The use of the isotropic orientation factor in fluorescence resonance energy transfer (FRET) studies of the actin filament

A Dale-Eisinger style analysis (R. E. Daleet al., Biophys. J.26, 161, 1979) is used to produce three-dimensional plots that display the limits on the average orientation factor k² that is required to calculate molecular distances in F-actin from fluorescence resonance energy transfer measurements. Maxima and minima plots are generated for the transfer of energy from a donor to a single acceptor and for transfer to multiple acceptors that are related by F-actin helical symmetry. The analysis is performed in terms of dipole cone half-angles rather than depolarization factors, in order to facilitate the modeling of the multiple acceptor problem. Calculations are carried out under the restrictive condition of a single electric dipole moment per fluorophore. In addition, both surface and volume averaging of the donor and acceptor dipoles are considered. Comparisons between the plots show that for the multiple acceptor cases with F-actin symmetry, there is a great reduction in the range for maxima and minima limits on k². The calculations also suggest guidelines for the choice of fluorescence label that will result in an average orientation factor occurring within acceptable limits, i.e., inside the limits for which k²=2/3 may be employed. Thus, without having detailed knowledge of the mean donor or acceptor dipole relative orientations, the use of k²=2/3 in radial coordinate studies of F-actin is more than reasonable and is fairly assured of being correct.