Heterogeneity and restricted state selection in FRET with fluorescent proteins

Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics.