Challenges and Solutions for Leave-One-Out Biosensor Design in the Context of a Rugged Fitness Landscape

Area of Science:

• Protein engineering
• Computational biology
• Biosensor development

Background:

• Split protein reconstitution offers a modular approach to biosensor design.
• Leave-one-out green fluorescent protein (LOO-GFP) biosensors can be computationally designed to detect specific target peptides.
• Reusable biosensors are crucial for early detection of emerging biological threats.

Purpose of the Study:

• To present biophysical inferences for nine LOO-GFP biosensor libraries targeting dengue, influenza, or HIV peptides.
• To address challenges in hit rate, library complexity, and protein dimerization encountered during LOO-GFP biosensor design.
• To characterize a detailed influenza hemagglutinin biosensor.

Main Methods:

• Computational design of LOO-GFP biosensors by modifying beta strands.
• Iterative "piecemeal" design strategy to overcome library complexity limitations.
• Fusion of LOO-GFP to *Drosophila* ultrabithorax to create biosensor fibers and mitigate dimerization.

Main Results:

• Initial low hit rates were attributed to energy function components over-rewarding tight side chain packing.
• A "piecemeal" iterative design strategy resolved the conflict between screening requirements and target peptide length.
• Fusion to *Drosophila* ultrabithorax successfully eliminated unwanted fluorescence in the unbound state.
• A characterized influenza hemagglutinin biosensor exhibited a shifted spectrum, micromolar affinity, and photo-switching ability.

Conclusions:

• LOO-GFP biosensors are a viable platform for detecting specific peptide targets.
• Computational design strategies and protein engineering solutions can overcome key development hurdles.
• The developed biosensor fiber technology shows promise for sensitive and reusable biological threat detection.