An SPR-based analysis of cGAS substrate K_D and steady-state K_M values

Surface plasmon resonance (SPR) is a standard method for evaluating direct protein-small molecule binding. While studying the catalytic mechanism of cyclic GMP-AMP synthase (cGAS), we developed an SPR-based method to measure steady-state K_M values that complements traditional SPR affinity measurements. The method relies on refractive changes to detect protein interaction with substrates and products, and takes advantage of stimulator of type 1 interferon genes (STING) binding to the cGAS product, 2',3'-cGAMP. The specific method described here uses co-immobilization of cGAS and double-stranded DNA through a biotin tag; it should be generally applicable to other proteins and protein complexes.