Structural dynamics of G i α protein revealed by single molecule FRET.

The heterotrimeric G proteins (Gαβγ) act as molecular switches to mediate signal transduction from G protein-coupled receptors to downstream effectors. Upon interaction with an activated receptor, G protein exchanges its bound GDP with GTP, stimulating downstream signal transmission. Release of GDP requires a structural rearrangement between the GTPase domain and helical domain of the Gα subunit. Here, we used single molecule fluorescence resonance energy transfer (smFRET) technique to study the conformational dynamics of these two domains in the apo state and in the binding of different ligands. Direct imaging of individual molecules showed that the Gi α subunit is highly dynamic, and at least three major conformations of Gi α could be observed in the apo state. Upon binding of GDP, Gi α becomes dramatically less dynamic, resulting in a closed conformation between the two domains. We postulate that changes between the three conformations are sequential, and the three conformations appear to have distinct affinities toward GDP.