Indeed, the only Trp present in the sequence of the protein (W243) is located at the C-terminal end of the protein far from the arginine binding pocket ( Figure 1). Unfortunately, no fluorescence signal is emitted by the protein due to the lack of Trp residues close to the arginine-binding site. Taking into account these properties, TmArgBP appears a promising system for developing an arginine biosensor. TmArgBP can be further manipulated by dissecting it into its constitutive D1 and D2 domains ( Figure 1). In particular, we have shown that a stable monomeric form may be obtained either though site-directed mutagenesis or through the deletion of the C-terminal end, the swapping element in the protein binding. We have exploited the extraordinary stability of TmArgBP to effectively manipulate the protein through mutation, truncation and dissection of its native structure. The biophysical characterization of the protein has also revealed that it is endowed with an unusual stability against temperature, chemical denaturants and pressure. The three-dimensional structure of the protein has also shown that the arginine binding does not only produce a remarkable tertiary structure variation of TmArgBP but also a radical rearrangement of its quaternary structure. The crystallographic structure of the protein has highlighted that its dimer is stabilized by swapping of the terminal C-terminal helix ( Figure 1). ![]() Although substrate-binding proteins are generally monomeric, TmArgBP is essentially dimeric, with the concomitant presence, as minor components, of higher oligomerization states. This protein presents a number of distinctive/unique features in the large class of SBP. In the last few years, we have characterized the arginine-binding protein isolated from the hyperthermophilic organism Thermotoga maritima (TmArgBP). Among these, substrate-binding proteins (SBP) that deliver arginine and other metabolites to the ABC cassette system for their transportation across the periplasmatic membrane appear to be particularly appropriate for their specificity and stability. Natural arginine binders are the evident candidates for developing this type of biosensors. In this scenario, the efficient detection and quantification of the arginine is a field of relevant biomedical/biotechnological interest. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. ![]() On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways.
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