ABSTRACT
Using polarization fluorimetry, we have investigated conformational changes of FITC-phalloidin-labeled F-actin in ghost muscle fibers. These changes were induced by myosin subfragment-1 (S1) in the absence and presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes of orientation and mobility of actin monomers during the ATPase cycle. The most pronounced differences in orientation (~4 degrees ) and in mobility (~43%) of actin were found between the actomyosin states induced by MgADP and MgATP.
Subject(s)
Actins/metabolism , Adenosine Triphosphate/metabolism , Muscle Contraction/physiology , Muscle Fibers, Skeletal/metabolism , Actins/chemistry , Actomyosin/chemistry , Actomyosin/metabolism , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Fluorescein-5-isothiocyanate/chemistry , Fluorescence Polarization , Hydrolysis , Myosin Subfragments/chemistry , Myosin Subfragments/metabolism , Phalloidine/chemistry , RabbitsABSTRACT
Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.
Subject(s)
Muscle Fibers, Skeletal/drug effects , Myosin Subfragments/chemistry , Nucleotides/pharmacology , Actins/metabolism , Actomyosin/metabolism , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Fluorescein-5-isothiocyanate/chemistry , Fluorescence Polarization , Muscle Fibers, Skeletal/ultrastructure , Myosin Subfragments/drug effects , Myosin Subfragments/ultrastructure , Phalloidine/chemistry , Protein Conformation , Tropomyosin/metabolismABSTRACT
Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.
