Direct-reversible binding of small molecules to G protein ?? subunits.

ABSTRACT: Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits ?, ?, ? mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to G?? and interfere with or enhance binding of select effectors with G??. To understand the molecular mechanisms of selectivity and assess binding of compounds to G??, we used biophysical and biochemical approaches to directly monitor small molecule binding to G??. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to G?? with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the k(off) was slow for most of the compounds in physiological buffers, they could be removed from G?? with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-G?? interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of G??-effector interactions is by specific direct non-covalent and reversible binding of small molecules to G??. This is highly relevant to development of G?? targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

SUBMITTER: Seneviratne AM 

PROVIDER: S-EPMC3140432 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature