L. Brown and B. Hambly
Department of Pathology, The University of Sydney, NSW 2006 Australia.
Fluorescence resonance energy transfer (FRET) spectroscopy was used to measure the distance between equivalent loci on adjacent myosin subfragment-1 (S-1) heads when bound to filamentous actin (F-actin) in rigor solution. The two distances measured were between (i) a fluorescent donor probe bound to the alkali light chain (ALC) of S-1 and an acceptor probe bound to the ALC of an adjacent S-1 and (ii) a fluorescent donor probe bound to the SH-1 residue of the motor domain of S-1 and an acceptor probe bound to the SH-1 residue of an adjacent S-1. Various ratios of donor and acceptor were prepared and the mixtures were bound to F-actin in rigor buffer. The distance between each locus was determined and compared to predicted distances from static electron micrograph reconstructions and other FRET spectroscopy of the acto myosin complex. For the measurement between corresponding sites in the light chain domain, both the donor (N[[(iodoacetyl)amino]ethyl]-5-napthylamine-1-sulphonic acid [IAEDANS]) and acceptor (5-iodoacetamide fluorescein [5-IAF]) fluorescent probes were bound stoichiometrically to Cys 177/Cys 136 of separate samples of ALC1/ALC3 and exchanged onto a-chymotryptic S-1 fragment of myosin. Likewise, the donor [IAEDANS] and acceptor [IAF] fluorescent probes were bound stoichiometrically to the SH1 residue of the a-chymotryptic S-1 fragment of myosin for the measurement between corresponding sites in the motor domain. No fluorescence quench occurred between fluorescent probes on corresponding sites of the light chain domain (single Cys of the alkali light chain) indicating a distance of >80Å which was substantially larger than the predicted distance (70Å). The distance between corresponding sites in the motor domain (SH1) was ~62Å which was close to the predicted distance (67Å) and in agreement with the measurement perfomed by Ishiwata et al. (1) of ~60Å. Sedimentation binding studies of the fluorescence samples showed that greater than 95% of S 1 had bound the F-actin. These results will be discussed in relation to the apparent flexibility between the motor domain and the light chain domain of the myosin head, when bound to F-actin in the rigor complex.
(1) Ishiwata S., Miki M., Shin I., Funatsu T., Yasuda K. & dos Remedios C.G. (1996) Biophys. J. 70:A14.