Article: article from journal or magazin.
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)
Amino Acid Sequence, Animals, Calcium/metabolism, Calcium-Binding Proteins/metabolism, Chordata, Nonvertebrate, Cysteine/analysis, Fluorescent Dyes, Models, Molecular, Molecular Sequence Data, Muscle Proteins/metabolism, Naphthalenesulfonates, Protein Binding, Protein Conformation, Sequence Homology, Amino Acid, Sulfhydryl Compounds/analysis, Thermodynamics
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