Revealing Structural Features And Affinities Of Protein Complexes In Living Cells By Mfis-Fret

Forster resonance energy transfer (FRET) due to its sensitivity of distance has been widely used to investigate the structure and interaction of biomolecules. Multiparameter fluorescence image spectroscopy (MFIS) provides particular advantages to FRET imaging because all the fluorescence parameters are monitored simultaneously with picosecond accuracy, which allows for a comprehensive analysis on biological systems. Traditionally, a reduction in average donor lifetime or an increase of average FRET efficiency was used as an indicator for molecular interaction. However, such changes observed in FRET-imaging can have two reasons: (1) the conformational change or (2) change in fraction of FRET-active species. To resolve this ambiguity, we introduce a new sub-ensemble analysis method to directly visualize and quantitatively analyze both factors. Characterization of true FRET efficiency enabled us to detect even subtle FRET variations and provided crucial information about the structural properties of molecular complexes. Furthermore, from determined fraction of FRET-active species, utilizing the intrinsic cell-to-cell variations of protein concentration, we show that dissociation constant (KD) of membrane-receptor interactions can be characterized in living cells.