title: Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein.
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 μM 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.
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA.
authors.forename: Teraya
authors.initials: T
authors.lastname: Donaldson
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA; Laboratory for Molecular Sensing, ISA-CNR, Via Roma 64, 83100 Avellino, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Iozzino
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA.
authors.forename: Lindsay J
authors.initials: LJ
authors.lastname: Deacon
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA.
authors.forename: Hilbert
authors.initials: H
authors.lastname: Billones
authors.affiliation: Laboratory for Molecular Sensing, ISA-CNR, Via Roma 64, 83100 Avellino, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ausili
authors.affiliation: Laboratory for Molecular Sensing, ISA-CNR, Via Roma 64, 83100 Avellino, Italy.
authors.forename: Sabato
authors.initials: S
authors.lastname: D'Auria
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA. Electronic address: jdattelb@richmond.edu.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Dattelbaum
mesh_terms: D001120:Arginine / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D020124:Thermotoga maritima / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D033902:Periplasmic Binding Proteins; D014364:Tryptophan; D001120:Arginine
keywords: Fluorescence; Hyperthermophilic bacteria; Periplasmic binding protein; Photo-induced electron transfer; Switch-based biosensor