Fluorescence resonance energy transfer using color variants of green fluorescent protein

This chapter discusses the fluorescence resonance energy transfer (FRET) using color variants of green fluorescent protein (GFP). Live cell FRET detection in yeast is in its infancy, and there are significant complications with the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) pair. Spectral overlap between excitation and emission spectra complicates the analysis. The extreme sensitivity of the current YFP to bleaching constrains image acquisition. The detection of a FRET signal is limited by background cellular autofluorescence and the relatively weak fluorescent signal intensities. Dynamic intracellular conditions—such as changes in pH or protein concentrations—can complicate the interpretation of experimental data. However, with careful controls, FRET is a powerful indication of protein–protein interaction. In instances where interactions give robust FRET signals, FRET is a valuable tool used to study the dynamic spatial and temporal behavior of protein–protein interactions in living cells. Future improvements in the spectral properties of CFP and YFP will increase the general applicability of FRET to study a broad range of protein–protein interactions in yeast.