A single myosin head can be cross-linked to the N termini of two adjacent actin monomers.
Myosin subfragment-1 (S1) can be cross-linked to two actin monomers by 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide only when F-actin is in excess over S1. Electron micrographs of the covalent actin2-S1 complex showed that S1 was cross-linked to two adjacent monomers of the same actin filament. Cross-linking experiments with pre-proteolyzed S1 derivatives in combination with a proteolytic dissection of the intact covalent actin2-S1 adduct (m = 265 kDa), revealed that two N-terminal segments of actin (residues 1-28) were covalently attached to a single S1 molecule. One was cross-linked to either the 20-kDa or the 50-kDa heavy chain fragments of S1, and the other only to the 50-kDa region. The doubly cross-linked product was formed under physiological ionic strength with S1 or with reconstituted myosin filaments, regardless of the presence of ADP or the regulatory proteins, tropomyosin and troponin. Finally, we found that this cross-linking could also take place within myofibrils in the rigor state. These results demonstrate that under nonsaturating conditions, the actin-S1 interface encompasses a much larger region than that recently proposed for the nonphysiological, fully saturated actin filaments.
Bonafé
N
N
Centre de Recherches de Biochimie Macromoléculaire du Centre National de Recherche Scientifique, Montpellier, France.
Chaussepied
P
P
D000199:Actins / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure*; D000205:Actomyosin / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D009119:Muscle Contraction / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure*; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D000205:Actomyosin; D005022:Ethyldimethylaminopropyl Carbodiimide