Rational design framework for fluorescent biosensors from periplasmic binding proteins.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Periplasmic binding proteins (PBPs) are promising scaffolds for fluorescent biosensors due to their ligand-induced conformational changes and specificity.
• Generalizable PBP biosensor design is limited by understanding how labeling site affects protein dynamics and signal transduction.

Purpose of the Study:

• To systematically investigate the impact of labeling positions on PBP-based fluorescent biosensor function using the Lysine-Arginine-Ornithine binding protein (LAO) as a model.
• To establish a framework for understanding the relationship between labeling site, protein dynamics, and biosensor performance.

Main Methods:

• Site-directed mutagenesis to label seven positions on the LAO protein with monobromobimane (mBBr).
• Characterization of labeled variants including quantum yields, fluorescence changes, ligand-binding affinities, and thermal stability.
• Molecular dynamics (MD) simulations to analyze protein conformational dynamics.

Main Results:

• Labeling position significantly influenced quantum yields, fluorescence intensity changes, ligand-binding affinities, and thermal stability.
• Functional positions (D51C, D53C, K228C, Y230C, E167C) maintained open-like conformations crucial for signal generation.
• A peristeric position (A89C) adopted a closed-like conformation, leading to inverse fluorescence and reduced affinity, highlighting the importance of dynamics.

Conclusions:

• Static structural criteria alone are insufficient for predicting PBP biosensor function; experimental validation is essential.
• The study provides a methodological framework for PBP biosensor engineering, emphasizing the critical role of conformational dynamics.
• Understanding labeling position-dependent dynamics is key to optimizing biosensor performance across different PBP scaffolds.