Exploring protein-protein interactions with large differences in protein expression levels using FLIM-FRET

Many molecular processes within a cell are carried out by molecular machines built from a large number of proteins organized by their protein-protein interactions (PPIs). Exploring PPIs in their cellular context is critical to better understand the proteins functions. Forster resonance energy transfer measured by fluorescence lifetime imaging (FLIM-FRET) enables to monitor PPIs and to map their spatial organization in a living cell with high spatial and temporal specificity. But both the accurate measurement and the interpretation of multi-exponential FLIM-FRET data associated to mixtures of interacting and non-interacting proteins are difficult. Here we show that a simple diagram plot can find interesting visualization properties by clustering pixels with similar decay signatures. FLIM diagram plot can be used to provide valuable information about stoichiometry and binding mode in PPIs, even in the presence of large differences in protein expression levels of the different interacting partners. The proposed FLIM diagram plot is a useful visual approach for a more straightforward interpretation of complex lifetime data. This approach was applied for revealing critical features of PPIs in live Pseudomonas aeruginosa.