Pierre D.J. Moens and Cristobal G. dos Remedios
Muscle Research Unit, Department of Anatomy and Histology, Institute for Biomedical Research, The University of Sydney, Sydney 2006, Australia.
Fluorescence resonance energy transfer (FRET) spectroscopy allows to measure changes in distance (of about 1 Å) between two probes bound to proteins in solution and therefore is suitable to study conformational changes of proteins. In helically symetrical structure like actin filament, the radial coordinate of a probe can be deduced from the distances separating the donor on one monomer to acceptors bound on adjacent monomers. Also, using two different labelling sites (e.g. cys-374 and the nucleotide site) it is possible (knowing the spatial relationship between the two sites) to obtain angular informations from the FRET data.
We used FRET to detect conformational changes in the C-terminus of actin when myosin, myosin head (S-1) bind to the filaments. Cys-374 on actin is labelled with 5-[[2-[(iodoacetyl)amino] ethyl]amino]-naphthalene-1-sulfonic acid as a donor probe and with N-(4-dimethyl-amino-3, 5-dinitrophenyl)-maleimide as the acceptor probe. The etheno ATP analog is used as a donor for the nucleotide site. The changes in radial coordinate of Cys-374 as well as the change in angle between Cys-374 and the nucleotide site and the changes in the intramonomer distance between these two residues are investigated when saturating amount of myosin S-1 is added to the F-actin solution. The combination of these three measurements allow us to draw a three dimensional picture of the changes occurring at the C-terminus of actin.
This research was supported by grants from the NH&MRC and ARC.