A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Solute-binding proteins (SBPs) are crucial for cellular processes like transport and sensing.
• Their ligand-induced conformational changes are valuable for biosensors but challenging for protein engineering.
• Balancing ligand affinity, thermostability, and dynamics is key for SBP function.

Purpose of the Study:

• To engineer a d-alanine-specific SBP into a fluorescence biosensor for the signaling molecule d-serine (D-serFS).
• To improve the sensor's affinity, specificity, thermostability, and dynamic range for practical applications.
• To demonstrate the sensor's utility in measuring physiologically relevant d-serine concentrations in biological samples.

Main Methods:

• Engineering a d-alanine-specific SBP through targeted mutations in the binding site and remote regions.
• Characterizing the engineered sensor (D-serFS) for ligand affinity (K_D), specificity, thermostability (T_m), and dynamic range.
• Utilizing two-photon excitation fluorescence microscopy to measure d-serine levels in rat brain hippocampal slices.

Main Results:

• The engineered D-serFS exhibited improved affinity (K_D = 6.7 ± 0.5 μM) and 40-fold increased specificity compared to glycine.
• Enhanced thermostability was observed, with a melting temperature (T_m) of 79 °C.
• A dynamic range of approximately 14% was achieved, allowing detection of physiologically relevant d-serine concentrations.

Conclusions:

• The engineered D-serFS functions as a sensitive and specific biosensor for d-serine.
• This work highlights the critical balance between protein dynamics, ligand affinity, and thermostability in protein engineering.
• The study demonstrates a successful strategy for developing complex, dynamic protein-based biosensors for biological research.