Fluorescent Sensors That Enable a General Method To Quantify Affinities of Receptor Proteins for Polyubiquitin...

In all eukaryotic cells, modifications of proteins by polymers of ubiquitin (polyUb) are signals used in diverse biological processes. To better understand how polyUb signals are read and promote their different functions, quantitative measurements of their interactions with receptor proteins are needed. However, affinities and selectivities of different forms of polyUb with various receptors have been difficult to determine because the availability of well-defined polyUb chains can be limiting and there is a lack of general, sensitive methods to assay their interactions. We have addressed this challenge by developing a series of fluorescent protein sensors for polyUb; by competition of the sensors against receptor proteins in vitro for limiting amounts of polyUb, receptor·polyUb affinities can be quantified. Due to the high affinities of the polyUb sensors (K_d ∼ 10^-9 M), binding assays using this competition format require much less polyUb (<0.1%) than would be needed in direct titrations of the polyUb ligands. Furthermore, the high sensitivity and large dynamic range of the sensor fluorescence readout allow for precise measurements even for very tight interactions (i.e., nanomolar K_d). Importantly, as demonstrated here with Ub₂ and Ub₃ ligands, the assay does not require labeling of either the receptor protein or the polyUb, and it can be used with polyUb ligands composed of virtually any Ub-Ub linkage type.