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Protein Science (2001), 10:771-778.
Copyright © 2001 The Protein Society

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves

Sibyl Baladi1, Philipp O. Tsvetkov2,3, Tatiana V. Petrova1, Takashi Takagi4, Hiroshi Sakamoto5, Vladimir M. Lobachov2, Alexander A. Makarov2 and Jos A. Cox1

1 Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland
2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
3 UPRESA CNRS 6032, Université de la Mediterranée, Marseille 13385, France
4 Biological Institute, Faculty of Science, Tohoku University, 980-8578 Sendai, Japan
5 Laboratoire de Chimie Structurale des Macromolécules, Department of Biochemistry and Molecular Genetic, Pasteur Institute, 75724 Paris Cedex 15, France

Reprint requests to: Jos A. Cox, Department of Biochemistry, 30 Quai Ernest Ansermet, 1211 Genève 4, Switzerland; e-mail: jos.cox{at}biochem.unige.ch; fax: 41-22-7026495.

(RECEIVED September 25, 2000; FINAL REVISION January 12, 2001; ACCEPTED January 12, 2001)

Sibyl Baladi and Philipp O. Tsvetkov contributed equally to this paper.

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.40601.


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44°C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 µM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+ forms melt in the 111°–123°C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

Keywords: Calcium-binding protein; EF-hand; thermal stability; molten globule state; domain structure

Abbreviations: CaVP, Ca2+ vector protein • CaVPT, Ca2+ vector protein target • N-CaVP, segment 1-86 of CaVP • C-CaVP, segment 81-161 of CaVP • CaM, calmodulin • TnC, troponin C • TML, trimethyllysine • DSS, dissucinimidyl suberate • CD, circular dichroism • DSC, differential scanning calorimetry • Td, denaturation temperature • NMR, nuclear magnetic resonance


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
The cephalochordate amphioxus is believed to constitute the evolutionary link between invertebrates and vertebrates. It contains a unique protein pair, Ca2+ vector protein (CaVP) and its target Ca2+ vector protein target (CaVPT). These proteins have not yet been identified in any other phylum (Cox 1986). CaVP and CaVPT form a 1:1 complex and are abundant in the muscle, the spinal chord, and the gonads of amphioxus. The physiological role of the CaVP/CaVPT protein pair is still unknown.

Although CaVP (18.3 kD) shares ~35% of sequence identity with calmodulin (CaM) and troponin C (TnC) (Kobayashi et al. 1987), it does not substitute for the latter proteins in functional assays (Cox 1986). Bona fide CaM and TnC have been isolated from amphioxus (Cox 1986; Takagi et al. 1994). CaVP possesses four EF-hands, but due to mutations of Ca2+-coordinating residues in sites I and II, it binds only two Ca2+ (Kobayashi et al. 1987). The protein contains an acetylated amino terminus, and two {varepsilon}-trimethyllysines (TML) in the third EF-hand (Fig. 1Go). CaVPT (26.6 kD) is constituted of three distinct domains (Takagi and Cox 1990) with a Pro–Ala–Lys-rich motif, an IQ motif and two immunoglobulin II (IgII) folds. Preliminary data indicate that only free CaVPT, but not the complex CaVP/CaVPT, interacts with a 106-kD protein related to paramyosin (T.V. Petrova, unpubl. data).



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  		Fig. 1. Amino acid sequence of CaVP including N-CaVP (arrow points to the last residue) and C-CaVP (arrow points to the first residue). EF-hand motifs are indicated in bold. Overlapping of both segments is emphasized in box. Relevant residues for this study are highlighted. Underlined lysines stand for TML. X, Y, and Z are the residues positioned at the vertices of the Ca2+-coordinating bipyramid.

 
In this study, we address questions related to the domain structure of CaVP and the interdomain interaction. On the basis of its structural homology with CaM and TnC, which are composed of two folding modules linked by a central {alpha}-helix, we hypothesized that the amino- and carboxy-terminal domains of CaVP constitute independent folding units (Cox et al. 1990). Therefore, CaVP, as well as its amino- and carboxy-terminal moieties were cloned, expressed in Escherichia coli, and purified. Because recombinant CaVP is not amino-acetylated and does not contain the two TML residues present in the wild-type protein, the interactive properties of the two forms were compared. We also analyzed the conformation and stability of the recombinant amino- and carboxy-terminal halves of CaVP and their interactions with ions, with each other and with CaVPT.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Expression and purification of recombinant proteins
On the basis of the homology of CaVP with CaM and TnC, the amino-terminal half (N-CaVP) was designed to end at residue 86, which includes the two first EF-hand motifs; the carboxy-terminal half (C-CaVP) starts at position 81, so as to include a Trp (Fig. 1Go), which is convenient for detection and quantification of the polypeptide. Full-length CaVP, N-CaVP, and C-CaVP were overexpressed in E. coli and purified. All three proteins show electrophoretic homogeneity in native and SDS-PAGE. Their exact molecular masses, determined by electrospray mass spectrometry, are 18,223 ± 1 Da (vs. 18,224 calculated) for CaVP, 9,768 ± 5 Da (vs. 9,773) Da for N-CaVP and 9,402 ± 2 Da (vs. 9,269) for C-CaVP. The discrepancy observed for C-CaVP is due to a nonprocessed amino-terminal Met (133 Da), which was confirmed by amino-terminal sequencing with the Edman method.

Comparative properties of recombinant and wild-type CaVP
Flow dialysis experiments demonstrated that recombinant CaVP binds two Ca2+ with the same affinity as the isolated wild-type protein (Fig. 2Go). Both proteins have similar Trp fluorescence spectra, with a red shift from 333 to 349 nm with denaturation, and denaturation curves, with half-maximal signal change at 1.4 M guanidine-HCl for the metal-free proteins.



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  		Fig. 2. Ca2+ binding to recombinant (open circles) and wild-type CaVP (closed circles), as well as C-CaVP (triangles). Ca2+ binding was measured by flow dialysis at 25°C in buffer C. The solid line represents the theoretical isotherm generated with the intrinsic binding constants 4.6 x 106 and 7.4 x 103 M-1 for K'Ca1 and K'Ca2, respectively.

 
To compare the binding affinity of recombinant and wild-type CaVP for CaVPT, the complex was reconstituted (Petrova et al. 1995) from the equimolar mixture of recombinant CaVP, wild-type CaVP, and CaVPT and submitted to gel filtration. Native gels (Durussel et al. 1993) allowed the discrimination between recombinant and wild-type CaVP, as the recombinant protein is more acidic. An equivalent distribution of the two CaVP forms was observed (data not shown). These data suggest that recombinant and wild-type CaVP possess similar conformations and are equipotent to form a complex with CaVPT.

Ca2+-binding properties of N-CaVP and C-CaVP
Equilibrium gel filtration in 50 mM Tris-HCl, pH 7.5, and up to 200 µM free Ca2+ revealed that N-CaVP does not bind Ca2+. The Ca2+-binding isotherm (Fig. 2Go), measured by flow dialysis, indicated that C-CaVP binds two Ca2+ with intrinsic binding constants of 4.6 x 106 M-1 and 7.4 x 103 M-1. These values are similar to those of entire CaVP (Petrova et al. 1995). The difference in the Ca2+ affinity of the two sites underlines the abundance and importance of the 1Ca2+ form under physiological conditions: the Ca2+ occupancy is 1.0 ± 0.1, from 2 to 25 µM free Ca2+ (Fig. 2Go).

Conformational properties of N-CaVP and C-CaVP monitored by circular dichroism
The far-UV circular dichroism (CD) spectrum of N-CaVP is not influenced by Ca2+ and corresponds to an {alpha}-helical content of 42% (data not shown). In contrast, the CD spectrum of C-CaVP shows a pronounced change with Ca2+ binding with an {alpha}-helical increase from 17% to 36% (Théret et al. 2000). In comparison, the {alpha}-helical structure of entire CaVP increases from 23% to 34%.

The near-UV CD spectra of C-CaVP, N-CaVP, and CaVP, in the presence of 5 mM Ca2+, are shown in Fig. 3GoA–C. The negative peaks at 262 and 269 nm are characteristic of Phe residues. The positive peaks at 284 and 291 nm, observed for N-CaVP and CaVP, are attributed to Tyr-47, Trp-74, and Trp-81 (see Fig. 1Go). The spectrum of C-CaVP is characteristic of a Phe-only protein, which indicates that Trp-81 is free and highly mobile. Heating of C-CaVP to 90°C produces only a slight decrease of intensity, suggesting that this half is very stable. N-CaVP displays only the Trp/Tyr peaks, which disappear with heating and reappear with cooling. Whole CaVP behaves as the sum of the individual halves (Fig. 3DGo). None of these spectra is influenced by removal of Ca2+.



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  		Fig. 3. Near-UV CD spectra of C-CaVP (A), N-CaVP (B), and CaVP (C) at pH 7.5 in the presence of 5 mM CaCl2 at different temperatures: 10°C (solid line), 90°C (dashed line), after heating to 90°C and cooling down to 10°C (dotted line). (D) Near-UV CD spectra of C-CaVP (dotted line), N-CaVP (dashed line), CaVP (solid line), and of the calculated sum of both segments (chain line).

 
Because some EF-hand proteins, such as parvalbumin or Nereis sarcoplasmic Ca2+-binding protein (NSCP), display extensive interactions between their amino and carboxyl domains, even after proteolytic separation (Permyakov et al. 1991; Durussel et al. 1993), we examined the additivity of optical properties of N-CaVP and C-CaVP. The far-UV CD and fluorescence spectra of an equimolar mixture of N-CaVP and C-CaVP, as well as the near-UV CD spectrum of CaVP (Fig. 3DGo), coincide with the sum of the components, indicating that the isolated halves do not influence each others secondary and tertiary structure. In addition, a mixture of the two polypeptides failed to produce band shifts or new bands on a native gel (data not shown). Thus, the recombinant halves of CaVP do not interact together, although an interdomain interaction exists in the entire CaVP (see below).

Thermal stability of CaVP and its halves
CaVP, N-CaVP, and C-CaVP solutions heated up to 90°C showed nearly complete reversibility in CD spectra (Fig. 3Go). At 222 nm, CaVP shows a single broad transition with denaturation temperatures, Td of 47°C for the metal-free and Ca2+-loaded forms (Fig. 4AGo; see also Table 1Go for a synopsis of the thermodynamic parameters). At 291 nm CaVP shows much sharper transitions with Td values of 37.2°C and 33.4°C for the apo and Ca2+ forms, respectively. The melting curves of isolated N-CaVP also show a broad far-UV CD transition and a sharp near-UV CD transition with a Td value of ~44°C (Fig. 4BGo). The isolated amino domain is thus stable, but the metal-free carboxyl domain in CaVP destabilizes the amino-terminal hydrophobic core (7°C decrease in Td) and Ca2+ binding to the carboxy-domain accentuates this (11°C decrease). The 222 nm melting curve of Ca2+-loaded C-CaVP did not display any transition (data not shown), whereas metal-free C-CaVP displays two transitions of about equal importance at 38.4°C and 68°C, respectively (Fig. 4CGo).



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  		Fig. 4. Temperature dependence of the CD intensity for CaVP, N-CaVP, and C-CaVP at pH 7.5. (A) CaVP in the presence of 1 mM EGTA at 291 (thin dotted line) and 222 nm (thick dotted line) and in the presence of 2 mM CaCl2 at 291 (thin solid line) and 222 nm (thick solid line). (B) N-CaVP at 222 nm (thick line) and 291 nm (thin line). (C) C-CaVP at 222 nm in the presence of 1 mM EGTA. The inset shows the first derivative of the CD melting curve.

 

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  		Table 1. Thermal denaturation parameters of CaVP, C-CaVP, and N-CaVP at pH 7.5
 
Because the three-dimensional structures of apo-, 1Ca2+-, and 2Ca2+-C-CaVP are different (Théret et al. 2000), the stabilities of the three states were compared for CaVP and C-CaVP by differential scanning calorimetry (DSC). Figure 5AGo shows that apo-CaVP produces only one heat absorption peak with a Td of 37.5°C (same as in CD experiments; see Table 1Go). If heating was stopped right after the peak, the reversibility was complete. The ratio of calorimetric versus effective enthalpy for this peak approaches one, indicating a simple two-state transition. 2Ca2+-CaVP melts in two very distant steps with Td of 31.5° and 117.9°C, respectively. 1Ca2+-CaVP also melts in two steps; the low temperature peak is composed of two components with Td of 28°C and 37°C, whereas the second step displays a unique Td of 111.7°C. Isolated N-CaVP displays a single two-state transition with a Td of 44.3°C (Fig. 5BGo), a value also found in CD experiments. The profile of apo-C-CaVP was flat without any clear melting transition (Fig. 5CGo). In contrast, 2Ca2+-C-CaVP and 1Ca2+-C-CaVP display single transitions with peaks at 123°C and 111.2°C, respectively. The denaturation peak of 1Ca2+-C-CaVP is well described by a two-state irreversible transition (data not shown) according to the model proposed in Sanchez-Ruiz et al. (1988). These data suggest that the low temperature transition for CaVP represents melting of the amino domain and that the high temperature transition corresponds to the carboxyl domain. The latter increases the amino domain melting sensitivity, which is progressively enhanced with binding of one and two Ca2+. In the high temperature range, the Td for CaVP and C-CaVP are similar: 111°C for the 1Ca2+ forms and 118°C and 123°C for 2Ca2+-CaVP and 2Ca2+-C-CaVP, respectively.



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  		Fig. 5. Temperature dependence of the partial molar heat capacity of CaVP, N-CaVP, and C-CaVP at pH 7.5. (A) apo form of CaVP in the presence of 0.1 mM EGTA (dotted line), 1Ca2+ form in 0.1 mM CaCl2 (solid line), and 2Ca2+ form in 2 mM CaCl2 (dashed line). The inset shows the temperature dependence of the excess heat capacity for CaVP low temperature peak. (B) N-CaVP in the presence of 5 mM CaCl2. (C) C-CaVP in the presence of 0.1 mM EGTA (dotted line), 0.1 mM CaCl2 (solid line), and 2 mM CaCl2 (dashed line).

 
Interaction between CaVP moieties and CaVPT
Complex formation between the CaVP halves and CaVPT was analyzed using three methods. Native PAGE of equimolar mixtures of CaVPT with either N-CaVP, C-CaVP, or CaVP in the presence of Ca2+ demonstrated the appearance of a single new band in the C-CaVP/CaVPT and CaVP/CaVPT mixtures, which likely corresponds to a 1:1 complex (arrows in Fig. 6A, Gotop). Complex formation was confirmed by cross-linking experiments as described by Durussel et al. (1993) (data not shown). Both in the presence and absence of Ca2+, a 1:1 complex was formed between CaVPT and C-CaVP or CaVP (of 40 and 50 kD, respectively), whereas no covalent adduct was formed with N-CaVP. The band intensities were stronger in the presence of Ca2+, suggestive of more efficient cross-linking. Finally, the gel filtration profile of an equimolar mixture of C-CaVP and CaVPT displays a single peak; SDS-PAGE shows that a 1:1 complex is formed (Fig. 6BGo, a). Addition of EDTA leads to the complex dissociation (Fig. 6BGo, b). A competition experiment with an equimolar mixture of CaVP, C-CaVP, and CaVPT suggests that the affinity of C-CaVP for CaVPT is weaker than the one of CaVP (Fig. 6BGo, d). In the presence of Ca2+, C-CaVP alone elutes at two elution volumes, corresponding to apparent molecular masses of 15 and 30 kD (Fig. 6BGo, c). Nevertheless, the comparison of the profiles of Figure 6Go, a and 6Go, c demonstrates the formation of a complex between C-CaVP and CaVPT. Under the same conditions, N-CaVP elutes alone with an apparent molecular mass of 10 kD (data not shown).



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  		Fig. 6. Interaction analysis of N-CaVP, C-CaVP, and CaVP with CaVPT. (A) N-CaVP, C-CaVP, recombinant CaVP, and an equimolar mixture of N-CaVP, C-CaVP, or recombinant CaVP with CaVPT, in the presence of 2 M urea, were microdialyzed 60 min against buffer A containing 100 mM NaCl plus 1 mM CaCl2, before loading on a native gel (12.5% acrylamide) in the presence of 10% glycerol. All samples contain 2.5 µg of each protein. Arrows indicate the mobility of the complex formed in the presence of Ca2+. Recombinant CaVP represents the positive control (same results with wild-type CaVP). This protein migrates in three bands. (B) Samples were chromatographed on a size-exclusion S-200 column under the same conditions as standards of known molecular masses (top), and analyzed by SDS-PAGE (15% acrylamide). An equimolar mixture of either C-CaVP and CaVPT (a), or of C-CaVP, CaVP, and CaVPT (d) was dialyzed against buffer A plus 100 mM NaCl and 1 mM CaCl2. Addition of 2 mM EDTA dissociates the C-CaVP/CaVPT complex (b). C-CaVP alone has a tendency to dimerize in 1 mM CaCl2 (c). At left, the positions of SDS-PAGE molecular markers are indicated (kD).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
This study provides a detailed analysis of the domain structure of CaVP, that is the Ca2 +- and CaVPT-binding domain, as well as the domain cross talk are characterized. For this purpose, recombinant CaVP was expressed and purified. We demonstrate here that two TML groups, which are present on wild-type CaVP, but absent on the recombinant protein, do not affect Ca2+ binding, conformational changes or interaction with CaVPT. A similar conclusion was reached previously in the case of CaM, which possesses one TML that may protect CaM against ubiquitin-dependent proteolysis (Gregori et al. 1985).

CaVP binds only two Ca2+ with its carboxyl domain and the isolated carboxy-terminal half binds two Ca2+ with the same binding pattern as whole CaVP with a high and a low affinity site, indicating that the amino-domain does not modify cation binding. Our DSC results also demonstrate that the carboxy-terminal domain in intact CaVP has similar Ca2+-dependent behavior as C-CaVP. Analysis of 1Ca2+-C-CaVP by nuclear magnetic resonance (NMR) indicates that site III binds the first Ca2+ ion and adopts a well-defined structure, whereas site IV remains highly fluctuating (Théret et al. 2000). In 2Ca2+-C-CaVP, both sites are well-structured and show a similar folding as the Ca2+-saturated carboxy-terminal half of CaM. Our data show that the isolated amino- and carboxy-terminal moieties of CaVP are independent folding units with well-defined secondary and tertiary structures and that only the carboxy-terminal segment displays pronounced Ca2+-dependent conformational changes. N-CaVP contains as much {alpha}-helical structure (~50%) as any functional EF-hand motifs, and possesses a well-formed hydrophobic core. The difference in the thermal stabilities of N-CaVP and C-CaVP (melting at 44°C and 123°C, respectively) is also valid for entire CaVP, which allowed us to identify the unfolding of each domain. These characteristics have not been observed in CaM or TnC and may impart a unique physiological significance.

Whereas the Ca2+-loaded forms of CaVP and C-CaVP are well structured, the carboxyl domain in both apo-CaVP and apo-C-CaVP displays the characteristics of a molten globule state (Ptitsyn et al. 1990): (1) the lack of a tertiary structure, as DSC experiments do not yield any partial molar heat capacity (Cp) signal change for this domain in the 10°–120°C range; (2) the presence of an {alpha}-helical structure (but with a content much lower than in the Ca2+-loaded forms). This {alpha}-helical structure is likely fluctuating along the polypeptide sequence, as it is not observed for C-CaVP by NMR (Théret et al. 2000). The far-UV CD signal shows a biphasic melting curve, suggesting that two groups of helical conformations with different stabilities are dominant in the apo-C-CaVP equilibria. Because NMR of apo-C-CaVP indicated no persistent dipolar interactions (Théret et al. 2000), we assume that its side chains are highly mobile and average out the nuclear Overhauser effect (NOE) signals, as in a state close to the molten globule (Ptitsyn et al. 1990). Recently, an increasing number of structurally disordered proteins in nondenaturating conditions have been described and the biological advantages for induced folding in molecular recognition discussed (for review, see Wright and Dyson 1999).

The absence of interaction between isolated CaVP halves suggests that they can interact with targets, as do CaM or TnC. However, the carboxyl domain of CaVP affects the thermal stability of the amino domain, which implies a crosstalk. Interestingly, the interdomain interaction is at the level of the tertiary structure. The melting temperatures for apo-CaVP and 2Ca2+-CaVP in far-UV CD coincide, whereas near UV-CD and DSC melting temperatures for the amino domain in these two states are different. N-CaVP has a Td of 44°C. The first transition in CaVP, which is attributed to the amino domain, occurs at 37.5°C in the metal-free form and at 31.5°C in the 2Ca2+ form. For the 1Ca2+ form, the amino domain shows biphasic thermal stability with one component similar to the metal-free state, and one close to the value of the 2Ca2+ state. These data clearly show that the carboxyl domain, and its degree of Ca2+ occupancy, modulates the stability of the amino domain. Because our data provide evidence that in intact apo-CaVP, the carboxy-terminal half also possesses a molten globule-like conformation, it may be postulated that this fluctuating structure has a lower destabilizing capacity than a well-formed Ca2+ saturated EF-hand pair. A crosstalk also occurs from the amino to the carboxyl domain, as the Ca2+-saturated carboxyl domain of CaVP melts 5°C earlier than C-CaVP. The thermal destabilization of an independent folding unit has also been described for CaM: the interdomain interaction is more pronounced in the metal-free than in the Ca2+-bound state (Protasevich et al. 1997; Sorensen and Shea 1998).

In this study, we showed that C-CaVP binds to CaVPT, whereas N-CaVP does not form a complex. Similar to full-length CaVP (Petrova et al. 1995), the C-CaVP/CaVPT complex is more stable in the presence of Ca2+. Although no complex was detected in dissociating methods such as native PAGE and gel filtration chromatography, some cross-linked complex can be detected even in the absence of Ca2+. A comparison can be made with the complex formation between CaM and its target peptide in myosin light chain kinase. In the absence of Ca2+, the carboxy-terminal lobe of CaM interacts with the amino-terminal part of the target peptide (Kd in the micromolar range; Tsvetkov et al. 1999). In the presence of Ca2+, the complex is strengthened by additional interaction of the amino-terminal lobe of CaM with the carboxyl-end of the peptide. Likewise, the carboxy-terminal domain of CaVP binds to CaVPT in vitro even in the presence of EDTA and our data suggest that the binding affinity of C-CaVP for CaVPT is weaker than the one of CaVP. We suggest a model where CaVP binds to CaVPT in a sequential process. In a first step the carboxy-terminal domain binds to CaVPT. This brings the amino-terminal domain close enough to also bind CaVPT. This last step could lead to a subsequent increase in the binding affinity.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Cloning and expression of whole CaVP and its moieties
Total RNA of adult Branchiostoma lanceolatum was prepared according to the acid guanidium thiocyanate method (Chomczynski and Sacchi 1987), and the mRNA was purified with an Oligotex dT-30 Super (Japan Roche). The single-stranded cDNA was synthesized using a First-Strand cDNA Synthesis Kit (Pharmacia). The redundant oligomer used for cDNA amplification by polymerase chain reaction (PCR) was 5'-GARGARAARGAYGAR-TGYATGAAR-3' (where R represents A or G, and Y represents C or T), a design based on the amino acid sequence EEKDECMK (residues 11–18) of B. lanceolatum CaVP. The oligo(dT) adaptor, 5'-GGGATCCGAATTCT17-3', was used as another primer. The 5' upstream of cDNA was determined as follows: the EcoRI-ended double-stranded cDNA was synthesized from mRNA using TimeSaver cDNA Synthesis Kit (Pharmacia). The EcoRI Cassette (Takara) was ligated at each end of cDNA. The 5' upstream region was amplified with PCR using the cassette-specific primer C1, 5'-GTACATATTGTCGTTAGAACGCG-3' and 5'-CGAAGACAACACAGACTTTATTAC-3' (complementary to the nucleotides position 1054–1078).

Recombinant proteins were overproduced using a vector under the control of T7 RNA polymerase. Open reading frames were inserted into the vector pET24a (Novagen) using both NdeI (with the 5'-CATATG-3' sequence at the 5' end) and HindIII (with 5'-AAGCTTA-3' at the 3' end) restriction sites. The vector-borne Shine-Dalgarno sequence was used for proper mRNA translation in E. coli. Cloning was done in strain NM554/pDIA17 (Raleigh et al. 1988). BL21(DE3)/pDIA17 strains were transformed with the plasmids pHSP312, pHSP272, pHSP315, coding for entire CaVP, N-CaVP, or C-CaVP, respectively. Cells were grown at 37°C in a twofold concentrated 2YT medium containing appropriated antibiotics, and induced with IPTG for 3 h.

Protein purification
E. coli cultures were resuspended with 20 mM Tris-HCl at pH 7.5, 1 mM dithiotreitol (DTT), 40 µM phenylmethane-sulfonyl fluoride (PMSF), and 0.2 g/mL pepstatin (buffer A), containing 10 µM CaCl2 and 0.1 mM of diisopropyl fluorophosphate. After sonication, the suspension was centrifuged and the pellet was reextracted. The supernatants of CaVP or C-CaVP were loaded on an ion exchange DEAE–cellulose column and eluted with a 0–350 mM NaCl gradient. CaVP- or C-CaVP-containing fractions were applied on a second DEAE–cellulose column equilibrated with buffer A plus 1 mM EDTA and 50 mM NaCl, and eluted with a 50–300 mM NaCl gradient. Proteins were chromatographed through Sephadex G-75 equilibrated in 20 mM HEPES at pH 7.5, 1 mM DTT, 40 µM PMSF, 0.2 µg/mL pepstatin (buffer B) plus 100 µM CaCl2. N-CaVP was purified by QAE ion exchange chromatography with a 0–250 mM NaCl gradient. Wild-type CaVP and CaVPT were purified as previously described (Petrova et al. 1995). The concentrations of CaVP, N-CaVP, C-CaVP, and CaVPT were determined by UV spectrophotometry, using extinction coefficients at 278 nm of 13,700, 12,660, 5,690 and 26,600 M-1cm-1, respectively.

Complex formation and analysis
Complexes between CaVPT and either N-CaVP, C-CaVP, or CaVP were formed and analyzed as previously described (Petrova et al. 1995).

Ca2+ removal and Ca2+ binding
Contaminating Ca2+ was removed as previously described (Cox 1996). The contamination was <0.05 Ca2+/protein. Ca2+-binding measurements were carried out by flow dialysis as previously described (Cox 1996).

Circular dichroism
CD spectra were acquired with a Jasco J-715 spectropolarimeter equipped with thermostated water-jacketed cells and Neslab RTE-111 programmable bath. Far-UV CD was performed in a 1-mm cell on 0.25 mg/mL protein in 5 mM Tris-HCl at pH 7.5 and in a 0.2-mm cell on 0.3–0.4 mg/mL protein in 50 mM cacodylate at pH 7.5; near-UV CD was done in a 10-mm cell on 1–1.3 mg/mL protein in 50 mM cacodylate at pH 7.5. Ellipticities were normalized to residue concentration using {theta}MRW = {theta}o Mr/l c ({theta}o = observed ellipticity in millidegrees; Mr = average molecular weight of an amino acid in the protein [i.e., 113.2, 113.6, 114.7 for CaVP, N-CaVP, and C-CaVP, respectively]; l = path length in millimeters; c = protein concentration in grams per liter). The results were also expressed as molar circular dichroic absorption using {Delta}{varepsilon} = {theta}o/3,300 l c (c = molar protein concentration). The secondary structure content was evaluated according to Johnson (1999). Continuous CD melting experiments were performed at a heating rate of 1 K/min. Denaturation temperatures were determined from the peak in the first derivatives of the melting profiles (accuracy 0.5°C).

Differential scanning calorimetry
Microcalorimetric measurements were carried out on a MicroCal VP-DSC instrument in 0.51 mL of cells and on a DACM-4 microcalorimeter (NPO Biopribor, Pushchino, Russia) in 0.48 mL of cells at a heating rate of 1 K/min on 1.3 mg/mL CaVP, 0.6–1.7 mg/mL N-CaVP and 0.9 mg/mL C-CaVP in 50 mM cacodylate at pH 7.5. The apo, 1Ca2+, and 2Ca2+ states were obtained by dialysis of CaVP and C-CaVP against the buffer containing 0.1 mM EGTA, 0.1 mM Ca2+ or 2 mM Ca2+, respectively. Curves were corrected for the instrumental baseline obtained by heating the solvent used for protein solution. The reversibility of denaturation was checked routinely by sample reheating after cooling in the calorimetric cell. The partial molar heat capacity of the protein (Cp), denaturation temperature (Td), calorimetric denaturation enthalpy ({Delta}Hcal) and effective or van't Hoff denaturation enthalpy ({Delta}Heff) were determined as described elsewhere (Privalov and Potekhin 1986), with the partial specific volume of 0.73 cm3g-1 calculated according to Makhatadze et al. (1990). To analyze functions of excess heat capacity, the MicroCal Origin (4.1) software was used. The accuracy of the calorimetric and effective enthalpies was 8%, that of Td within 0.2°C.


   Acknowledgments
 
This work was supported by the Swiss National Science Foundation (Grant 3100–053710.98), by International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union-Russian Foundation for Basic Research Grant 97–105, Joint Grant 7SUPJ062334, by grants from Centre National de la Recherche Scientifique (France) URA2185, and by the Swiss National Science Foundation Joint Grant 7SUPJ062334.00/1. We are grateful to Professor Claudette Briand for her interest in this work and helpful discussions.

The publication costs of this article were defrayed in part by payment of page charges.This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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