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 SH1 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.