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 conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads 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 absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.
Borovikov
Y S
YS
Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg, Russia. boroviko@link.cytspb.rssi.ru
Moraczewska
J
J
Khoroshev
M I
MI
Strzelecka-Gołaszewska
H
H
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001718:Birefringence; D003850:Deoxyribonuclease I / Q000737:chemistry*; D005454:Fluorescence Polarization; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry*; D009282:Naphthalenesulfonates; D010590:Phalloidine / Q000031:analogs & derivatives; D011487:Protein Conformation; D011817:Rabbits; D012235:Rhodamines; D020860:Subtilisin / Q000737:chemistry*
D000199:Actins; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D012235:Rhodamines; C067497:rhodamine B isothiocyanate phalloidin; D010590:Phalloidine; C006421:1,5-I-AEDANS; D003850:Deoxyribonuclease I; D020860:Subtilisin