From the
Calponin is a thin filament-associated smooth muscle protein
that has been suggested to play a role in the regulation of smooth
muscle contraction. We have used circular dichroism spectroscopy,
electron microscopy, and analytical ultracentrifugation to study the
physical properties of recombinant chicken gizzard
Calponin (CaP)
The
The in vivo function of CaP has not been settled to date.
Although phosphorylation of myosin regulatory light chain has been
firmly established as the primary mechanism for regulation of smooth
muscle contraction (Adelstein and Eisenberg, 1980; Hartshorne, 1987),
thin filament-associated proteins such as caldesmon (Sobue and Sellers,
1991) have been suggested to play an auxiliary role that might be
important for the maintenance of tension under low light chain
phosphorylation levels. The known properties of CaP strongly suggest
that it too might play a role in thin filament-based regulation of
smooth muscle contraction (Winder and Walsh, 1993). Other cellular
functions that involve the actin cytoskeleton also have been suggested
(Lehman, 1991; Mabuchi, 1994; North et al., 1994; Takahashi
et al., 1993).
To achieve a better understanding of how CaP
interacts with actin and possibly with other actin-associated proteins,
we have undertaken a physical characterization of CaP using the
techniques of circular dichroism spectroscopy, electron microscopy, and
analytical ultracentrifugation. Our results indicate that CaP is a
flexible, elongated molecule with a low
Materials Reagents for polyacrylamide gel electrophoresis were from Bio-Rad.
Buffer components were from Research Organics (Cleveland, OH).
Restriction enzymes and other recombinant DNA materials were from Life
Technologies, Inc. Common laboratory reagents were from Sigma. Proteins Chicken gizzard CaP was purified according to Takahashi et al. (1986). Recombinant chicken gizzard
All specimens were observed with Philip 300 electron
microscope at 60 kV. Analytical Ultracentrifugation
The specific absorbance of recombinant
The circular dichroism spectrum of
Our goal in carrying out this work was to investigate the
gross size and shape of CaP and its mode of interaction with actin. We
used recombinant chicken gizzard
Both
recombinant
It is interesting
to note that, although substantially elongated, the
The
length of
In conclusion, our studies
indicate that CaP is a flexible elongated molecule with a low
We thank Dr. Sen Liu and Yude Qian for assistance in
the ultracentrifugation and circular dichroism studies, respectively.
We are grateful to Drs. John Gergely and Sherwin S. Lehrer for critical
reviews of the manuscript and helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-calponin. The
-helix content of
-calponin was estimated from its circular
dichroism spectrum to be
13%.
-Calponin melts with a single
sharp transition at
57 °C. Rotary shadowing electron
micrographs of
-calponin reveal diverse shapes ranging from
elongated rods to collapsed coils. The lengths of the rod-shaped
structures are
18 nm. Analytical ultracentrifugation studies found
-calponin to be homogeneous with a monomer molecular mass of 31.4
kDa, and a s
value of 2.34
S. These data could be used to model
-calponin as a prolate
ellipsoid of revolution with an axial ratio of 6.16, a length of 16.2
nm, and a diameter of 2.6 nm. Taken together, our results indicate that
calponin is a flexible, elongated molecule whose contour length is
sufficient to span three actin subunits along the long pitch helix of
an F-actin filament.
(
)
is a smooth
muscle-specific protein (Gimona et al., 1990; Takahashi et
al., 1987; Takahashi and Nadal-Ginard, 1991b) that has been
isolated from a variety of organs (Abe et al., 1990b; Marston,
1991; Takahashi et al., 1986; Walsh et al., 1993). It
appears to be thin filament-associated based on biochemical (Lehman,
1991; Nishida et al., 1990) and localization studies (Gimona
et al., 1990; North et al., 1994; Parker et
al., 1994; Takeuchi et al., 1991; Walsh et al.,
1993). In vitro studies have shown that CaP can bind to actin
(Makuch et al., 1991; Takahashi et al., 1986; Winder
and Walsh, 1990), tropomyosin (Childs et al., 1992; Takahashi
et al., 1988a, 1988b), Ca
-calmodulin
(Takahashi et al., 1986, 1988b), and myosin (Lin et
al., 1993; Szymanski and Tao, 1993) and that it inhibits the
actin-activated ATPase activity of myosin (Abe et al., 1990a;
Horiuchi and Chacko, 1991; Makuch et al., 1991; Winder and
Walsh, 1990). Phosphorylation of CaP in vitro has been
demonstrated (Naka et al., 1990; Winder and Walsh, 1990);
phosphorylated CaP no longer binds actin, nor does it inhibit
actomyosin ATPase (Winder and Walsh, 1990). It is not clear whether CaP
is phosphorylated in vivo, as conflicting results have been
reported by various workers (Bárány and
Bárány, 1993; Bárány et al., 1991;
Gimona et al., 1992; Winder et al., 1993).
- and
-isoforms of chicken gizzard calponin have been cloned
(Takahashi and Nadal-Ginard, 1991b). The cDNA-derived amino acid
sequence of
CaP reveal regions of similarity with smooth muscle
sm22
, Drosophila melanogaster mp20, and vav proto-oncogene product (Adams et al., 1992; Takahashi and
Nadal-Ginard, 1991a, 1991b). More recently, an acidic isoform of CaP
has been cloned and characterized (Applegate et al., 1994).
-helical content. Its
contour length is sufficient to span three actin subunits if it binds
to F-actin along its long pitch helix.
CaP was produced and
purified according to Gong et al. (1993). Rabbit skeletal
actin was prepared according to Spudich and Watt (1971). Circular Dichroism Spectroscopy Spectroscopy was carried out on a AVIV associates (Lakewood, NJ) model
62DS spectropolarimeter equipped with a Hewlett-Packard 89100A
temperature controller as described previously (Lehrer and Qian, 1990).
Electron Microscopy
Visualization of CaP Molecules
Recombinant CaP and
purified chicken gizzard CaP were diluted to 15 µg/ml in a solution
containing 0.1 M ammonium acetate and 30% glycerol, pH 7.2
(dilution buffer), and sprayed onto a surface of freshly cleaved mica
for visualization by the rotary shadowing technique according to
Mabuchi (1990).
Visualization of
Monoclonal anti-CaP (Sigma, C-6047, purified by CaP
affinity chromatography) and recombinant CaP-anti-CaP
Complexes
CaP (290 µg/ml) were
mixed at a molar ratio of 1:1.5 in phosphate-buffered saline (Life
Technologies, Inc.) and incubated overnight at 4 °C. The mixture
was then diluted to 0.6 µg/ml in
CaP concentration in the
dilution buffer, then processed and visualized as described by Mabuchi
(1991).
Sedimentation Equilibrium
Experiments were carried
out on a Beckman Instruments model E analytical ultracentrifuge
equipped with a real-time video-based data acquisition system and
Rayleigh optics (Liu and Stafford, 1992; Yphantis et al.,
1994). The video-based system automatically converts each digitized
Rayleigh pattern onto a computer disk file of fringe displacement
versus radius using a Fourier analysis similar to the one
originally described by DeRosier et al. (1972). The optics
were aligned according to the procedures described by Richards et
al. (1971a, 1971b, 1972). The camera lens was focused at the 2/3
plane of the cell. The cells were equipped with sapphire windows and
12-mm, 6-channel external loading centerpieces (Ansevin et
al., 1970). Other details and methods of data analysis were as
described previously (Brenner et al., 1990; O'Shea
et al., 1989).
Sedimentation Velocity
Patterns were acquired with
the on-line Rayleigh system and converted into concentration versus radius every 20 s. Sedimentation coefficients were determined by
following the rate of movement of the peak in the time derivative curve
as described previously (Stafford et al., 1990).
CaP was
previously determined to be 0.89 (mg/ml)
cm
using quantitative amino acid analysis to
measure the protein concentration (Gong et al., 1993). In this
work, we redetermined this quantity and obtained a value of 0.74
(mg/ml)
cm
. This was done using
the Rayleigh interferometric optical system of the analytical
ultracentrifuge to measure the refractive index of the protein
solution, from which the protein concentration was obtained (Graceffa
et al., 1988). For comparison, the value predicted by the
aromatic amino acid content of
CaP is 0.773 (mg/ml)
cm
. We consider the value of 0.74
(mg/ml)
cm
obtained by the
refractive index method to be more accurate, and was used throughout
this work for spectrophotometric determination of
CaP
concentration.
CaP is shown
in Fig. 1. The spectrum was analyzed using the basis spectra and
algorithm of Greenfield and Fasman (1969), yielding 13%
-helix,
32%
, and 55% other structures. The decrease in ellipticity at 222
nm was monitored as a function of temperature (Fig. 2). It can be
seen that
decreases sharply with a single
transition at the relatively high temperature of 57 °C.
Figure 1:
Circular dichroism spectrum of
recombinant CaP. [
CaP] = 0.36 mg/ml (11.3
µM), in a buffer containing 20 mM Tris-HCl, 0.1
M NaCl, 1 mM EGTA, pH 7.5. The spectrum was taken
using a 1-cm path length cuvette, at a temperature of 25
°C.
Figure 2:
Thermal stability of CaP. The
fractional change in ellipticity at 222 nm (see Fig. 1) was monitored
as a function of increasing temperature from 15 to 65 °C in 0.2
°C steps. Other conditions are specified in the legend for Fig.
1.
Sedimentation equilibrium measurements (Fig. 3) found CaP
to be essentially monodisperse at a concentration of 1.2 mg/ml (37
µM) in a buffer containing 0.1 M NaCl. The
monomer molecular mass was found to be 31.4 ± 1.0 kDa compared
to a value of 32.3 kDa calculated from the amino acid sequence. The
sample exhibited a slight tendency to dimerize with an equilibrium
constant on the order of 1
10
M
. The data from two loading
concentrations and two speeds were analyzed simultaneously using the
program NONLIN (Johnson et al., 1981).
Figure 3:
Equilibrium ultracentrifugation of
CaP. The equilibrium runs were carried out at 34,000 rpm at 4
°C for 24 h. Plot of the number ( circles), weight
( open squares), and z-average ( closed
squares) molecular mass averages versus local cell
concentration for a single cell loading concentration of 1.2 mg/ml (37
µM). The buffer contains 20 mM Hepes, 0.1
M NaCl, 0.5 mM dithiothreitol, pH
7.5.
Sedimentation
velocity measurements (Fig. 4) yielded a
svalue of 2.34 S. The size
and overall shape of calponin was determined as follows: using a value
of 0.366 g of H
0/g of protein for the hydration, and a
partial specific volume of 0.732 cm
/g estimated from the
amino acid composition, the frictional ratio f/f
was calculated to be 1.32. Using these data we modeled the
CaP molecule as a prolate ellipsoid of revolution with a
relatively high axial ratio of 6.16, having a length of 16.2 nm and a
diameter of 2.6 nm. These results are summarized in .
Figure 4:
Apparent
sedimentation coefficient distribution ( i.e. uncorrected for
diffusion) for CaP. The velocity runs were carried out at 56,000
rpm at 20 °C. Buffer conditions are specified in the legend for
Fig. 3. The x axis, s
, is the
apparent sedimentation coefficient computed as described by Stafford
(1994). Thirty-two Rayleigh patterns were averaged to compute the
g( s
) curve. The solid curve is
the least squares fit of the data to a Gaussian function. The small
deviations from the fitted curve are consistent with the small degree
of dispersity shown by sedimentation equilibrium. The sedimentation
coefficient (Table I) was determined from the rate of movement of the
peak in the time derivative curve as described under
``Experimental Procedures.''
The gross shape of CaP was also studied by rotary shadowing electron
microscopy. Micrographs of both purified chicken gizzard CaP
(Fig. 5 A) and recombinant CaP
(Fig. 5 B) reveal a diversity of shapes, ranging from
extended rods (marked with 1), to bent or folded rods (marked
with 2) and to globules (marked with 3). The average
length of 51 extended rodlike structures was 22 ± 3 nm. Since
this length includes the thickness of the metal, the true length of the
extended molecule is likely to be
18 nm.
Figure 5:
Visualization of CaP molecules by
rotary shadowing electron microscopy. The images of
CaP molecules
with variable shapes are arbitrarily divided into three categories:
rodlike (objects circled and labeled as 1), collapsed rods
(labeled as 2), and globular (labeled as 3). No
distinctive difference was observed between CaP purified from chicken
gizzard ( Panel A) and recombinant
CaP ( Panel B).
The images of monoclonal anti-CaP ( Panel C) are typical of IgG
molecules. Images of the anti-CaP-
CaP complex ( Panel D)
reveal the presence of appendages ( arrows) at the apices of
the antibody molecules. Bars indicate 50
nm.
To ascertain that the
structures observed in Fig. 5 B are CaP molecules,
monoclonal anti-CaP was used to label the
CaP. Individual anti-CaP
molecules appear as triangularly shaped objects
(Fig. 5 C), consistent with the overall shape of IgG
molecules. Micrographs of the anti-CaP-
CaP complex reveal
additional structures at the apices of the triangular IgG molecules
(Fig. 5 D). On closer inspection it can be seen that
these structures have the same range of shapes exhibited by the
specimens prepared from
CaP alone. Thus, it seems clear that all
the variably shaped objects are recognized by anti-CaP and are
authentic
CaP molecules.
CaP for the bulk of the work to
avoid potential complications that might arise from the presence of
isoforms and/or heterogeneity in phosphorylation. The sedimentation
equilibrium measurements clearly show that at an ionic strength of
120 mM,
CaP is essentially monomeric. Sedimentation
velocity measurements reveal that monomeric
CaP is elongated.
Using reasonable estimates for the degree and hydration and partial
specific volume, and assuming a prolate ellipsoidal geometry, the
length of the
CaP molecule was estimated to be 16.2 nm.
CaP and chicken gizzard CaP when visualized by rotary
shadowing electron microscopy display a variety of shapes, ranging from
rods to globules. The length of the fully extended rodlike images could
be estimated to be
22 nm. If one takes into account of the fact
that the shadowing material may add
2 nm at each end of the
protein, the actual length of the rodlike objects are likely to be
18 nm, in good agreement with the length estimation obtained by
sedimentation velocity. A simple and reasonable interpretation of the
ultracentrifugation results in conjunction with those obtained by
electron microscopy is that
CaP is an elongated molecule with
several flexible hinges. The sedimentation velocity measurements are
relatively insensitive to flexibility in a macromolecule and will
essentially yield information on the contour length (Iniesta et
al., 1988). During sample preparation for electron microscopy,
however, the molecule may collapse onto itself to various degrees,
giving rise to the observed distribution in shape.
-helix content
of
CaP is relatively low, only
13%. For comparison, sequence
analysis also yields an
-helix content of 13%, with the helical
regions localized within the N-terminal segment of
CaP (Takahashi
and Nadal-Ginard, 1991b). Our finding that
CaP melts with a single
sharp transition suggests that the helix or helices are in a distinct
structural domain that unfolds cooperatively. That
CaP melts at
the relatively high temperature of 57 °C indicates a high thermal
stability, which is consistent with the early finding that during its
purification chicken gizzard CaP is readily recovered from heat treated
chicken gizzard extracts (Takahashi et al., 1986).
CaP (16.2 nm) is such that it can span the length of
three actin subunits along the long pitch helix of an F-actin filament
(16.4 nm, taking 2.73 nm as the subunit rise along the genetic helix).
These results suggest that the actin:CaP binding stoichiometry might be
3:1, although a lower actin:CaP ratio is possible if overlap between
successive CaP molecules can occur. The actual actin:CaP binding
stoichiometry has not been studied in detail; it was reported by Winder
et al. (1991) to be 3.1-3.3:1 but binding curves
reported by other workers (Makuch et al., 1991; Noda et
al., 1992) suggest that it might be closer to 1-2:1. Further
studies on the interaction between CaP and actin will be required to
examine the validity of our proposal.
-helix content and a contour length of
16.2 nm. It may
interact with actin in a side-to-side fashion with a stoichiometry of 3
actin subunits or less per CaP.
Table:
Size and
shape parameters of CaP determined from sedimentation analysis
CaP, recombinant chicken
gizzard
-isoform of CaP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.