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A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine.

Vanessa Vongsouthi1, Jason H Whitfield1, Petr Unichenko2

  • 1Research School of Chemistry, Australian National University, Canberra 2601, Australia.

ACS Sensors
|November 16, 2021
PubMed
Summary
This summary is machine-generated.

Researchers engineered a d-alanine-specific protein into a d-serine biosensor (D-serFS). This engineered protein shows improved affinity, specificity, and thermostability, enabling detection of d-serine in rat brain slices.

Keywords:
FRET biosensorcomputational designd-serineneuroimagingprotein engineeringrational design

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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.