Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors

Foerster (or fluorescence) resonance energy transfer (FRET) is a powerful tool for investigating protein-protein interactions, in both living cells and in controlled environments. A typical hetero-FRET pair consists of a donor and acceptor tethered together with a linker. The corresponding energy transfer efficiency of a hetero-FRET pair probe depends upon the donor-acceptor distance, relative dipole orientation, and spectral overlap. Because of the sensitivity of the energy transfer efficiency on the donor-acceptor distance, FRET is often referred to as a “molecular ruler”. Time-resolved fluorescence approach for measuring the excited-state lifetime of the donor and acceptor emissions is one of the most reliable approaches for quantitative assessment of the energy transfer efficiency in hetero-FRET pairs. In this contribution, we provide an analytical kinetics model that describes the excited-state depopulation of a FRET probe as a means to predicts the time-resolved fluorescence profile as a function of excitation and detection wavelengths. In addition, we used this developed kinetics model to simulate the time-dependence of the excited-state population of both the donor and acceptor. These results should serve as a guide for our ongoing studies of newly developed hetero-FRET sensors (mCerulean3–linker–mCitrine) that are designed specifically for in vivo studies of macromolecular crowding. The same model is applicable to other FRET pairs with the careful consideration of their steady-state spectroscopy and the experimental design for wavelength- dependence of the fluorescence lifetime measurements.