The side chains
of Trp-78 and Trp-154 were subsequently built from the topology and
parameter internal coordinates. The following residues were allowed to
be free during the simulations: 20, 24, 35, 40, 43, 44, 47, 71, 73, and
80 (F78W); 98, 101, 102, 105, 113, 149, 151, 155, 157, and 158 (F154W).
All nonpolar hydrogens were treated as extended heavy atoms, and the
dielectric constant was equal to the distance in angstroms between
interacting atoms. The charge on ionized side chains was reduced by
80%, and nonbonded interactions were switched off over the range
7-11Å. Calculations were performed on a Silicon Graphics, Inc.
Indigo-2 computer using executable code derived from CHARMM (15)
version 22 with topology and parameter files taken from
CHARMM version 19.
Minimum perturbation mappings were computed on a 5° grid of Trp-78
and Trp-154
1 ×
2 torsion space (13).
During minimization,
1 and
2 were
constrained with a harmonic constraint energy constant of 400 kcal
mol-1 rad2. At each of the 72 × 72 grid
points, the free atoms were minimized using 40 steps of the steepest
descent method followed by 240 steps with the Powell (16) method. SHAKE
(17) was applied to all bonds involving hydrogen, and the system was
minimized for an additional 40 steps by the Powell method. Grid points
for which the final minimized energy exceeded 50 kcal
mol
1 were considered outliers, and the map was
interpolated at these points using bivariate interpolation (18).
Protein Preparation--
The expression and purification of the
TnC mutants has been described
previously.2 Metal-free
protein samples were prepared by dialysis (twice) against a medium
composed of 120 mM MOPS, 90 mM KCl, 2 mM EGTA at pH 7.0. After dialysis, the volume was adjusted
with dialysate to yield a protein concentration of 7-10
µM for fluorescence or 16 µM for
ultracentrifugation. Protein concentrations were calculated using the
previously determined molar absorptivity values of 6.6 mM
1 cm
1 for F78W and 5.5 mM
1 cm
1 for F154W.2
Calcium-ligated states of the mutants were obtained by adding aliquoits
of CaCl2 (Orion) so that the free calcium ion
concentrations, determined as described in Robertson and Potter (19),
were pCa 6.8 and pCa 3.5, corresponding to the 2-calcium and 4-calcium states. Additionally, the magnesium-saturated state was obtained by the
addition of 2 mM MgCl2. The high concentration
of MOPS used ensured that the pH changes accompanying metal ion binding to TnC were negligible.
Time-resolved Fluorescence--
The time-resolved anisotropy
decay of the Trp fluorescence was measured using time-correlated single
photon counting (20). Protein samples of approximately 7 µM concentration were excited at 300 nm using the
frequency-doubled output of a cavity-dumped synchronously pumped
Coherent 700 rhodamine 6G dye laser (Coherent), which was itself pumped
by the frequency-doubled output of a Coherent Antares YAG laser. The
emission was isolated using an interference filter (351 nm; 4 nm
bandpass) and detected with a Hamamatsu R2809 microchannel plate
photo-multiplier tube (Hamamatsu, Japan). The instrument response
function (full width at half maximum) obtained by collecting scattered
light from a suspension of nondairy creamer was 50 ps. Data channels
(2048) were acquired using a Nucleus PCA-II data acquisition card
(Oxford Instruments). Data acquisition was automated, and the parallel
(I
) and perpendicular
(I
) components were obtained by alternately
integrating the respective data sets for 30 s, to minimize the
effects of laser instability, until the peak count in the parallel
channel was approximately 2 × 104. The parallel and
perpendicular components of the intensity decay are related to the
anisotropy, r(t), and the total fluorescence intensity I(t) is related by
|
(Eq. 1)
|
|
(Eq. 2)
|
with I(t) being modeled by a
multiexponential function (20). Molecules lacking spherical symmetry
are usually modeled as prolate or oblate ellipsoids, and under such
circumstances, the anisotropy is expected to decay as a sum of
exponentials given by
|
(Eq. 3)
|
where r0 is the fundamental or zero-time
anisotropy, and the pre-exponential factors,
i, and the rotational correlation times,
i, are functions of the rates of rotation about the
molecular axes of the molecule and the orientation of the absorption
and emission dipoles relative to these axes (21-23).
Data analysis of the time-resolved anisotropy decay was performed using
the program
ANISO,4 employing
cubic discretization of the instrument response function. The
parameters were estimated by the maximum-likelihood method (24), which
minimizes the Poisson deviance,
|
(Eq. 4)
|
Here ck
and
ck
denote the measured counts for
the parallel and perpendicular polarized components, and
Fk
and
Fk
are the corresponding theoretical expressions for the number of counts, that is,
|
(Eq. 5)
|
where s denotes the parallel (
) or
perpendicular (
) component, and the integrals are discretized as
explained in Bajzer et al. (25). R is the
instrument response function with
|
(Eq. 6)
|
is the zero-time shift,
is a light scattering
correction, and b is the presumed constant background
parameter. The criteria for "goodness of fit" included f
tests applied to the Poisson deviances (that are distributed as
2) for models having different numbers of rotational
components and the required randomness of the residuals (26). The
steady-state anisotropy, rss, which is the average
of r(t) weighted by the total intensity
I(t) (27), was calculated using the
following.
|
(Eq. 7)
|
The experimentally obtained rotational correlation times were
compared with those expected for an idealized dumbbell using equations
developed by Garcia de la Torre and Bloomfield (28-30). This procedure
considers a dumbbell to be composed of two large spheres of diameter
1 connected by smaller spheres of diameter
2. The equation describing the rotational diffusion
coefficient for rotation about an axis perpendicular to the main
symmetry axis, Dr
, is
|
(Eq. 8)
|
where
is the viscosity at temperature T, and
kB is the Boltzmann constant. The coefficients
cijare given by Table I of Garcia de la Torre and
Bloomfield (30) and are valid for L/
1 < 10 and
1/
2 < 1. To calculate the anisotropy decay of the model dumbbell, the rotational diffusion
coefficient for rotation about the main symmetry axis,
D1, given by
|
(Eq. 9)
|
is also required (31). The functional form for the decay of the
fluorescence anisotropy in terms of the diffusion coefficients for
rotation about D1 and
Dr
for a system with three
rotational correlation times is similar to Eq. 3 with
1 = (4D1 + 2Dr
)-1,
2 = (D1 + 5Dr
)-1, and
3 = (6Dr
)-1 (31).
The expected rotational correlation times for the TnC mutants were
calculated using hydrated volumes of 27,973 Å3 and 34,045 Å3, corresponding to a commonly used value for the degree
of hydration for globular proteins of 0.2 g of H2O/g
of protein (27) and the value of 0.4 g of H2O/g of
protein suggested by Hubbard et al. (32). In all
calculations, the value of
2 was 3.5 Å.
Ultracentrifugation--
Equilibrium sedimentation experiments
were performed at 23.3 °C using short column (0.7 mm) cells (33)
and Rayleigh interference optics on a Beckman Model E analytical
ultracentrifuge equipped with a electronic speed control, RTIC
temperature controller. and a pulsed laser diode light source (670 nm).
Data were acquired at speeds of 10,000, 20,000, 30,000, and 40,000 revolutions/minute using a television camera-based online data
acquisition and analysis system (34). Identical experiments were also
conducted in a Beckman XL-I ultracentrifuge at 20 °C. Sample
concentrations were approximately 16, 8, and 5 µM, and
data was collected at intervals after the estimated time to equilibrium
and tested for equilibrium by subtracting successive scans (35). Data
within the optical window were selected and analyzed to estimate the
molecular weight of the various species using the program NONLIN (36).
Sedimentation velocity experiments were performed on a Beckman XL-I
instrument equipped with absorption or Rayleigh interference optics
(37), a 4-hole titanium rotor, 6-channel, 12-mm-thick charcoal-filled epon centerpieces, and appropriate windows. Experiments were conducted at 60,000 revolutions/minute at 20 °C. Data were acquired at 1-s intervals to produce sedimentation coefficient distributions according to the method described by Stafford (38). Estimates of the shape of the
TnC mutants were obtained by calculating Perrin shape factors and the
corresponding axial ratio of an ellipsoid. The Perrin shape factor,
F, is defined as
|
(Eq. 10)
|
where sobs is the measured sedimentation
coefficient, and s0, that of an equivalent
spherical molecule having a radius
R0·s0, is calculable
from
|
(Eq. 11)
|
where M is the mass of the particle,
, the calculated partial specific volume (0.72 g
1 cm3) based on the amino acid sequence,
is the solvent viscosity, NA is Avogadro's number,
R0 = [3M(1 +
)
/4
NA]1/3,
is the
density and
is the degree of hydration.
 |
RESULTS |
Minimum Perturbation Mapping--
The minimum perturbation
approach is based on the assumption that the overall structure of a
stable mutant differs from the wild-type protein only in the positions
of the backbone and side chain atoms that are neighbors of the mutant
side chain. Consequently, the method provides simple estimates of the
possible conformation of the mutant side chain (12). Minimum
perturbation maps for Trp-78 and Trp-154 are shown in Fig.
1. The map for Trp-78 has two wells, and
the trans-perpendicular (tp) well is about 11 kcal/mol more stable than
the trans-antiperpendicular well. The Trp-154 map has one distinct well
(tp). The maps predict that in both mutants, the trans-perpendicular
tryptophan conformation predominates over other side chain
conformations. Because the structure of neither mutant has been solved
crystallographically, the possibility that other tryptophan
conformations are significantly populated or even predominate cannot be
absolutely excluded. We investigated the possibility that alternate
packing of neighboring side chains might stabilize other tryptophan
conformations by calculating minimum perturbation maps for each
tryptophan mutant with the side chains neighboring truncated at the
-carbon position (maps not shown). The Trp-78 map with the indole
ring-truncated neighbor side chains has the gauche-antiperpendicular
well stabilizing to within 4 kcal/mol of the tp well and has
both the tp and ga wells splitting into two
sub-wells of similar stability. The Trp-154 map with truncated neighbor
side chains also has a ga well that is about 4 kcal/mol less
stable than the tp well. Although these results confirm that
crystal structures of the TnC mutants are required in order to be
certain about the predominant tryptophan side chain conformations, they
imply that there may be multiple conformations of the Trp residue, and
the interconversion between these conformations may perhaps influence
the anisotropy decays of the Trp residue.

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Fig. 1.
Minimum perturbation maps of Trp-78
(left) and Trp-154 (right)
1 × 2
isomerization in the mutants of chicken skeletal TnC. The
map for Trp-78 has wells in the trans-perpendicular (tp) and
trans-antiperpendicular (ta) orientations, whereas that for
Trp-154 has one well (tp). The contour lines within a well
are 1, 3, 5, 7, 9 kcal/mol (dashed) and 2, 4, 6, 8, 10 kcal/mol (solid) apart. Subsequent contours are separated by
10 kcal/mol.
|
|
Time-resolved Fluorescence--
We have used the time-resolved
fluorescence anisotropy decay of the Trp moiety in the TnC mutants to
obtain information about the rotational dynamics of TnC. Fig.
2 shows typical time-resolved intensity
decay data, which upon analysis, yielded the parameters given in Table
I. The time-resolved fluorescence
anisotropy decay of the Trp fluorescence from F154W was, except for the
metal-free state, dominated by the "long" rotational component,
1, which accounted for approximately 72-81% of the
total anisotropy decay under the sample conditions listed in Table I.
In the metal-free state of F154W at 20 °C, the recovered rotational
correlation times are 3.1 ns and 0.57 ns, with the zero-time
(r0) and steady-state (rss)
anisotropy being 0.23 and 0.07, respectively. Metal ion binding to the
high affinity sites in the C-terminal domain increases the recovered
1 and rss values relative to that
obtained in the metal-free state, whereas the r0
value remains unaltered. However, the
1 value is
sensitive to the type of metal ion occupying the C-terminal domain
sites. Thus, the recovered
1 value for the
2·Ca2+ state of F154W was 4.1 ns, and that for the
2·Mg2+ state was 3.4 ns. This suggests that the structure of
the 2·Ca2+ and 2·Mg2+ states of TnC are different
and is consistent with the fluorescence emission spectral behavior of
F154W under conditions where the high affinity sites are saturated by
calcium or magnesium ions.2 TnIp binding to
2·Ca2+-F154W results in a recovered
1 value of
8.1 ns and an rss value of 0.12. In the
calcium-saturated state of F154W, the recovered
1 value
is 11.7 ns, and the value of the short rotational component is almost
an order of magnitude less than those obtained in the apo-,
2·Ca2+, 2·Mg2+, or
2·Ca2+-TnIp states. The recovered
r0 value for the calcium-saturated state is
0.29, which is close to the value of 0.31 obtained by steady-state
emission anisotropy measurements in 67% glycerol at
46 °C.5
Additionally, the value of the recovered steady-state anisotropy for
the 4·Ca2+ state was approximately 44% higher than that for
the metal-free state. In the presence of saturating concentrations of
calcium and TnIp, the recovered parameters are almost
identical to those of the calcium-saturated state.

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Fig. 2.
Perpendicular (A) and
parallel (B) polarized components of the intensity
decay of F154W at 20 °C. The
insets are the residuals (R) and auto-correlation
(AC). The last 48 channels from this data set were truncated
before fitting. Each channel represents a time point of approximately
10.9 ps. Protein concentration was 7 µM, and the solution
composition was 100 mM MOPS, 90 mM KCl, 2 mM EGTA, pH 7.0, at 20 °C.
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Table I
Parameters recovered from the analysis of the time-resolved
fluorescence anisotropy decay data of the TnC mutants at 20 °C
The two rotational correlation times 1 and 2 and
their corresponding amplitudes, 1 and 2 as well
as the steady-state anisotropy (rss) and the
anisotropy at zero time (r0) are shown. The
uncertainties in the recovered parameters were obtained from 100 Monte-Carlo simulations. Protein concentrations were approximately 7 µM, and the solution composition was 100 mM
MOPS, 90 mM KCl, 2 mM EGTA, pH 7.0, at
20 °C.
|
|
The time-resolved fluorescence anisotropy decays of F78W were dominated
by
1, which accounted for 72-86% of the total
anisotropy decay. In the metal-free state, the recovered
1 value of 7.04 ns for this mutant was approximately 2×
greater than the corresponding value for F154W. This suggests that the
motional components of TnC that are sensed by Trp-78 and Trp-154 are
different. Magnesium binding to the high affinity sites results in a
reduction of the
1 value relative to that of the
metal-free state. This implies that magnesium binding to sites III and
IV alters the conformation of the C-terminal domain such that the
motional component detected by Trp-78 is different than that of the apo
state. When calcium replaces magnesium at the high affinity sites, the
recovered
1 value is the same at that obtained for the
metal-free state. The r0 and
rss values for the metal-free, magnesium-saturated, and 2·Ca2+-bound states are almost identical.
TnIp binding to 2·Ca2+-F78W increases the
recovered
1 value by approximately 3 ns. The
r0 and rss values are similar
to those of the metal-free, 2·Ca2+ and 2·Mg2+
states. The
1 and
2 values for the
calcium-saturated state of F78W are 11.47 ns and 0.09 ns, respectively.
The recovered r0 value is identical to that
obtained by steady-state emission anisotropy measurements in 67%
glycerol at
46 °C,5 and the rss
value was 0.16. The parameters recovered for 4·Ca2+ state of
F78W are remarkably similar to those obtained for the analogous state
of F154W as well as the 4·Ca2+-TnIp state of
F154W and suggest that Trp-78 and Trp-154 are detecting the same
components of TnCs motion.
The available high resolution structures have established that TnC
adopts a dumbbell shape in the 2·Ca2+ and 4·Ca2+
states (1, 7, 8). While high resolution structural data of the
metal-free state of TnC is currently unavailable, such data exists for
the homologous protein CAM (39). Metal-free CAM exists as a dumbbell,
and it is plausible that metal-free TnC is similarly shaped. To compare
the recovered rotational correlation times to those expected for TnC,
we have calculated the rotational diffusion coefficients for the
dumbbell shape depicted in the x-ray and NMR high resolution structures
of TnC using the analytical procedures developed by Garcia de la Torre
and Bloomfield (28-30). Fig. 3 shows the
predicted rotational correlation times of a dumbbell as a function of
its length, assuming hydration values of 0.2 and 0.4 g of
H2O/g of protein, and Fig. 4
depicts some of the possible motions of TnC. As expected, the values of
the rotational correlation time depend on the presumed degree of
hydration. Assuming that the length of TnC in solution is 75Å, which
is the value observed in the 2·Ca2+ crystal structure (2),
the predicted rotational correlation times at 20 °C would be 17, 14, and 10 ns for a hydration of 0.4 g of H2O/g of protein
and 14, 12, and 8 ns for a hydration of 0.2 g of H2O/g
of protein. For 2·Ca2+-F154W, the recovered
1
value of approximately 4 ns is substantially less than the shortest
expected rotational correlation time
(6Dr
1). This
also holds for the metal-free and 2·Mg2+-bound states, if the
shape of TnCs under these conditions is similar to that of the
2·Ca2+ state. For the 4·Ca2+ and
4·Ca2+-TnIp states of F154W and the
4·Ca2+ state of F78W, the
1 values recovered
from the r(t) data are consistent with those
calculated from (D1 + 5Dr
)
1, assuming
that the degree of hydration is 0.2 g of H2O/g of
protein. The
1 values recovered from the
r(t) data for the metal-free, 2·Ca2+,
and 2·Mg2+ states of F78W are shorter than
1/6Dr
, whereas that for the
2·Ca2+-TnIp state of F154W is close to this value
for a degree of hydration of 0.2 g of H2O/g of
protein.

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Fig. 3.
Predicted rotational correlation times
( ia = 1-3) of a rigid dumbbell as a
function of total length and the degree of hydration. ,
( = 0.4 g of H2O/g of protein; - - -,
= 0.2 g of H2O/g of protein.
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Fig. 4.
Possible rotational motions of TnC.
A, rotational diffusion about an axis parallel to the main
symmetry axis (D1) and perpendicular to the main
symmetry axis (Dr );
B, motion about a hinge in the central helix.
|
|
The low
1 value obtained for the apo state of F154W
prompted us to examine the effect of viscosity on the rotational motion of Trp-154. Fig. 5 shows the effect of
varying the external viscosity by additions of sucrose on the long and
short rotational components of the apo state of F154W. The
1 value scales linearly with the external viscosity,
whereas the short rotational component,
2, does not.
Similar results were also observed when the external viscosity was
altered by changes in temperature. Thus, although the magnitude of the
1 value for the apo state of F154W is perhaps not
indicative of the overall rotational motion of TnC, its dependence on
the external viscosity suggests that is probably represents "hinge
bending" motions of the two domains of TnC (40).

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Fig. 5.
Reciprocal of the recovered rotational
correlation times for F154W as a function of
T/ . The top graph shows the
data ( ) and fit ( ) to a straight line for the long rotational
component, whereas the variation of the short rotational component
( ) is shown below. The external viscosity was adjusted by additions
of sucrose (10-60%). cp 1, centipoise.
|
|
Analytical Ultracentrifugation--
It is difficult to ascribe
physical meaning to the rotational correlation times recovered from the
r(t) data and make inferences regarding the shape
that TnC adopts in solution from the fluorescence anisotropy data only
(41). To obtain information about TnC shapes, especially under solution
conditions for which high resolution structural data is unavailable, we
have measured sedimentation coefficients by velocity sedimentation.
Fig. 6 shows typical results for F154W in
the metal-free, 2·Ca2+, 2·Mg2+-states and also in
the presence of saturating concentrations of calcium (in the absence of
trifluoroethanol). In general, the g(s)
distributions had a single Gaussian component, except in those
experiments conducted in trifluoroethanol that usually had a small
component at 0.95-1.0 S. The experimentally obtained sedimentation coefficients, the calculated Perrin shape factors, and the resulting axial ratios are presented in Table
II.

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Fig. 6.
Sedimentation coefficient distribution of
F154W in the metal-free (lightface solid line),
2·Mg2+ (dashed line), and
2·Ca2+ (boldface solid line) states at
20 °C. Protein concentrations were 5-16
µM, and the solution composition was 100 mM
MOPS, 90 mM KCl, and 2 mM EGTA, pH 7.0.
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Table II
Calculated sedimentation coefficients of F154W obtained from
sedimentation velocity measurements at 20 °C
The Perrin shape factor (F) and the corresponding axial
ratio as well as the predicted hydrodynamic shapes are also given.
F was calculated assuming a hydration of 0.4 g of
H2O/g of protein (32) and a calculated of 0.7213 cm3g 1 based on amino acid sequences. The error in the
calculated sedimentation coefficients is approximately 10%. Protein
concentrations were 16, 8, and 5 µM, and the solution
composition was 100 mM MOPS, 90 mM KCl, 2 mM EGTA, pH 7.0, at 20 °C.
|
|
In the metal-free and calcium-saturated states, the sedimentation
coefficient is 1.6 S. From this value, an axial ratio of 4-5 can be
calculated that is at the higher limit of axial ratios normally
associated with globular proteins (42), suggesting that the molecule is
in an extended conformation. The addition of either calcium or
magnesium to the high affinity sites results in a contraction of the
axial ratio to a value of 3. An axial ratio of 3 for the
2·Ca2+ state of TnC is consistent with that observed in the
x-ray crystal structure (2) and suggests that the overall shape of
molecule when the high affinity sites are saturated with calcium or
magnesium is similar. The addition of TnIp to
2·Ca2+-F154W results in a further reduction of the axial
ratio to 1.0, indicative of a spherically symmetric molecule.
 |
DISCUSSION |
The primary objectives of this work are interpretation of the
parameters recovered from an analysis of time-resolved fluorescence anisotropy decay data of the single Trp mutants of TnC and to make
inferences regarding the effect of ligand binding on the dynamic
behavior of TnC. The x-ray crystal structures of 2·Ca2+ TnC
at pH 5.1 (2) and of calcium-saturated TnC at pH 7.2 (8) as well as the
NMR solution structure of calcium-saturated TnC at pH 7 (7) reveal that
TnC adopts a dumbbell shape. We have calculated the rotational
correlation times expected of an ideal dumbbell having the dimensions
of TnC using the analytical procedure developed by Garcia de la Torre
and Bloomfield (29-30) and Small and Anderson (31). Ideally, a
comparison of the theoretically predicted and experimentally obtained
rotational correlation times should allow one to ascribe each measured
correlation time to rotational diffusion about a particular molecular
axis. A comparison of the predicted rotational correlation times with
those obtained from an analysis of the time-resolved anisotropy decays
reveals that, although the model predicts three rotational correlation times (
1-
3) for the dumbbell-shaped TnC,
only two are recovered experimentally. The inability to recover more
than two-rotational components from time-resolved anisotropy decays is
consistent with previous reports in the literature (43-45).
Although the magnitude of the recovered
1 values, under
some conditions (the calcium saturates states, for example) are
consistent with those expected for the shortest predicted times
(1/6Dr
), the
2
values are much too short to represent overall rotational motions of
TnC. The simplest and perhaps most intuitive explanation for these
findings is that in solution, TnC is not the rigid dumbbell depicted by
the crystal structures. Indeed, the finding that there is detectable
fluorescence energy transfer between Tyr-10 and Tyr-109 of rabbit
skeletal TnC (46), a condition which necessitates that the separation
between these residues be less than or equal to 10 Å, supports this
idea, or at the very least, that there is flexibility, presumably,
about the central helix of the molecule. Alternatively, it may be
argued that motions leading to the predicted long correlation times
exist but are undetected by the fluorescence anisotropy decay
measurements or that current methods of data analysis are incapable of
resolving more than two rotational components. An inspection of Eq. 3
reveals that the magnitude of pre-exponential factor,
i,
will dramatically influence the ability to recover a particular
rotational correlation time. Specifically, if
i
0, that
rotational component,
i, would not be recovered. Given the
angle (
) between the absorption and emission dipoles, the dependence
of the anisotropy decay amplitudes on the angle between a chosen
symmetry axis and the dipoles can be determined. Such an analysis
reveals, as was the case for the dityrosine derivative of CAM (31),
that there are only a few possible orientations of the dipoles where
one might recover more than one correlation time describing the
rotational motion of the molecule.
At 20 °C, the recovered
1 value for metal-free F78W
is approximately two times greater than the corresponding value for
F154W, implying that the motion "sensed" by the Trp moiety in these
two mutants are different. Although not supported by available data (47), the glycine residue in the central helix of TnC has been suggested to be a possible "hinge region" (1) that would allow the
N- and C-terminal domains to move relative to each other. In an NMR
study of the backbone and methyl dynamics of the regulatory domain of
chicken skeletal TnC (residues 1-90), Gagné et al. (48) report an overall rotational correlation time for the metal-free state of 4.86 ± 0.15 ns at 29.6 °C. From our measurements, the
1 value of Trp-78 in the metal-free state at 30 °C is
5.12 ± 0.05 ns. This lends support to the notion that Trp-78
senses the motion of the N-terminal domain. Also, the 3 ns
1 value recovered for the apo state of F154W likely
represents the rotational motion of the C-terminal domain. This value
is somewhat smaller than the 4 ns that would be expected of a spherical
C-terminal domain based on molecular weight and most likely reflects
the less ordered nature of this domain relative to the N-terminal
domain in the apo state (49, 50). This interpretation is supported by
the fact that the recovered
1 values for the
2·Ca2+ state of F154W, where presumably, the C-terminal
domain is more ordered, is approximately 4 ns.
Additional support for the idea that the values of the long correlation
times of F154W reflect domain motion, particularly in the apo,
2·Mg2+, and 2·Ca2+ states of these mutants is
obtained when the effect of viscosity on the recovered
1
values are examined. Because the frictional coefficient is proportional
to solvent viscosity, protein motions consisting of large scale
fluctuations (the hinge-bending motions of two domains, for example)
will be influenced by the solvent viscosity (40). Thus, the component
representing the overall motion is expected to scale with the external
solvent viscosity, whereas those representing local motions should be
relatively insensitive to the external viscosity. As is evident from
Fig. 5, the
1 values of Trp-154 in the apo state of
F154W scale with external viscosity in contrast to the
2
values. The recovered
2 values are significantly greater
than the approximately 40 ps expected for the local motion of the Trp
residue. Given our interpretation that the mutants are ostensibly
sensing the independent motions of the N- and C-terminal domains, we
believe that the
2 values actually represent the
superposition of the domain motion of TnC on the localized motion of
the Trp residue.
We have used sedimentation velocity measurements to obtain estimates
regarding the shape TnC adopts when various ligands are bound, as this
is not readily obtained from the r(t) data alone. Although a distinction between an axial ratio of 4 and 5 cannot be
made, the sedimentation data (see Table II) clearly suggests that in
the metal-free, 4·Ca2+, and 4·Ca2+-TnIp
states, TnC is in an extended conformation. The increased steady-state
anisotropy values for the Ca4+ and
Ca4+-TnIp states relative to the metal-free state
implies a decrease in the "flexibility" of the Trp residue TnC, and
this is perhaps responsible for the longer
1 values
obtained under these conditions. For the
2·Ca2+-TnIp state, however, TnC has approximately
spherical symmetry, and consequently, the
1 value should
truly represent the rotational motion of the whole molecule. The
compact globular shape that TnC apparently adopts in the presence of
TnIp and calcium bound to the high affinity sites as
suggested by the sedimentation data disagrees with interpretations of
the x-ray scattering data of Blechner et al. (51), who infer
that the molecule was extended in the 2·Ca2+-TnIp
state. The recently solved crystal structure of TnC bound to a TnI
peptide (residues 1-47 of TnI) (52) revealed a complex having a
compact globular shape in contrast to the dumbbell structure of the
2·Ca2+ and 4·Ca2+ states and suggests that peptide
binding to TnC can result in structures similar to that seen with CAM
(53).
The data presented here may have important implications regarding the
function of the troponin complex, assuming that the behavior of the
TnC-TnIp complex mirrors that of TnC-TnI. These are: (i)
within the troponin complex, under conditions where the high affinity
sites of TnC are occupied by calcium ions, TnC has a compact,
essentially spherical shape; ii) calcium binding to the regulatory
sites, which provides the signal for muscle contraction, results in TnC
becoming elongated. This latter observation is consistent with previous
reports (7) and the results of Stone et al. (54), who found
that in a complex consisting of calcium-saturated TnC and TnI, TnC was
elongated. Thus, the conformational switch that signals muscle
contraction may involve not only the exposure of the hydrophobic patch
in the N-terminal domain but alterations in the shape of TnC, which
either allow or prevent its interactions with other members of the
troponin complex.
 |
ACKNOWLEDGEMENT |
We thank Dr. Enoch W. Small for helpful
discussions and for providing the FORTRAN program used to calculate the
rotational diffusion coefficient
Dr
and Mr. Peter Callahan for
his assistance in producing Fig. 4.
 |
FOOTNOTES |
*
This work was supported by National Institute of Health
Grants GM34847 (to F. G. P.) and AR37701 (to J. D. P.).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.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Mayo Foundation, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-3753; Fax: 507-284-9349; E-mail: prendergast{at}mayo.edu.
2
M. C. Moncrieffe, S. Yu Venyaminov, T. E. Miller, G. Guzman, J. D. Potter, and F. G. Prendergast, submitted
for publication.
3
J. D. Potter, J. D. Guzman, J. Zhao, and T. Miller, unpublished information.
4
Z. Bajzer, I. Penzar, M. C. Moncrieffe, and F. G. Prendergast, manuscript in preparation.
5
M. C. Moncrieffe and F. G. Prendergast,
unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
TnC, troponin C;
CAM, calmodulin;
TnIp, troponin I peptide (residues
96-116);
MOPS, 4-morpholinepropanesulfonic acid;
tp, trans-perpendicular;
ta, trans-antiperpendicular.
 |
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.