Protein flexibility as revealed by fluorescence resonance energy transfer: an extension of the method for systems with multiple labels

The temperature profile of the normalized fluorescence resonance energy transfer efficiency is capable of monitoring the relative change of flexibility and/or conformational state of macromolecules [Biochemistry 23 (1984) 3403]. The method described earlier for one donor–one acceptor systems is extended to multiple fluorophore systems when the energy transfer occurs between either one donor–m acceptors, or n donors–one acceptor or n donors–m acceptors (where n and m are integer values). It is shown that the normalized energy transfer efficiency obtained for systems containing multiple labels is a linear combination of the normalized transfer efficiency assigned to individual donor–acceptor pairs of the system, thus its temperature profile is capable of monitoring the change of intramolecular flexibility and/or conformational state.