PROJECT SUMMARY/ABSTRACT GPCRs form the largest membrane protein family and also dominate the therapeutic market as targets for more than 30% of FDA-approved drugs. These drugs act on a broad spectrum of indications from cancer to inflammatory, cardiovascular, respiratory and gastrointestinal disease. GPCRs are archetypical allosteric proteins that translate extracellular ligand stimulation into an intracellular response. In contrast to the binary “on or off” response of most signaling molecules, GPCRs possess a ligand-independent basal activity that can be increased or decreased upon ligand binding, and then further regulated by allosteric modulators. Activated receptors transduce signals through G protein and arrestin proteins equally (balanced signaling) or selectively (biased signaling). Taken together, a single receptor may specifically recognize several ligands and respond uniquely to each, creating a complex conformational landscape. The last decade has seen nearly 300 X-ray and cryo-EM structures have greatly expanded our view of GPCR architecture and function; however, the molecular mechanisms of basal activity, partial agonism, biased signaling, and allosteric modulation can only be partially derived from these structures. The principles of class Monod-Wyman-Changeux (MWC) and dynamically-drive (DD) allostery can significantly enhance our understanding of, and ability to tune, GPCR signaling. Whereas classic MWC conformational allostery is relatively simple to infer from X-ray or cryo-EM structural models, DD allostery is far more difficult to measure experimentally. Advances in NMR spectroscopy relaxation measurements have empirically-demonstrated that sidechain methyl dynamics can be used as a proxy for conformational entropy (i.e. DD allostery). In addition, it remains the only technique capable of quantifying atomic-resolution motions across the picosecond to second timescale and in many cases can detect states populated as little as 0.5%. We propose two research projects aimed at exploring the allosteric role of MWC and DD allosteric mechanisms in the activation of 1) peptide-binding GPCRs and 2) arrestin. There is immense therapeutic potential in the ability to tune receptor signaling using partial or biased agonists in contrast to full agonists/antagonists – and it remains virtually untapped. Our proposal provides a much- needed complement to decades of functional mutant screens, EPR, fluorescent labels, and high-resolution structures. In the long-term, the goal of our research program is to describe the conformational transitions and dynamics of the greater GPCR superfamily from the inactive state to the active state unhindered by crystal contacts or stabilizing proteins.