Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Significance Weak, transient protein complexes are emerging as vital to cell function and regulation, with roles ranging from stabilization of a protein structure to driving cytoplasmic phase separations. Because of their fleeting nature and sensitivity, such complexes are notoriously hard to characterize. We develop and validate a method to quantify such “quinary interactions” inside the cell itself, and apply it to the putative interaction of the glycolytic enzymes GAPDH and PGK. Our method can characterize weak protein–protein interactions in live cells, with future application to other biomolecular species, such as RNAs.