A pipeline for screening small molecule‐enhanced protein stability in a bacterial orphan receptor

Bacterial one-component signaling proteins integrate sensory and gene regulation functions within the same polypeptide, creating powerful natural sensors of environmental conditions which can also be adapted into powerful tools for synthetic biology and biotechnology. A key sensor motif within many of these proteins is the Per-ARNT-Sim (PAS) domain, known for its conserved fold yet highly divergent sequences, allowing for a broad range of ligands to control PAS protein function by changes in small molecule binding occupancy or configuration. This diversity introduces a challenging step-identification of ligands for "orphan" PAS proteins which show signatures of ligand binding but no copurifying high-affinity bound small molecules-into characterization and engineering of such proteins. In this study, we characterized CU228, a putative PAS-HTH transcription factor from Candidatus Solibacter usitatus, as a novel model system for searching for novel ligands by small molecule stabilization. Bioinformatics and structural analyses predicted a PAS domain with a Trp-rich internal cavity, suggesting potential small molecule interactions. Using a ~760 compound fragment library, differential scanning fluorimetry identified three ligands (KG-96, KG-408, and KG-484) that substantially increased CU228 thermal stability with ΔT_m values up to +10°C. Microfluidic modulation spectroscopy revealed ligand-induced preservation of α-helical and β-sheet integrity under thermal stress. Saturation transfer difference NMR confirmed direct binding of all three ligands and enabled estimation of micromolar-range dissociation constants, consistent with expected fragment-level affinity. Our findings expand the analytical toolbox for probing protein-ligand interactions in flexible, signal-responsive systems, laying the groundwork for designing synthetic chemogenetic variants of one-component transcription factors.