Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 °C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 mM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima. 2. Molecular organization and structural stability

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscope observations with specific secondary structural elements. The data determined the presence of a buried ẞ-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding bacterial binding protein family members and their potential biotechnological applications

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima. 2. Molecular organization and structural stability

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscope observations with specific secondary structural elements. The data determined the presence of a buried ẞ-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding bacterial binding protein family members and their potential biotechnological applications