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

Area of Science:

• Cellular biology
• Biophysics
• Molecular imaging

Background:

• Molecular processes rely on protein-protein interactions (PPIs) within cellular machinery.
• Understanding PPIs in vivo is crucial for deciphering protein functions.
• Förster resonance energy transfer (FRET) via fluorescence lifetime imaging (FLIM-FRET) offers high spatiotemporal resolution for PPI monitoring.

Purpose of the Study:

• To develop a simplified method for interpreting complex FLIM-FRET data.
• To enhance the visualization and analysis of PPIs in living cells.
• To accurately assess PPI stoichiometry and binding modes, even with varying protein expression levels.

Main Methods:

• Utilized a novel FLIM diagram plot for visualizing and clustering pixels based on fluorescence decay signatures.
• Applied the FLIM diagram plot to analyze Förster resonance energy transfer (FRET) data.
• Investigated protein-protein interactions (PPIs) in live Pseudomonas aeruginosa.

Main Results:

• The FLIM diagram plot effectively clusters pixels with similar decay characteristics.
• This visualization approach provides valuable insights into PPI stoichiometry and binding modes.
• The method demonstrates robustness even with significant differences in interacting protein expression levels.
• Critical features of PPIs in Pseudomonas aeruginosa were successfully revealed.

Conclusions:

• The FLIM diagram plot offers a straightforward and valuable visual approach for interpreting complex lifetime data.
• This method facilitates a more accurate understanding of protein-protein interactions in their native cellular environment.
• The technique holds promise for advancing research in cellular molecular machines and bacterial interactions.