©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Physical Characterization of Calponin
A CIRCULAR DICHROISM, ANALYTICAL ULTRACENTRIFUGE, AND ELECTRON MICROSCOPY STUDY (*)

Walter F. Stafford III (1) (2), Katsuhide Mabuchi (1), Katsuhito Takahashi (3), Terence Tao (1) (2) (4)(§)

From the (1) Muscle Research Group, Boston Biomedical Research Institute, Boston Massachusetts 02114, the (2) Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, the (3) Department of Medicine, Center for Adult Diseases, Osaka, Osaka 537, Japan, and the (4) Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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. 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 svalue 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.


INTRODUCTION

Calponin (CaP)() 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).

The - 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).

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 -helical content. Its contour length is sufficient to span three actin subunits if it binds to F-actin along its long pitch helix.


EXPERIMENTAL PROCEDURES

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 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 CaP-anti-CaP Complexes

Monoclonal anti-CaP (Sigma, C-6047, purified by CaP affinity chromatography) and recombinant 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).

All specimens were observed with Philip 300 electron microscope at 60 kV. Analytical Ultracentrifugation

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).


RESULTS

The specific absorbance of recombinant 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.

The circular dichroism spectrum of 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 H0/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.


DISCUSSION

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 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.

Both recombinant 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.

It is interesting to note that, although substantially elongated, the -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).

The length of 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.

In conclusion, our studies indicate that CaP is a flexible elongated molecule with a low -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



FOOTNOTES

*
This work was supported by the National Institutes of Health Grant P01-AR41637. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114. Tel.: 617-742-2010; Fax: 617-523-6649. E-mail: Tao@BBRI.ERI.Harvard.edu.

The abbreviations used are: CaP, calponin; CaP, recombinant chicken gizzard -isoform of CaP.


ACKNOWLEDGEMENTS

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.


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