One-photon versus two-photon excited fluorescence resonance energy transfer of GFP fusion proteins

Understanding the function of a protein by following its dynamic interplay with other proteins in a living cell can contribute fundamentally to the overall cellular process or disease in which it participates. The, principles of fluorescence resonance energy transfer serve as the basis for the development of new methodology which utilizes mutants of the green fluorescent protein (GFP). A major drawback in utilizing FRET as a means of determining protein interaction has been the overlap in spectra between the donor and acceptor GFP fluorophores and attempts to separate them by filters. To circumvent this issue, one-photon spectral data were generated for the FRET pairs expressed in living cells. To validate the protein-protein interaction we applied dequenching techniques whereby bleaching the acceptor fluorophore would lead to an increase or dequenching of the donor fluorescence. The FRET spectra were quantitatively compared as ratios of the donor and acceptor emission peaks (arbitrary intensities). In comparison, two-photon generated fluorescence of the FRET pairs provides for direct ratioing of the intensity peaks, since at 810nm the donor is efficiently excited with the acceptor minimally excited. Furthermore, bleaching of the GFP molecules is negligible. Together, one-photon and two-photon excited FRET complimentarily provides proof of protein-protein interaction in living cells.