Abstract
Periplasmic display in yeast is a promising but underdeveloped method for screening and studying genetically encoded biomolecules. We present PIONEER, a modular platform that localizes peptides, proteins, and nanobodies to the membrane-proximal periplasmic space of S. cerevisiae, enabling direct interrogation of membrane protein function. By optimizing secretion signals and display scaffolds, PIONEER ensures stable ligand retention and robust autocrine signaling. The ability to assess peptide and protein ligand activity through signaling, rather than binding alone, marks a key advance toward function-first screening. Applied to human G protein-coupled receptors (GPCRs), the system enables detection of surface expression, ligand activity profiling, and classification of nanobody regulators, including antagonists and conformational stabilizers. It also distinguishes intrabodies based on their functional effects and chaperone activity. Although demonstrated with GPCRs, PIONEER is adaptable to a wide range of membrane and soluble protein targets. As AI-driven design expands the space of candidate ligands and binders, this platform offers a scalable method for linking predicted sequences and their structures to biological function.