Facile Assessment of Protein Binding Events Using a Colorimetric Dye

Colorimetric techniques and their associated dyes can offer lower-cost and more accessible alternatives than their fluorescent and luminescent counterparts for many screening applications. Here, we sought to identify colorimetric dyes suitable for a presumptive assay probing protein–ligand binding, with a particular focus on conformationally dynamic proteins that are often recalcitrant to standard biophysical methods. From an array of 23 commercially available colorimetric dyes, we identified three candidates that exhibited concentration-dependent absorbance changes in the presence of two structurally distinct proteins of interest, Med25 AcID and CBP KIX. Follow-up studies of protein–ligand binding events narrowed the focus to erythrosin B, which was the most sensitive reporter of Med25 and CBP binding events with existing small-molecule ligands. Notably, binding data for Med25–norstictic acid and CBP–garcinolic acid complexes exhibited clustering patterns consistent with orthogonally measured thermostability data for each complex. Thus, colorimetric dyes such as erythrosin B may serve as more broadly accessible tools for presumptive screening assays of high-value protein targets, including those with a high degree of conformational malleability.