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

Citation
S. Baladi et al., Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves, PROTEIN SCI, 10(4), 2001, pp. 771-778
Citations number
23
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
4
Year of publication
2001
Pages
771 - 778
Database
ISI
SICI code
0961-8368(200104)10:4<771:FUICVP>2.0.ZU;2-S
Abstract
Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding p rotein, 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 halv es (C-CaVP). The interactive properties of recombinant and wild-type CaVP a re very similar, despite three post-translational modifications in the wild -type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic c ore, and melts at 44 degreesC. C-CaVP binds two Ca2+ with intrinsic dissoci ation constants of 0.22 and 140 muM (i.e., very similar to the entire CaVP) . The metal-free domain in CaVP and C-CaVP shows no distinct melting transi tion, whereas its 1Ca(2+) and 2Ca(2+) forms melt in the 111 degrees -123 de greesC range, suggesting that C-CaVP and the carboxy- domain of CaVP are na tively unfolded in the metal-free state and progressively gain structure up on binding of 1Ca(2+) and 2Ca(2+). Thermal denaturation studies provide evi dence for interdomain interaction: the ape, 1Ca(2+) and 2Ca(2+) states of t he carboxy-domain destabilize to different degrees the amino-domain. Only C -CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest th at the carboxy-terminal domain of CaVP interacts with CaVPT and that the am ino-terminal lobe modulates this interaction.