Site-directed labeling of ß-arrestin with monobromobimane for measuring their interaction with G protein-coupled receptors.

ß-arrestins (ßarrs) are multifunctional proteins that interact with activated and phosphorylated G protein-coupled receptors (GPCRs) to regulate their signaling and trafficking. Understanding the intricate details of GPCR-ßarr interaction continues to be a key research area in the field of GPCR biology. Bimane fluorescence spectroscopy has been one of the key approaches among a broad range of methods employed to study GPCR-ßarr interaction using purified and reconstituted system. Here, we present a step-by-step protocol for labeling ßarrs with monobromobimane (mBBr) in a site-directed fashion for measuring their interaction with GPCRs and the resulting conformational changes. This simple protocol can be directly applied to other protein-protein interaction modules as well for measuring interactions and conformational changes in reconstituted systems in vitro.