INFLUENCE OF TIGHTLY BOUND MG2-ACTIN( AND CA2+, NUCLEOTIDES, AND PHALLOIDIN ON THE MICROSECOND TORSIONAL FLEXIBILITY OF F)

To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of ac tin filaments as a function of the type of tightly bound divalent cati on (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic p hosphate and analogues, KCI concentration, and the level of phalloidin . Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (r(FA) ) 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 influen ce of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibi lity of F-actin polymerized in the presence of ATP; filaments with Mg2 + bound (r(FA) = 0.066) at the active site cleft were more flexible th an those with Ca2+ bound (r(FA) = 0.083). Filaments prepared from G-ac tin in the presence of MgADP were more flexible (r(FA) = 0.051) than t hose polymerized with MgATP; the addition of exogenous inorganic phosp hate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the p resence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the bin ding of phalloidin decreased the torsional flexibility of all filament s regardless of the type of cation or nucleotide bound at the active s ite. These results emphasize the dynamic malleability of the actin fil ament, the role of the cation-nucleotide complex in modulating the tor sional flexibility, and suggest that the structural differences that h ave previously been seen in electron micrographs of actin filaments ma nifest themselves as differences in torsional flexibility of the filam ent.