Summary: Title: Structural dynamics underlying GPCR-G protein selectivity Upon binding to agonists with different efficacies, G protein-coupled receptors (GPCRs) mediate multiple signaling pathways by coupling to different subtypes of G proteins. Certain agonists exhibit selectivity in their efficacy to specific G-protein signaling pathways. Such ligands provide precise therapeutic benefits with fewer side effects as drugs compared to today's GPCR-targeted drugs. There are very few selective ligands known for specific G-protein signaling pathways because designing such agonists is a daunting experimental challenge. This is due to serious lack of understanding of how GPCRs modulate their functional selectivity for their cognate G-protein when bound to different type of agonists and in cells. In the past three years using a combination of multiscale molecular dynamics (MD) method and genetically coded FRET sensors we have shown that GPCRs have latent intracellular cavities that can reshape and couple to different G-proteins. By reengineering G-proteins and GPCRs we have identified the hotspot residues in the GPCR-G-protein interface that serve as the “QR code” for G-protein selective coupling by GPCRs. Although it is clear that allosteric communication from the ligand binding site to the G-protein coupling site plays a role in G-protein selectivity very little is known about the mechanism of this communication and the residues involved in this process. Here we propose to (1) use the computational method Allosteer developed in our lab in combination with FRET sensors, NanoBRET assays and cell based downstream assays to delineate the allosteric network of GPCR residues involved in regulating G-protein selectivity in 9 different Gs, Gi and Gq coupled class A GPCRs in cellular conditions. The second aim is to understand the effect of partial agonists in comparison to the full agonists, on the GPCR-G-protein selective coupling. We will identify the hotspot residues on the G-protein and GPCRs when bound to partial agonists and compare the similarities and differences with full agonists. The outcome of the proposed work will provide the role of allosteric network of residues in G-protein selectivity and how partial agonists modulate the receptor conformations. This will push forward our understanding of the biological process of how GPCRs recognize their cognate G-proteins in live cells.