Project Summary Roughly one third of all FDA approved drugs target G protein coupled receptors (GPCRs), and this protein family holds myriad therapeutic opportunities yet to be discovered. GPCRs signal through allosteric changes in protein structure, and there is a large unmet need for effective, highly generalizable tools for conformational control of these receptors. This project has two Specific Aims, both of which employ computational protein design with Rosetta to build proteins de novo that bind and modulate GPCR conformation. Upon ligand binding and activation, GPCRs associate with and signal through G proteins. In the first Aim, we design proteins de novo which mimic the interaction domain of the endogenous Gα protein subunit. Because the interaction between receptor and Gα is highly conserved, the protein tools we propose to develop can be applied broadly. Synthetic genes encoding de novo Gα mimetics have been expressed, and protein was purified from Escherichia coli. Binding to several different detergent-solubilized GPCRs was demonstrated via pull-down and Western blotting. Functional Gα mimetic proteins will be structurally characterized and used as templates for re-engineering a small suite of mimetics for all Gα subtypes. Additionally, we propose to leverage the Gα mimetics to enhance the expression and purification of GPCRs. To demonstrate this system, we will attempt to produce three different GPCRs for the first time: super conserved receptor expressed in brain 1-3. In the second aim, we will design disulfide constrained peptides to serve as agonists and antagonists for all structure-enabled family B GPCRs. This receptor family recognizes endogenous peptide ligands, and they are difficult to drug with small molecules. Structures that detail ligand recognition are available for 60% of family B receptors; we will use these as the basis for de novo design of high-affinity antagonist ligands based on disulfide-constrained peptides. We will then screen hundreds of thousands of rationally designed de novo peptides using yeast display, fluorescence activated cell sorting, and deep sequencing. Our unique design approach enables us to custom tailor the peptide topology so that we can readily convert our antagonists into high affinity agonists by extending the peptide N-terminus. The efficacy of designed ligands identified via the high-throughput screen will be assessed individually using a luciferase reporter assay for GPCR signaling. The research proposed here represents the first time that computational de novo protein design will be applied to control GPCR conformation. This work has the potential to create valuable tools to empower the GPCR research community and accelerate discovery of lead therapeutics for many diseases.