Influence of tightly bound Mg2+ and Ca2+, nucleotides, and phalloidin on the microsecond torsional flexibility of F-actin

To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of actin filaments as a function of the type of tightly bound divalent cation (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic phosphate and analogues, KCl concentration, and the level of phalloidin. Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (rFA) of the triplet probe erythrosin-5-iodoacetamide covalently bound to Cys-374 of skeletal muscle actin; extrapolations to an infinite actin concentration corrected the measured anisotropy values for the influence of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibility of F-actin polymerized in the presence of ATP; filaments with Mg2+ bound (rFA = 0.066) at the active site cleft were more flexible than those with Ca2+ bound (rFA = 0.083). Filaments prepared from G-actin in the presence of MgADP were more flexible (rFA = 0.051) than those polymerized with MgATP; the addition of exogenous inorganic phosphate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the presence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the binding of phalloidin decreased the torsional flexibility of all filaments regardless of the type of cation or nucleotide bound at the active site. These results emphasize the dynamic malleability of the actin filament, the role of the cation-nucleotide complex in modulating the torsional flexibility, and suggest that the structural differences that have previously been seen in electron micrographs of actin filaments manifest themselves as differences in torsional flexibility of the filament.