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

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.

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 • T_d, denaturation temperature • NMR, nuclear magnetic resonance

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