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 spectroscopic observations with specific secondary structural elements. The data determined the presence of a buried beta-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 of bacterial binding protein family members and their potential biotechnological applications.
Scirè
Andrea
A
Department of Biochemistry, Biology, and Genetics, Università Politecnica delle Marche, Ancona, Italy.
Marabotti
Anna
A
Staiano
Maria
M
Iozzino
Luisa
L
Luchansky
Matthew S
MS
Der
Bryan S
BS
Dattelbaum
Jonathan D
JD
Tanfani
Fabio
F
D'Auria
Sabato
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D040681:Structural Homology, Protein; D049490:Systems Biology; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D001120:Arginine