Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1.

The fast-reacting thiol (SH1) of myosin subfragment-1 (S-1) was covalently and specifically labeled with (iodoacetamido)fluorescein (IAF), while Cys-373 of actin was also covalently and preferentially labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (1,5-IAEDANS). The method of fluorescence energy transfer was used to examine the spatial proximity between the two sites, i.e., SH1 and Cys-373, in the rigor complex of acto-S-1. Approximately 30% fluorescence energy transfer was observed from the 1,5-IAEDANS on actin as a donor to the IAF on S-1 as an acceptor in their rigor complex; under certain assumptions this corresponds to a distance of ca. 6.0 nm.