Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves.
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 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (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 degrees -123 degrees 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.
Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.
S
S
Baladi
P O
PO
Tsvetkov
T V
TV
Petrova
T
T
Takagi
H
H
Sakamoto
V M
VM
Lobachov
A A
AA
Makarov
J A
JA
Cox
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D020832:EF Hand Motifs / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D009124:Muscle Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
C050084:CAVP protein, Amphioxus; C066481:CaVP target protein, amphioxus; D002135:Calcium-Binding Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins