Two-step FRET as a structural tool

The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.