Using Analytical Ultracentrifugation to Characterize Ric8:Gαi Protein Binding

G-protein coupled receptors (GPCRs) are a target of 35% of FDA-approved drugs and play a major role in cellular communication. GPCRs activate various Gα proteins, such as Gαi, which activate downstream effector proteins and result in intracellular signaling. Ric-8A is a molecular chaperone protein required for Gαi biogenesis and has cytosolic guanine nucleotide exchange factor activity. Understanding proteins which regulate GPCR pathways, like Ric-8A, may reveal novel therapeutic targets in G-protein signaling. As an undergraduate volunteer in the Analytical Ultracentrifugation (AUC) core, my goal is to use AUC to characterize the binding behavior of Gαi and Ric-8A, independently verify association constants found using isothermal titration calorimetry, and compare this behavior to mutant Ric-8A proteins. This will provide important information on the binding surface, mechanism, and character of the Gαi- Ric-8A interaction. We hypothesize that Gαi and Ric-8A bind with micromolar range affinity and mutations on the binding surface of Ric-8A will disrupt this interaction. In AUC, individual molecules in mixtures are hydrodynamically separated under extreme centrifugal force and identified by their rate of sedimentation. Preliminary experiments included collecting UV-Vis spectra of Gαi, GDP, and Ric-8A at various concentrations to create a reference dataset. Our initial AUC experimental results indicate an increase in binding at increasing ratios of Gαi:Ric-8A. At a 4:1 ratio, excess Gαi is observed. This preliminary work verifies our method and the purity of our proteins. Our future AUC work will evaluate Gαi and wildtype/mutant Ric-8A at physiologically relevant concentrations and define essential amino acids on their interaction surface.