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

Abstract

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.

A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from Thermotoga maritima

“…In line with previous reports [7], [9], gel filtration analysis indicates that the protein adopts different aggregation states (data not shown). Although aggregates with higher molecular masses were detected, we focused our attention on the main component.…”

“…In this case, however, tertiary structure variations associated with ligand binding are very limited. The oligomerization through domain swapping exhibited by TmArgBP also provides a rationale for the observed ability of the protein to form higher aggregates [7], [9]. These states may be achieved through a mutual swapping of the C-terminal helix that is not limited to two subunits but involves three (trimers) or four (tetramers) protein molecules, as found in RNase A [13], [21], [22].…”

“…Although this may appear in contrast with previous reports [7], the analysis of the 3D structure reconciles all data. Indeed, SPR experiments indicated a much lower affinity (K D of 17 µM) of the protein for a rather complex arginine-containing peptide [9]. The lower affinity for this peptide can be easily explained by considering that the protein strongly grasps both charged amino and carboxyl ends of the aminoacid.…”

“…We identified the arginine binding protein form Thermatoga maritima (TmArgBP) as an ideal system for arginine detection [6], [7], [8], [9], [10]. Arginine sensing is extremely important since argininemia is a debilitating inherited condition, characterized by a gradual accumulation of arginine and ammonia in the blood, whose diagnosis is crucial for effective medical intervention [11].…”

“…The biochemical and biophysical characterization of the protein has shown that TmArgBP presents a number of remarkable properties. These include an extraordinary stability to both temperature and chemical denaturants [9]. Additionally, in contrast to the vast majority of SBP that operate as monomers, TmArgBP forms multimeric assemblies at room temperature [7], [9].…”