Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein

摘要: Abstract The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic superfamily. As highly thermostable protein, TmArgBP has been investigated for potential to serve as scaffold development fluorescent biosensors. To establish relationship between structural dynamics and ligand capabilities, we constructed single tryptophan mutants probe pocket. Trp residues placed around pocket reveal strong dependence on fluorescence emission with all but one the mutants. Using these data, calculated dissociation constants 1.9–3.3 μM arginine. Stern–Volmer quenching analysis demonstrated that undergoes large conformational change upon binding, which common feature this While still active at room temperature, time-resolved intensity anisotropy decay data suggest exists rigid structure under conditions. Interestingly, dimer temperature in both presence absence arginine, determined by asymmetric flow field fractionation (AF4) supported native gel-electrophoresis anisotropy. Our stability will contribute our understanding hyperthermophilic proteins their biotechnological applications.