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 degrees 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 muM 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.
Luchansky
Matthew S
MS
Department of Chemistry, University of Richmond, Gottwald Center for the Sciences, 28 Westhampton Way, Richmond, VA 23173, USA.
Der
Bryan S
BS
D'Auria
Sabato
S
Pocsfalvi
Gabriella
G
Iozzino
Luisa
L
Marasco
Daniela
D
Dattelbaum
Jonathan D
JD
D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002942:Circular Dichroism; D013058:Mass Spectrometry; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D020124:Thermotoga maritima / Q000378:metabolism*
D001426:Bacterial Proteins; D033902:Periplasmic Binding Proteins; D001120:Arginine