BPS2026 – Single-molecule visualization of function-related dynamics of the human stimulatory G-alpha protein

G proteins are nucleotide exchange factors that initiate intracellular signaling cascades. Upon interaction with their cognate receptors, the Gα subunit, composed of a GTPase domain and an α-helical domain (AHD), undergoes a large structural rearrangement that separates the two domains and exposes the nucleotide-binding pocket. Although numerous crystal structures of G proteins alone, as well as crystal and cryo-EM structures of receptor-G protein complexes, are available, the mechanism of complex formation, Gαs structural rearrangement, and nucleotide exchange remains incompletely understood. To address this, we employed single-molecule fluorescence resonance energy transfer (smFRET) to directly visualize the dynamics of Gαs nucleotide exchange. This approach uniquely resolves individual conformations and reveals the sequence of molecular events underlying nucleotide exchange. Our experiments identified FRET states of Gαs corresponding to both fully open and closed conformations, along with two intermediate states not previously observed by crystallography or cryo-EM. Unexpectedly, we discovered a pronounced effect of magnesium on the conformational equilibrium of both GTP- and GDP-bound Gαs. Complementary biophysical and biochemical assays confirmed that magnesium stabilizes specific conformational states and modulates functional activity. Dwell-time analysis further showed that Gαs predominantly adopts the closed conformation in the presence of magnesium, with fewer transitions to the open state. Together, our results demonstrate that magnesium allosterically regulates the dynamic interplay between the GTPase and AHD domains, thereby inhibiting nucleotide exchange.