Proton nuclear magnetic resonance and electron paramagnetic resonance studies on skeletal muscle actin indicate that the metal and nucleotide binding sites are separate

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Abstract
The distance separating the high-affinity binding sites on [rabbit] actin for a divalent metal ion and nucleotide was evaluated by using high-resolution proton NMR and EPR spectroscopy. Replacement of the Ca2+ or Mg2+.sbd.bound to the high-affinity divalent cation site of G-actin by trivalent lanthanide ions such as La3+, Eu3+ or Gd3+.sbd.results in an increase in the mobility of the bound ATP as observed in the NMR spectra of G-actin monomers. Little difference was observed between the spectra obtained in the presence of the diamagnetic La3+ control and the paramagnetic ions Eu3+ and Gd3+ which, respectively, shift and broaden the proton resonances of amino acids in the vicinity of the binding site. Analysis of the NMR spectra indicates that the metal and nucleotide binding sites are separated by a distance of at least 16 .ANG.. In the past, the metal and ATP were assumed to bind as a complex. Further verification that the 2 sites on actin are physicially separated was obtained by using an ATP analog with a nitroxide spin-label bound at the 6''position of the purine ring. An estimate of the distance was made between the site containing the ATP analog and the paramagnetic ion, Mn2+, bound to the cation binding site. These EPR experiments were not affected by the state of polymerization of the actin. The cation and nucleotide sites on either G- or F-actin are apparently well separated.