Comp. Biochem. Physiol. Vol. 94B, No. 4, pp. 769-773, 1989 Printed in Great Britain
0305-0491/89 $3.00+ 0.00 © 1989Pergamon Press pie
TROPONIN C OF THE ANTARCTIC ICEFISH (CHAMPSOCEPHALUS GUNNARI) WHITE MUSCLE G. FELLERand CH. GERDAY Laboratoire de Biochimie Musculaire de l'Universit6 de Li6ge, Institut de Chimie (B6), Sart Tilman, B-4000 Liege, Belgium (Tel: 041 56 33 40) (Received 24 April 1989) Abstract--1. The troponin C (TN-C) from the Antarctic icefish (Champsocephalus gunnari) has been isolated to homogeneity by a procedure involving extraction from acetone powder, DEAE-Sepharose 4B and AcA 54 column chromatography. 2. The calcium-induced conformational changes of apo TN-C have been studied by absorption difference spectroscopy, circular dichroism and intrinsic fluorescence. 3. The results indicate that the overall characteristics of icefish TN-C (such as amino acid composition, modifications of helix content and of the microenvironment of aromatic residues as a function of calcium binding) are quite similar to those of rabbit TN-C. 4. The intrinsic fluorescence properties are close to those reported for pike and carp TN-C.
INTRODUCTION The Ca 2+ control of contraction in striated muscles involves the troponin complex, a proteinic component of the thin filament, consisting of three subunits present in equimolar ratio: TN-T which binds the complex to tropomyosin, TN-I which inhibits actomyosin ATPase and TN-C, the Ca2+-binding subunit. According to the generally accepted model (Ebashi et al., 1969), interactions between myosin heads and actin monomers are sterically controlled by a shift of tropomyosin, induced by the binding of Ca 2÷ to TN-C (Potter and Gergely, 1974; Zot and Potter, 1987). Four Ca2+-binding sites homologous to the EF hand site of parvalbumin, another intracellular calcium-binding protein, have been recognized in TN-C (Collins et al., 1973) and numbered I-IV starting from the N-terminus (Kretsinger, 1976). Among these sites, two possess a high affinity for Ca 2÷ but bind Mg 2+ competitively (Ca2÷-Mg 2+ sites, III and IV) and two bind Ca 2÷ specifically with a 100-fold lower affinity (Ca 2÷-specific sites, I and II) as revealed by equilibrium dialysis. However, the affinity of these sites for Ca 2÷ is enhanced about 10-fold in the whole troponin complex, or when TN-C is complexed with TN-I (Potter and Gergely, 1975). Taking into account not only the intracellular concentration of Ca 2÷ and Mg 2÷ but also the dissociation rates of these cations from TN-C, one would expect to have the high affinity sites always saturated by Mg 2÷, even during relaxation, suggesting a structural function of these sites in the TN-C conformation. On the other hand, the Ca2÷-specific sites would only be occupied by Ca 2÷ during contraction; they seem therefore involved in the regulation of the excitationcontraction coupling (Wnuck, 1988). Several spectroscopic studies showed that the binding of Ca 2÷ to TN-C, in a physiological range of concentration of the cation, induces significant
modifications of the secondary structures (Johnson and Potter, 1978; Nagy and Gergely, 1979), allowing the molecule to adopt a more compact and rigid conformation (Murray and Kay, 1972). Following calcium titration, changes in helical content, tyrosine fluorescence and the environment of aromatic residues mainly occur during binding of the cation to Ca2+-Mg 2+ sites. This suggests that only the subtle conformational changes observed for a calcium: TN-C ratio of 3 and 4 could be related to the removal of troponin inhibitory effect after Ca 2÷ release from sarcoplasmic reticulum (for review see Wnuk, 1988). The three-dimensional structure of TN-C from avian fast skeletal muscle has been recently elucidated (Herzberg and James, 1985a,b; Sundaralingam et al., 1985a,b). The shape of the TN-C molecule resembles a dumb-bell with a long helical arm joining two globular parts where the Ca2÷-binding loops I and II are located in the N-terminal head and the loops III and IV in the C-terminal head. The present work is part of an analysis devoted to the striated muscles of the hemoglobin- and myoglobin-free Antarctic icefish. As the functional properties of the calcium-binding sites are thought to evolve with the level of animal organisation and in view of the phylogenic distance between Channichthyidae and other vertebrates commonly used as TN-C source, we have undertaken a spectroscopic study of the calcium-binding sites of icefish TN-C. MATERIALS AND METHODS
Purification of TN-C Icefish (Champsocephalus gunnari), were caught near the Kerguelen Archipelago (49°30'S, 70°E) and were kept frozen until further treatment in Belgium. The trunk white muscles were dissected free of any pigmented material and extracted twice at 4°C with a low ionic strength buffer containing 10 mM Tris-HC1, 1% (v/v) Triton X-100, pH 7.5. The centrifuged pellet was dried by addition of 6 vol of cold acetone ( - 30°C). Troponin C was
769
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G. FELLER and CH. GERDAY
then extracted from the resulting acetone powder and purified as described (McCubbin et al., 1982), except that the ion exchange chromatography step was achieved on a DEAE-Sepharose 4B column (2.5 x 40cm) equilibrated with 50 mM Tris, 6 M urea, 5 mM EDTA, 5 mM /~-mercaptoethanol, pH 8, and eluted with a linear KCI gradient from 0 to 0.6 M. Molecular sieving of the TN-C containing fraction on an AcA 54 column (2.8 × 82 cm) equilibrated with 50mM NH4HCO3, 5 m M EDTA, 5mM fl-mercaptoethanol was also carried out as the final purification step. Apo TN-C was prepared by desalting the concentrated protein solution on a Sephadex G25 plexiglass column (2 x 50cm) equilibrated in a 50 mM NH4HCO 3 solution previously decalcified on a Chelex 100 column.
Analytical procedures Amino acid analyses were performed on native and carboxymethylated proteins hydrolysed for 24, 48 and 72 h using a Dionex DC300 amino acid analyzer, equipped with a Waters dual wavelength colorimeter Model 440 and a HPLC polystyrene sulfonic column Waters (0.4 x 25 cm). Tryptophan was determined by N-bromosuccinimide titration of S-carboxymethylated protein (Spande and Witkop, 1967). Titration of -SH groups were made in denaturing conditions before and after reduction of the proteins with sodium borohydride (Habeeb, 1973). Ultraviolet absorption and difference spectra were recorded with a Beckman DU 8 spectrophotometer using proteins solubilized in 50mM NH4HCO3 or 20mM HEPES, 25 mM KCI, pH 7.5. Fluorescence measurements were made in the above buffers using a spectrofluorimeter Kontron SFM 23. Absorbance of solutions were kept below or close to 0.1 at the excitation wavelength. Circular dichroism spectra were recorded in a 0.1 cm path length cell using a Dichrograph Jobin-Yvon Mark V. The ~ helical content of TN-C was calculated according to the equation proposed by Chen et al. (1972) where 0222= Ac × 3300 and f~ = (0222 + 2340)/-30300. In all the above experiments, concentration of TN-C solutions were measured by amino acid analysis. Calcium titration was achieved by adding suitable aliquots of adequately diluted standard solution of 25 mM calcium nitrate (BDH No. 14136). Table 1. Amino acid composition of icefish TN-C, compared with those of some other fish species. Results are expressed as residues per
Amino acids Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Mr = lcefish 8.0 0.0 8.8 24.5 9.1 :~ 7.1~; 31.8 3.3 14.3 9.7 1.3§ 7.1 4.7 8.1 II 14.5 1.6 8.5 0.0
18,000 Carp* 9.7 0.0 7.1 22.5 7.9 9.7 30.2 2.7 14.0 10.6 0.8 6.3 7.7 7.7 11.5 2.1 9.5 0.0
*Gerday et al. (1984). tMcCubbin et al. (1982). :~Extrapolated at zero time hydrolysis. §Determined as cysteie acid. IlValue for 72 hr hydrolysis.
Eel* 7.3 0.0 3.6 25.5 6.3 7.3 32.8 3.6 17.5 9.9 1.0 5.8 10.1 9.0 10.3 0.0 10.0 0.9
Piker 8.2 0.0 7.3 24.9 6.1 7.3 32.9 1.0 13.0 10.1 1.0 5.8 8.8 7.8 14.0 1.9 9.2 0.0
RESULTS AND DISCUSSION
Purification o f icefish T N - C C h r o m a t o g r a p h y of the t r o p o n i n complex on D E A E - S e p h a r o s e 4B c o l u m n in d e n a t u r i n g conditions allowed us to o b t a i n icefish white muscle T N - C in a fairly pure state as judged by the well resolved peak o f the c h r o m a t o g r a m , polyacrylamide gel electrophoresis (not shown) a n d a m i n o acid analysis (no histidyl a n d t r y p t o p h a n residues). A p o T N - C prepared for calcium-induced c o n f o r m a t i o n a l change studies, exhaustively dialyzed against appropriate buffers m a d e with l0 m M E D T A a n d subsequently desalted on a Sephadex G25 column, contains, as s h o w n by atomic absorption, a residual calcium c o n c e n t r a t i o n corresponding to a Ca2+ : T N C m o l a r ratio ranging from 0.06 to 0.1, depending on the preparation. The a m i n o acid compositions of C. gunnari a n d of o t h e r T N - C from various fish species are given in Table 1. Icefish T N - C a m i n o acid c o m p o s i t i o n is closely c o m p a r a b l e to those of pike ( M c C u b b i n et al., 1982) a n d carp (Gerday et al., 1984). Like other vertebrates T N - C , it shows high ratio of glutamic + aspartic acid residues to lysine + arginine residues, a n d of phenylalanine to tyrosine residues. Thiol titration by D T N B in the presence of 8 M urea a n d after reduction with sodium b o r o h y d r i d e shows a s p o n t a n e o u s exposure of 0.97 mol thiol/mol T N - C , in good agreement with a m i n o acid analysis. However, the reactivity of this single thiol g r o u p is reduced by a b o u t 50% in the absence of sodium borohydride.
U.V. absorption spectra The typical u.v. a b s o r p t i o n spectrum of T N - C , as s h o w n in Fig. 1, is mostly due to n - - , n * electronic transitions of phenylalanine residues. A b s o r p t i o n b a n d s o f this c h r o m o p h o r e are visible at 253,259, 265 a n d 269 nm, whereas the shoulder at 276 n m corres p o n d to tyrosine residues. Ultra-violet a b s o r p t i o n difference spectra have been recorded after addition o f increasing a m o u n t s of calcium up to saturation of
0.6-
0.3
0
. 240
-
. 28,0
'l 320
A.(nm)
Fig. 1. Ultra-violet absorption spectrum of icefish apoTN-C taken in 50 mM NH4HCO3, pH 7.5. Protein concentration: 127 ,aM.
Antarctic icefish troponin C
T•15]
771
~50 v
× t,0
20
~ 0 ~
ol
. . . . 2
3
4
~
6
~
10.
MOLE RATIO Co2~/TNC Fig. 2. Calcium titration of icefish TNC by u.v. absorption difference spectra. Absorption changes, expressed as the difference between maximum signal at 265 nm and minimum signal of phenylalanine bands at 267 nm are plotted vs the molar ratio of Ca2+ to TNC. Conditions as in Fig. 1. the Ca2+-Mg 2+ and Ca 2+ sites. Binding of Ca 2+ to icefish TN-C results in an increase of the amplitude of the absorption bands of phenylalanine residues giving rise to maximal bands at 259, 265 and 269 nm, with a marked shoulder around 261 nm, and minima bands at 257, 263 and 267 nm. With the exception of an increase in the amplitudes of phenylalanine and tyrosine absorption bands, the profile is reminiscent of those obtained with rabbit and pike TN-C (McCubbin et al., 1982). Calcium titration as followed by perturbations of the phenylalanine absorption bands (Fig. 2) indicates that the binding of 2 Ca 2+ induces about 80-90% of the overall absorption changes. However, some additional effects occur up to 4 Ca 2+ bound. The increase in the amplitude of the signal associated with Phe chromophore has been related to the transfer of some or all of these residues to a more hydrophobic environment during saturation of high affinity sites (Nagy et al., 1978). Indeed, on the basis of the known tridimensional structure of bird TN-C
o
10
i
2
~
~, 6 6 ~ B ~ MOLE RATIOCa2+/TNC
Fig. 4. Calcium dependence of far u.v. CD spectra for icefish TNC. The ellipticity changes at 222 nm are expressed as a function of f, (~ helix fraction) x 100. Conditions as in Fig. 3. (Herzberg and James, 1985a; Sundaralingam et al., 1985a) one can observe that 4-5 Phe residues are located in helices E and H flanking sites III and IV, the structure of which is believed to be folded by calcium-binding. Circular dichroism spectra Typical far-u.v, circular dichroism spectra of icefish TN-C are shown in Fig. 3. Contamination by calcium of apo TN-C, deduced from ellipticity at 222 nm after addition of EGTA, parallels the values obtained by atomic absorption. Apo TN-C displays the usual ellipticity value at 222 nm of -10,5000 + 380 deg cm2/dmol. When the four sites are occupied by calcium, the ellipticity rises to 16,000 + 380 deg cm2/dmol. Figure 4 illustrates the variations in ~ helical content of TN-C plotted against calcium concentration. Saturation of the four Ca2+-binding sites induces a 40% increase of the helicity which reaches a value of f~ = 0.45. Modifications of the secondary structures are apparently completed after the binding of 3 mol Ca2+/mol TN-C, while 70% of the increase in helicity is induced by the binding of the first two calcium ions. The calcium-loaded TN-C adopts a more rigid conformation as already suggested by the absorption difference spectra. According to Sundaralingam et al. (1985a), the folding of the ~ helices E and H, and of the helical arm D/E during the binding of the first two calcium ions are responsible for the secondary structure modifications recorded by circular dichroism. Helical portions in the N-terminal domain would be preformed in the metal-free state and would undergo only minor rearrangement during regulatory site saturation. F6-
15
42-
200
220
240
2 (nrn) Fig. 3. Far u.v. circular dichroism spectra of icefish TNC taken in 50 mM NH4HCO3, pH 7.4 for Ca2+:TNC ratios of 0 (curve a), 2.3 (curve b) and 4.6 (curve c). Protein concentration: 22/~M. [(9] represents the mean residue ellipticity.
3io 29o
~(nm)
Fig. 5. Fluorescence emission spectra of icefish TNC in 20 mM HEPES, 25 mM KC1, pH 7.5. Protein concentration: 30#M. Curve a, in absence of Ca2+; curve b, Ca2÷:TNC = 4. Excitation wavelength, 276 nm.
772
G. FELLERand Ca. GERDAY
;0,°]\ /
0.8~_ MOLE RATIO CJ*/TNC Fig. 6. Relative fluorescence of icefish TNC as a function of calcium concentration. F0, fluorescence of apo-TNC; F, fluorescence in presence of various amounts of Ca 2+. Conditions as in Fig. 5. Intrinsic tyrosine fluorescence In the absence of any tryptophan residue in icefish TN-C, intrinsic tyrosine fluorescence of the protein has been used to evaluate the calcium-induced perturbations of the protein conformation near the tyrosyl residues. The fluorescence spectra of icefish T N - C , in the Ca2+-free and Ca2+-loaded forms are illustrated in Fig. 5. A p o T N - C display upon excitation at 276 nm an emission maximum near 310 nm. On the other hand, under saturating calcium concentration, the fluorescence relative value of Ca2+-loaded T N - C is 15% lower than that of a p o - T N - C while the maximum emission is slightly shifted towards longer wavelengths around 312 nm. Following Ca 2÷ titration at 310 nm (Fig. 6) one can observe that most of the fluorescence quenching is reached for a Ca 2+ : T N C ratio of 1 and is almost complete when 2 Ca 2÷ are bound. The fluorescence properties of icefish T N - C strongly diverge from that of rabbit (Leavis and Lehrer, 1978) and eel T N - C (Gerday et al., 1984), both showing a marked increase in fluorescence intensity upon calcium-binding. However, the fluorescence quenching recorded with icefish T N - C has also been reported in the case of pike (McCubbin et al., 1982) and carp T N - C (Gerday et al., 1984). In the former work, the quenching has been related to a buried tyrosyl residue and extra negative charges shielding the fluorophore whereas Gerday et al. (1984) consider that position 109 is not occupied by a tyrosine residue, the latter being in a position unaffected by calcium-binding. The proper answer to these questions will obviously be given only when the primary structure of one of these proteins will be known. Addition of E G T A , simulating calcium-free T N - C , does not restore the initial fluorescence value, but on the contrary produces a dramatic quenching o f the intrinsic tyrosine fluoresence. A c a l c i u m - E G T A complex is not involved in this effect. Rather, we think that an E G T A - p r o t e i n interaction, as reported in the case of some parvalbumins (Parello et al., 1979) or calmodulin (Haiech et al., 1980), is responsible for the decrease in fluorescence emission. C,~NCLUSIONS Despite the phyl genetic distance and the fact that the Channich~ayidae family has evolved in an extreme and almost closed environment for a long period of time, the icefish troponin-C shows
physiochemical and functional properties quite comparable to those of troponin-C from other teleosts. Acknowledgements--We thank the Territoire de Terres Australes et Antarctiques Franqaises for the facilities offered at Port-Aux Francais Station. We also thank N. GerardinOtthier and S. Collin for excellent technical assistance. This work was supported by the "Fonds de la Recherche Fondamentale et Collective-Initiative Ministerielle, Belgium" and by the "Fonds de la Recherche Scientifique et M4dicale, Belgium".
REFERENCES Chen Y. H., Yang J. T. and Martinez H. M. (1972) Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry N Y 11, 4120-4131. Collins J. H., Potter J. D., Horn M. J., Wilshire G. and Jackman N. (1973) The amino acid sequence of rabbit skeletal muscle troponin C: gene replication and homology with calcium-binding proteins from carp and hake muscle. FEBS Lett. 36, 268-272. Ebashi S., Endo M. and Ohtsuki I. (1969) Control of muscle contraction. Qt. Rev. Biophys. 2, 351-384. Gerday Ch., Francois J. M. and Dubois I. (1984) Troponin C from eel (Anguilla anguilla) skeletal muscle: a protein containing one single tryptophan residue. Comparison with carp skeletal muscle troponin C. Mol. Physiol. 6, 43-54. Habeeb A. (1973) A sensitive method for localisation of disulfide containing peptides in column effluents. Analyt. Biochem. 56, 60~55. Haiech J., Vallet B., Aquaron R. and Demaille J. G. (1980) Ligand binding to macromolecules: determination of binding parameters by combined use of ligand buffers and flow dialysis; application to calcium-binding proteins. Analyt. Biochem. 105, 18-23. Herzberg O. and James M. N. G. (1985a) Structure of the calcium regulatory muscle protein troponin C at 2.8A resolution. Nature, Lond. 313, 653~59. Herzberg O. and James M. N. G. (1985b) Common structural framework of the two Ca2+/Mg 2+ binding loops of troponin C and other Ca 2+ binding proteins. Biochemistry N Y 24, 5298-5302. Johnson J. D. and Potter J. D. (1978) Detection of two classes of Ca 2+ binding sites in troponin C with circular dichroism and tyrosine fluorescence. J. biol. Chem. 253, 3775-3777. Kretsinger R. H. (1976) Calcium binding proteins. A. Rev. Biochem. 45, 239-266. Leavis P. C. and Lehrer S. S. (1978) Intrinsic fluorescence studies on troponin C. Arch. Biochem. Biophys. 187, 243-251. McCubbin W. D., Oikawa K. Sykes B. D. and Kay C. M. (1982) Purification and characterization of troponin C from pike muscle: a comparative spectroscopic study with rabbit skeletal muscle toponin C. Biochemistry N Y 21, 5948-5956. Murray A. C. and Kay C. M. (1972) Hydrodynamic and optical properties of troponin C. Demonstration of a conformational change upon binding calcium ion. Biochemistry N Y 11, 2622-2627. Nagy B. and Gergely J. (1979) Extend and localization of conformational changes in troponin C caused by calcimn binding. Spectral studies in the presence and absence of 6M urea. J. biol. Chem. 254, 12732-12737. Nagy B., Potter J. D. and Gergely J. (1978) Calciuminduced conformational changes in a cyanogen bromide fragment of troponin C that contains one of the binding sites. J. biol. Chem. 253, 5971-5974.
Antarctic icefish troponin C Parello J., Reimarsson P., Thulin E. and Lindman B. (1979) Na + binding to parvalbumins studied by 23Na NMR. FEBS Lett. 100, 153-156. Potter J. D. and Gergely J. (1974) Troponin, tropomyosin and actin interactions in the Ca 2÷ regulation of muscle contraction. Biochemistry N Y 13, 2697-2703. Potter J. D. and Gergely J. (1975) The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J. biol. Chem. 250, 4628-4633. Spande T. F. and Witkop B. (1967) Determination of the tryptophan content of proteins with N-bromosuccinimide. Meth. Enzym. 11, 498-506. Sundaralingam M., Bergstrom R., Strasburg G., Rao S. T., Roychowdhury P., Greaser M. and Wang B. C, (1985a)
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Molecular structure of troponin C from chicken skeletal muscle at 3 angstrom resolution. Science N Y 227, 945-948. Sundaralingam M., Drendel W. and Greaser M. (1985b) Stabilization of the long central helix of troponin C by interhelical salt bridges between charged amino acid side chains. Proc. nam. Acad. Sci. USA 82, 7944-7947. Wnuck W. (1988) Calcium binding to troponin C and the regulation of muscle contraction: a comparative approach. In Calcium and Calcium Binding Proteins. Molecular and Functional Aspects (Edited by Gerday Ch., Bolis L. and Gilles R.), pp. 44458. Springer, Berlin. Zot A. S. and Potter J. D. (1987) Structural aspects of troponin tropomyosin regulation of skeletal muscle contraction. A. Rev. Biophys. Chem. 16, 247-281.