Thermodynamic and molecular properties of the interaction between amphioxus calcium vector protein and its 26 kDa target.
Calcium vector protein (CaVP) of amphioxus shares some common structural features with Ca(2+)-dependent activators such as troponin C and calmodulin, and is associated in vivo with a 26 kDa (CaVPT), a multidomain protein with one IQ- and two IgII-motifs. Isolated CaVP binds two Ca2+ ions with very different intrinsic affinity constants: K'Ca1 = 4.9 x 10(6) M-1 and K'Ca2 = 7.3 x 10(3) M-1, respectively. In the complex with CaVPT, CaVP also binds two Ca2+, but with strong positive cooperativity (nH = 1.9) and with distinctly higher affinity: K'Ca1 = 2.4 x 10(5) M-1 and K'Ca2 = 1.0 x 10(8) M-1. Since neither in the isolated CaVP nor in the complex Ca2+ binding is influenced by 2 mM MgCl2, both sites can be considered as Ca(2+)-specific. In the absence of Ca2+, the complex is stable under physiological conditions, but the interaction is governed by the principle of linked functions and Ca2+ binding to CaVP reinforces the affinity between CaVP and CaVPT 70-fold. Both proteins interact with the hydrophobic probe 2 p-toluidinylnaphthalene-6-sulfonate (TNS), but CaVPT enhances the fluorescence 45-fold, CaVP-Ca2 and metal-free CaVP only 10- and 5-fold, respectively. Complex formation between CaVPT and CaVP leads to a 3-fold reduction of the fluorescence enhancement, suggesting that a strong solvent-shielded hydrophobic core is formed. CaVP contains two highly reactional thiols (kSH > 0.3 s-1) for 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); CaVPT contains three thiols, two of them also with kSH > 0.3 s-1 in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)
Petrova
T V
TV
Department of Biochemistry, University of Geneva, Switzerland.
Comte
M
M
Takagi
T
T
Cox
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002816:Chordata, Nonvertebrate; D003545:Cysteine / Q000032:analysis; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000032:analysis; D013816:Thermodynamics
C050084:CAVP protein, Amphioxus; C066481:CaVP target protein, amphioxus; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009124:Muscle Proteins; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D003545:Cysteine; D002118:Calcium