Abstract
Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. 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). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. 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. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. 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. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.
Highlights
Since we were interested in designing simplified Thermotoga maritima arginine-binding protein (TmArgBP) variants, we used the structure of the truncated monomeric variant TmArgBP20−233 as a scaffold
We manually modelled the Trp side chain inside the binding pocket by exploring the most frequent rotameric states of the residue [22]. Most of these rotamers could not be allocated into the binding pocket without major steric clashes, the one characterized by χ1 and χ2 dihedral angles of 178◦ and 105◦ did not produce any local strain within the protein and presumably did not hamper the arginine binding (Figure S3)
We exploited the extraordinary stability of TmArgBP to generate novel and simplified scaffolds whose tryptophan fluorescence depends on their arginine binding state
Summary
Introduction l-arginine, thereafter denoted as arginine, is a fundamental nutrient of living organisms [1] This amino acid, which is semi-essential for mammals, is required in protein synthesis and in other important metabolic pathways. Taking into account the relative inhomogeneity of the wild-type TmArgBP that presents trimeric and higher oligomeric forms along with the dimeric state and the virtually irreversible arginine binding to the protein, we here attempted to generate novel TmArgBP-derived scaffolds using simplified variants. Through combined dissections and mutations of TmArgBP, we were able to generate distinct protein scaffolds whose tryptophan fluorescence was sensitive to the arginine binding (Figure S1). Our data demonstrate that extremely stable substrate-binding proteins, which are very promising systems for developing biosensors, may be effectively manipulated to obtain simplified scaffolds that reversibly bind the ligand
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