Complete Kinetic Theory of FRET

Forster resonance energy transfer (FRET) can be used to measure distances and infer structures at the molecular level. However, the flexible linkers with which the fluorophores are attached to a macromolecule introduce a lack of knowledge. Both the dye’s geometry and kinetics give rise to uncertainties. Whereas the impact of the geometry is already well understood, the real extent of the kinetics has not been investigated thoroughly. Here, we present a single-molecule (sm)FRET theory that defines the kinetics of dye movements in a complete form. We introduce a formal nomenclature and provide a recipe for the calculation of the corresponding FRET efficiency. We further analyze experimental data in order to obtain parameters characterizing the geometry and kinetics of the FRET dyes and use them to resimulate the FRET efficiencies by diffusion of fluorophore and linker movement. We show in a real case scenario of dye molecules attached to dsDNA that when making geometrical and kinetic assumptions commonly us...