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A Pipeline for Screening Small Molecule-Enhanced Protein Stability in A Bacterial Orphan Receptor
James J Siclari1,2, Denize C Favaro1, Richard H Huang3
1Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY 10031.
Biorxiv : the Preprint Server for Biology
|July 9, 2025
Summary
Researchers identified novel small molecule ligands for bacterial Per-ARNT-Sim (PAS) proteins, enhancing thermal stability and providing new tools for synthetic biology. This study advances understanding of ligand discovery for orphan PAS domains.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Bacterial one-component signaling proteins, featuring Per-ARNT-Sim (PAS) domains, act as natural sensors and tools in biotechnology.
- Identifying ligands for "orphan" PAS proteins is challenging due to sequence divergence and lack of bound molecules.
Purpose of the Study:
- To characterize CU228, a PAS-HTH transcription factor, as a model system for discovering novel small molecule ligands.
- To expand the analytical methods for probing protein-ligand interactions in signal-responsive systems.
Main Methods:
- Bioinformatics and structural analysis to predict ligand-binding sites.
- Differential scanning fluorimetry with a fragment library to screen for stabilizing ligands.
- Microfluidic modulation spectroscopy (MMS) and saturation transfer difference NMR to confirm binding and affinity.
Main Results:
- CU228, a PAS-HTH protein, was identified as a model for ligand discovery.
- Three novel ligands (KG-96, KG-408, KG-484) were found to stabilize CU228, increasing thermal stability by up to 10°C.
- Ligand binding was confirmed by MMS and NMR, with micromolar dissociation constants.
Conclusions:
- The study successfully identified novel ligands for an orphan PAS protein using small molecule stabilization.
- The findings provide new tools for characterizing and engineering one-component transcription factors.
- This work lays the foundation for developing synthetic chemogenetic variants of bacterial signaling proteins.
Keywords:
NMR spectroscopybacterial one-component signalingligand bindingmicrofluidic modulation spectroscopythermal shift assayMore Related Videos
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