Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding

Effects of subtilisin cleavage of actin between residues 47 and 48 on the c onformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) wen investigated by measuring polarized fluorescence fro m rhodamine-phalloidin- or 1,5-IAEDANS-labeled actin filaments reconstructe d from intact or subtilisin-cleaved actin in myosin-free muscle fibers (gho st fibers). In separate experiments, polarized fluorescence from 1,5-IAEDAN S-labeled S1 bound to non-labeled actin filaments in ghost fibers was measu red. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-ph alloidin-labeled filaments, orientation and mobility of the C-terminus of a ctin, filament flexibility, and orientation and mobility of the myosin head s bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the abs ence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in ma intaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be e ssential for the generation of force and movement during actin-myosin inter action. (C) 2000 Elsevier Science B.V. All rights reserved.