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Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein.

Teraya Donaldson1, Luisa Iozzino2, Lindsay J Deacon1

  • 1Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA.

Analytical Biochemistry
|March 6, 2017
PubMed
Summary

A new fluorescent biosensor was developed using Thermotoga maritima arginine-binding protein (TmArgBP) for detecting arginine. The photo-induced electron transfer (PET) method offers superior quantitative detection compared to traditional probes.

Keywords:
FluorescenceHyperthermophilic bacteriaPeriplasmic binding proteinPhoto-induced electron transferSwitch-based biosensor

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Biosensor Development

Background:

  • Thermotoga maritima arginine-binding protein (TmArgBP) is a key protein for arginine detection.
  • Traditional fluorescent biosensors often lack sensitivity and quantitative capabilities.
  • Developing reagentless biosensors is crucial for efficient molecular detection.

Purpose of the Study:

  • To engineer a reagentless fluorescent biosensor for arginine detection using TmArgBP.
  • To compare two distinct biosensor design strategies: solvent accessibility and photo-induced electron transfer (PET).
  • To optimize the PET-based biosensor for sensitive and quantitative arginine detection.

Main Methods:

  • Site-directed mutagenesis of TmArgBP to create cysteine (K145C) and quencher (D18X) sites.
  • Labeling TmArgBP mutants with environmentally sensitive fluorescent probes.
  • Utilizing photo-induced electron transfer (PET) between aromatic residues (tyrosine, tryptophan) and the fluorescent probe for signal generation.
  • Characterizing arginine binding affinity using dissociation constants (Kd).

Main Results:

  • TmArgBP mutants labeled with environmentally sensitive probes showed limited fluorescence changes.
  • The PET-based biosensor demonstrated significant fluorescence quenching upon arginine binding.
  • Tyrosine and tryptophan residues effectively quenched probe emission (>80%) via PET.
  • Quantitative detection of arginine was achieved with dissociation constants ranging from 0.87 to 1.5 μM.

Conclusions:

  • The PET-based strategy provides a highly effective and broadly applicable method for small molecule detection.
  • This approach can be adapted for any protein exhibiting ligand-dependent conformational changes.
  • The developed biosensor offers a sensitive and quantitative alternative for arginine analysis.