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Proton magnetic resonance spectroscopy has been used to study the cation(Mg2+ , Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the biding reaction. Complex formation with troponin I and the drug intercative conformational response to cation coordination and the binding of the drug and troponin I are discussed. [ABSTRACT FROM AUTHOR]

Exchangeability of actin-bound ADP and calcium in actomyosin at low ionic strength has been studied using F-actin labelled with [14C]ADP or 45Ca and measuring release of radioactivity into solution. Low-speed centrifugation, ultracentrifugation and ultrafiltration were used to separate protein from the medium. Comparison of the results obtained with these three separation procedures has revealed that the release of [14C]ADP and 45Ca into the medium in the presence of millimolar concentrations of MgATP is largely due to the release under these conditions of actin itself retaining its bound ADP and calcium. The real exchange of the bound nucleotide and calcium, even under the most favourable conditions, was in our experiments limited to about 20 %. Detailed examination of the dependence of both the release of actin and the exchange of actin-bound ADP and calcium on the free divalent cations present, the kind and concentration of the added nucleotide, and temperature of incubation indicates that there is no correlation between the exchange and superprecipitation of actomyosin. The results presented support the view that the limited enhancement by myosin of the exchange of nucleotide and cation bound to actin under certain conditions results from accidental disruption of bonds between actin monomers due to a mechanical stress exerted on actin filaments upon their interaction with myosin filaments. [ABSTRACT FROM AUTHOR]

Two tryptic fragments of the Ca2+-binding protein calmodulin have been studied by high-resolution 1H NMR. TR1C (residues 1 – 77) spans the first two domains of the protein and TR2C (residues 78 – 148) spans the second two domains. The spectra indicate that each of the two-domain peptide assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modeled after the two EF-Hand Ca2+-binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+ -binding protein. [ABSTRACT FROM AUTHOR]

The relaxation rate enhancements of the 23Na nuclei for NaHCO3 solutions of troponin C and its tryptic peptides TR-1 and TR-2 indicate true binding of Na+ ions to these biomolecules. The low-affinity sites 1 and II of TR-1 and troponin C are the sites of competitive Na+/Ca2+ binding, below one calcium ion per molecule, with log KNa ≈ 2. At low calcium content Na+ ions bind to TR-2 and to troponin C non-competitively with Ca2+ ions; binding of Ca2+ ions to the high-affinity sites III and IV allosterically affects the binding of the Na+ ions: even when sites I and II, located on TR-1 or sites I, II, III, IV of troponin C, are saturated with Ca2+ ions, Na+ ions continue to bind weakly at secondary binding sites. [ABSTRACT FROM AUTHOR]

The effect of various divalent cations on the state of aggregation of actin monomers has been studied at pH 7.6 by means of viscosity measurements, determination of the protein sedimenting at high and low centrifugal forces, dephosphorylation of the actin-bound ATP and by observation of the negatively stained preparations under the electron microscope. The metal concentration dependence of the degree of actin polymerization in the presence of Ca2+, Mg2+, Sr2+ and Mn2+ is the same. All these cations produce typical double-stranded F-actin filaments. Ni2+ and Zn2+ induce polymerization at lower concentrations than Mn2+ and alkaline earth metals, but the resultant polymers have lower viscosities. Examination in the electron microscope has shown that Ni2+ produces typical F-actin filaments, which, however, tend to brake into short fragments. In the presence of Zn2+ globular aggregates coexisting with the filaments have been observed. In the presence of Mn2+ or alkaline earth metals at millimolar concentrations the F-actin filaments assemble into net-like paracrystals which are transformed into side-by-side aggregates when the cation concentration is increased. The cation concentration dependences of polymerization and of paracrystal formation suggest that these two processes occur upon binding of these cations to distinct classes of sites and that the order of affinities to sites of weaker binding, involved in the paracrystal formation, is as follows: Mn2+ > Ca2+ > Mg2+ = Sr2+. Unlike the other cations, Zn2+ at concentrations higher than that necessary for maximum polymerization causes precipitation of G-actin without formation of any ordered structures. [ABSTRACT FROM AUTHOR]

The interaction of actin with various cations has been studied at pH 7.6, at 20 °C, by means of gel filtration and centrifugation techniques. In addition to the single high-affinity site, five low-affinity sites with apparent association constants of 5-6 x 10³ M-1 for Ca2+, Mn2+ and Sr2+ and of 1.6 x 104 M-1 for Ni2+ have been found in the actin molecule. Unlike the other cations, Zn2+ appears to have three high-affinity sites from which it is not removed by Dowex 50 treatment, and eight low-affinity sites with an apparent association constant of 1.3 x 104 M -1. Competition experiments suggest that Ca2+, Sr2+, Mn2+ and Ni2+ are bound to the same low-affinity sites, some of which are also available for Zn2+. Zn2+ also binds to additional sites which are not available for the other cations. Binding of 4 mol of either Ca2+, Mn2+, Sr2+, Ni2+ or Zn2+ per mol of actin monomer at the low-affinity sites characterized above causes a complete transformation of the monomeric actin into polymers. The nonidentity of some of the 2+-binding sites with those for the other cations seems to explain structural differences between the polymers formed in the presence of this cation and those produced upon binding of Mn2+, Ni2+ or alkaline earth metals, described in the preceding paper in this journal. The results suggest that formation of the double-stranded F-actin filaments requires the neutralization of negative charge at specific sites in the monomer molecules. The aggregation of F-actin filaments into paracrystals observed at pH 7.6 in the presence of Ca2+, Mg2+, Sr2+ or Mn2+ at millimolar concentrations seems to involve the binding of these cations to a third class of binding sites with apparent association constants of the order of 10² M-1. The binding parameters to this class of sites could not be determined by the methods used in this work. [ABSTRACT FROM AUTHOR]