Intermolecular dynamics and function in actin filaments Original Research Article

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38–52), the hydrophobic plug (residues 262–274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal α-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved α-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38–52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 Å) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38–52, plug 262–274 and the C-terminus may account for the observed cross-linking of these sites with reagents <18 Å. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41–Cys-374 cross-linked actin filaments.