Subcellular Localization Defects Characterize Ribose-Binding Mutant Proteins with New Ligand Properties in...

Area of Science:

• Synthetic biology and biosensor engineering.
• Protein engineering and computational design.
• Bacterial cell biology and protein localization.

Background:

• Periplasmic binding proteins (PBPs) are utilized as scaffolds for engineering novel biosensors with specific ligand-binding capabilities.
• These engineered proteins are integrated into bacterial bioreporter chassis to generate signals upon target compound detection.
• Current computational design methods often overlook crucial general protein properties like translocation and receptor interactions.

Purpose of the Study:

• To investigate the role of general periplasmic binding protein properties in signaling behavior.
• To compare the subcellular localization of wild-type ribose-binding protein (RbsB) with engineered mutants in Escherichia coli.
• To understand how localization defects impact the sensing performance of engineered biosensors.

Main Methods:

• Utilized C-terminal mCherry fluorescent protein fusions as proxies for subcellular localization.
• Calibrated and deployed fluorescent protein fusions to track RbsB and its evolved mutants in E. coli.
• Analyzed localization patterns, including periplasmic accumulation and potential mislocalization, in wild-type and mutant proteins.

Main Results:

• Wild-type RbsB-mCherry localized correctly to the periplasm, often accumulating at polar regions.
• Engineered RbsB mutants exhibited high cell-to-cell fluorescence variability, with increased instances of mislocalization and aggregation.
• Analysis of directed evolution libraries indicated improvements in mutant characteristics, aligning with wild-type behavior and structure predictions.

Conclusions:

• Defects in the periplasmic localization of engineered RbsB variants contribute significantly to their impaired sensing performance.
• Functional biosensor design necessitates considering protein folding, translocation, and receptor interactions beyond computational binding pocket modifications.
• Future engineering efforts should incorporate selection for secondary mutations that restore proper cellular localization and signaling.