Quantitative analysis of multi‐protein interactions using FRET: Application to the SUMO pathway

Protein–protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant Kd of protein–protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady‐state and time‐resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus‐YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free‐solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi‐well plate format. Most importantly, our FRET‐based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.