FRET at the Single Molecule Level using Molecular Brightness and Fluorescence Correlation Spectroscopy

Abstract

Förster resonance energy transfer (FRET) is a noninvasive, quantitative method to study intermolecular interactions and conformational changes. The energy transfer efficiency of FRET pairs are routinely quantified using steady-state fluorescence spectroscopy or time-resolved fluorescence for which the main challenge of these approaches is the averaging over a larger ensemble of molecules. Here, we present a new approach for determining the energy transfer efficiency of hetero-FRET probes at the single-molecule level, using time-resolved fluorescence correlation spectroscopy (FCS) and molecular brightness (i.e., the number of fluorescence photons per molecule emitted while diffusing in the open observation volume). In this approach, the molecular brightness of the donor, in the presence and absence of the acceptor, is related to the energy transfer efficiency. We used the engineered hetero-FRET probe, RD, which consists of a mCerulean-linker-mCitrine construct, as the model system. Laser intensity-dependent FCS experiments were carried out on both the intact and cleaved RD indicating an energy transfer efficiency of (50 ± 10)% for RD in buffer, corresponding to a distance of 7 ± 2 nm between the donor and acceptor. These results demonstrate that our FCS approach is applicable to hetero-FRET constructs in well-controlled environments. Further it should be useful in living cells because it has the added advantage of requiring low expression levels of the FRET sensor, in contrast to more conventional FRET methods.