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

Abstract

The distance separating the high-affinity binding sites of 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+ bound to the high-affinity divalent cation site of G-actin by trivalent lanthanide ions such as La3+, EU3+, or Gd3+ 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 A. In the past, the metal and ATP have been widely assumed to bind as a complex. Further verification that the two sites on actin are physically separated was obtained by using an ATP analogue 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 analogue 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 data obtained by using this technique support the conclusion stated above, namely, that the cation and nucleotide sites on either G- or F-actin are well separated.

Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation

“…FRET measurements with the fluorescent cation Tb(III) bound to the Me site and FTP as the acceptor at the NUC site suggested that these sites were about 16~ apart (Barden & dos Remedios/1984). 1H n.m.r, data using lanthanide ions support this conclusion (Barden et al, 1980;. On the other hand, other FRET data (using e-ATP/Co(II) or Ni(II) as the donor-acceptor pair) have demonstrated that the distance between Me and NUC sites is no more than 104 (Miki & Wahl, 1985) and there are 31p n.m.r, data to support this conclusion (Brauer & Sykes, 1982).…”

Proteolysis and structure of skeletal muscle actin.

“…Assuming that Tb3' has the highest affinity for the M2+-binding site, they sought perturbations of aromatic residues using NMR (27) and UV spectroscopy (28); on finding none they reasoned that this portion of the primary sequence (the first 20 residues) was uniquely distant from aromatic residues. On also failing to find effects of bound Tb3+ on the ring protons of bound nucleotide, they concluded that the two sites were more than 1.6 nm distant (22); this observation too fitted the idea that the M2+-site was in the NH2-terminal residues. It should be noted that Brauer and Sykes (29) found the phosphorus nuclei of bound nucleotide to be near the M2'-binding site, but the finding may not be contradictory in as much as intranucleotide distances can be considerable.…”

“…A proteolytic removal of residues eliminates the ability of actin to bind M2+ (4,22). The calcium-binding protein from carp, the "CBP" (23), has been investigated, and there exist homology studies (24) that have compared the amino acid sequences involved in various calcium-binding sites.…”

Fluorescence resonance energy transfer between sites in G-actin. The spatial relationship between Cys-10, Tyr-69, Cys-374, the high-affinity metal and the nucleotide

“…This suggests that either the Tb3' site is located a short distance from the Co2+ site as has been claimed [49, 501 or the replacement of the bound divalent cation with a trivalent lanthanide ion may increase the separation between the two sites by about 0.5 nm. This may be achieved either by the trivalent cation mobilizing the nucleotide base [22] so that the trivalent and divalent cation sites are actually identical but the base is translocated when a trivalent cation binds. Alternatively, a conformational change in the actin may be induced which involves a widening of the cleft [5], or a combination of the two processes may apply.…”