Measuring the rupture force between adenosine A2A G-protein coupled receptors and G-proteins using axial optical tweezers.

Abstract

G-protein-coupled receptors (GPCRs) are the largest group of membrane receptors. Here we used axial optical tweezers to measure the rupture force between the adenosine A2A receptor in a nanodisc and its associated G-protein. We also measured the interaction between the receptor and an engineered G-protein, mini-Gs, which is a truncated Gs alpha-subunit that can increase agonist affinity and stabilize the active state of GPCRs. These measurements required us to extend the force range accessible with axial optical tweezers to approximately 50 pN. This involved strengthening the DNA handles to which we attach the G-protein or mini-Gs and improving the force calibration of the axial optical tweezers by employing a twistable worm-like chain (tWLC) model. These advances enabled us to measure the rupture force between the adenosine A2A receptor and the G-proteins under various physiological conditions.