Advances in methodologies for quantifying protein–protein interactions in living cells

Abstract Protein–protein interactions (PPIs) occur in most cellular processes, and characterizing PPIs is essential for understanding biological function and regulation. One of the most important parameters is the dissociation constant ( K d ), which reflects the strength of PPIs. A range of in vitro methods, including isothermal titration calorimetry, fluorescence resonance energy transfer (FRET), surface plasmon resonance, and single‐molecule fluorescence assays, have been developed to determine K d values under controlled conditions. However, the living cell environment differs markedly from dilute buffer systems due to macromolecular crowding, compartmentalization, and regulatory complexity, often leading to discrepancies between in vitro and in‐cell K d values. In mammalian cells, advances in fluorescence‐based techniques, including fluorescence lifetime imaging microscopy‐FRET and fluorescence cross‐correlation spectroscopy, have enabled quantitative measurements of PPI affinity in living systems. In contrast, accurately measuring K d in bacterial cells has remained a challenge. Recently, in‐cell NMR and quantitative FRET approaches have been developed for measuring K d in bacterial cells. This review highlights the principles, capabilities, and limitations of representative in‐cell K d measurement techniques and provides guidance for selecting appropriate approaches to quantitatively characterize PPIs in physiologically relevant contexts.