In-depth analysis of Gs protein activity by probing different fluorescently labeled guanine nucleotides

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the G alpha subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both G alpha s and G alpha i have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of G alpha s is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of G alpha s, G alpha s(short) and G alpha s(long), is still pending. Since the previous experimental evaluation of the activity and function of the G alpha s isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human G alpha s by applying an established methodological setup developed for G alpha i characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTP gamma S exhibited the highest binding affinity towards the G alpha s subunit. This work provides a deeper understanding of the G alpha s subunit and provides novel information concerning the differences between the two protein variants.