Fluorescence energy transfer between probes on actin and probes on myosin.
The structural relationship between F-actin filaments and the biologically active fragments of myosin (either as myosin subfragment-1 or heavy meromyosin) has been investigated using the technique of fluorescence energy transfer. Donor and acceptor probes were used to obtain the following inter- and intramolecular distances. Energy transfer was measured: (1) from the SH1 groups of the myosin 'heads' to the nucleotide sites of F-actin (in the absence of free nucleotide); (2) from the SH1 groups of myosin to multiple probes on the surface of the actin filament; (3) from the nucleotide-binding sites of F-actin to the ATPase sites of myosin; (4) from the ATPase sites of myosin to the nucleotide-binding sites of F-actin; (5) from the SH1 sites of myosin to the nucleotide-binding sites of F-actin; and (6) from the Cys-373 residues of F-actin to the nucleotide binding sites of F-actin. We observed very little energy transfer between the probes on actin and the probes on myosin (10% or less) and we observed a large transfer between the actin Cys-373 and the actin nucleotide. These data strongly suggest that both the SH1 moiety and the ATPase site of myosin are located more than 6 nm from the actin sites. When these distances are combined with similar measurements by other authors and inserted into the most recent three-dimensional reconstruction of electron micrographs of the acto-subfragment-1 complex, it is apparent that the SH1 and the ATPase sites on myosin are not located adjacent to actin and are most probably located in the half of the myosin head that is distal from actin in the actomyosin complex.
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004735:Energy Transfer; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D009218:Myosins; D003545:Cysteine