Resonance energy transfer evidence for two attached states of the actomyosin complex.
Resonance energy transfer measurements were made between a donor fluorophore, N-(bromacetyl)-N'-(1-sulpho-5-naphthyl)ethylenediamine, located on the single cysteine of the Al light chain of myosin (S1(A1), and an acceptor fluorophore, 5-(iodoacetamido)fluorescein, sited on Cys-374 of actin. In the binary rigor complex a transfer efficiency of 24% was noted, representing a spatial separation of about 6 nm. When the same measurements were made using a stable analogue of S1 X ATP, in which the fast reacting SH1 thiol group is crosslinked to another thiol group in the 20 kDa domain of S1, the 2 fluorophores were found to have moved closer together by greater than or equal to 3 nm. This provides, for the first time, direct experimental evidence for a change in structure of the myosin crossbridge that could account for tension generation.
D000205:Actomyosin; D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014335:Tropomyosin; D014336:Troponin; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C015296:5-iodoacetamidofluorescein; C039087:N-bromoacetyl-N'-(1-sulfo-5-naphthyl)ethylenediamine; D000205:Actomyosin