Direct-reversible binding of small molecules to G protein βγ subunits

► An SPR method is presented for examining small molecule binding to G protein βγ subunits. ► SPR analysis demonstrates direct reversible binding of small molecules to Gβγ. ► Rate constants for small molecule binding and dissociation are surprisingly slow. ► Mass spectrometry demonstrates a predominantly 1:1, non-covalent binding mode for gallein. ► Radioligand binding supports a 1:1 binding mode of gallein to Gβγ.