Resonance energy transfer evidence for two attached states of the actomyosin complex

Abstract 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·ATP, in which the fast reacting SH 1 thiol group is crosslinked to another thiol group in the 20 kDa domain of S 1, the 2 fluorophores were found to have moved closer together by ⩾ 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.