title: Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein.
abstract: The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9-3.3 μM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.
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: Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Anne A
authors.initials: AA
authors.lastname: Galyean
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA 23173, USA.
authors.forename: Teraya
authors.initials: T
authors.lastname: Donaldson
authors.affiliation: Laboratory for Molecular Sensing, IBP-CNR, Via Pietro Castellino 111, 80131 Napoli, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Pennacchio
authors.affiliation: Department of Chemistry, University of Richmond, Richmond, VA 23173, USA; Laboratory for Molecular Sensing, IBP-CNR, Via Pietro Castellino 111, 80131 Napoli, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Iozzino
authors.affiliation: Laboratory for Molecular Sensing, IBP-CNR, Via Pietro Castellino 111, 80131 Napoli, 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 / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D005453:Fluorescence; D005454:Fluorescence Polarization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D055503:Protein Multimerization; D020124:Thermotoga maritima; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*
chemical_list: D033902:Periplasmic Binding Proteins; D014364:Tryptophan; D001120:Arginine
keywords: Anisotropy decay; Asymmetric flow field flow fractionation; Fluorescence; Periplasmic binding protein; Tryptophan-scanning mutagenesis