Accurate FRET Measurements and Testing of the Theory for Multimeric Complexes Using Reference Fluorescence Standards

Forster Resonance Energy Transfer (FRET) is a process in which a donor (D) in the excited state transfers its energy nonradiatively to an acceptor (A) in the ground state. The underlying theory has been confirmed countless times, particularly with regard to the dependence of the FRET efficiency on the sixth power of the distance between D and A. In contrast, a complete FRET theory for multiple donors and acceptors in oligomeric complexes has been developed only recently (Raicu, 2007, J. Biol. Phys. 33:109-127), in parallel with technology of sufficient accuracy for tests in living cells (Raicu et al., 2009, Nature Photon. 3:107-113). This novel approach now has been applied to linked fluorescent proteins located in the cytoplasm and at the plasma membrane. The cytoplasmic probes were fused combinations of a donor (Cerulean, C), an acceptor (Venus, V), and a chromophore-deficient, Venus-like molecule that cannot absorb or transfer energy (Amber, A) (Koushik et al., 2009, PLoS ONE 4(11):e8031): namely, ACVA, ACAV, VCAA, and VCVV. The membrane-bound probes were fused dimers and trimers of eGFP2 (G2) and eYFP (Y): namely, G2Y, YG2, G2YG2, and YG2Y. According to the theory (Raicu, 2007), the FRET efficiency of a tetramer such as VCVV can be predicted from that of analogues that contain a single acceptor (e.g., ACVA, ACAV, VCAA); also, the apparent FRET efficiency of a trimer such as G2YG2 or YG2Y can be predicted from the pair-wise efficiency that corresponds to that of dimers such as G2Y and YG2. These predictions have been confirmed for FRET efficiencies measured by means of two-photon microspectroscopy (Raicu et al., 2009), in accord with the theory and underlying assumptions for FRET within multimers.