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Istituto di scienza dell'alimentazione

Torna all'elenco Contributi in rivista anno 2017

Contributo in rivista

Tipo: Articolo in rivista

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

Anno di pubblicazione: 2017

Autori: Donaldson, Teraya; Iozzino, Luisa; Deacon, Lindsay J.; Billones, Hilbert; Ausili, Alessio; D'Auria, Sabato; Dattelbaum, Jonathan D.

Affiliazioni autori: Univ Richmond; CNR

Autori CNR:

  • SABATO D'AURIA

Lingua: inglese

Abstract: The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 mu M across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

Lingua abstract: inglese

Pagine da: 60

Pagine a: 66

Pagine totali: 7

Rivista:

Analytical biochemistry Elsevier.
Paese di pubblicazione: Stati Uniti d'America
Lingua: inglese
ISSN: 0003-2697

Numero volume: 525

DOI: 10.1016/j.ab.2017.02.021

Stato della pubblicazione: Published version

Indicizzato da: ISI Web of Science (WOS) [000398878700009]

Parole chiave:

  • Periplasmic binding protein
  • Fluorescence
  • Switch-based biosensor
  • Hyperthermophilic bacteria
  • Photo-induced electron transfer

Strutture CNR:

 
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