Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells

Abstract Quantitative measurement of protein–protein interactions (PPIs) within living cells is vital for understanding their cellular functions at the molecular level and for applications in synthetic biology, protein engineering, and drug discovery. Although several techniques have been developed to measure PPI strength in vitro, direct measurement of PPI strength within living bacterial cells remains challenging. Here, a method for quantitatively measuring PPIs by determining the dissociation constant (Kd) in living E. coli using fluorescence resonance energy transfer (FRET), a technique termed KD‐FRET, is reported. It is found that the direct excitation of the acceptor fluorophore among spectral crosstalks primarily results in non‐interacting pairs exhibiting an apparent Kd, leading to false‐positive signals. KD‐FRET proves highly effective in quantifying various PPI Kd values, inclu…