RESUMEN
Translation initiation is a highly regulated, multi-step process which is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of E. coli 70S initiation complexes containing an fMet-tRNAfMet G30-C40 variant paired to noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact start codon selection.
RESUMEN
Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3' tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU'-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < -13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.
Asunto(s)
Proteínas Bacterianas , Flavobacterium , Proteínas Ribosómicas , Ribosomas , Flavobacterium/citología , Flavobacterium/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , Proteínas Bacterianas/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.
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Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Mutación , Biosíntesis de Proteínas , Células de Purkinje/enzimología , Células de Purkinje/patología , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Animales , Dominio Catalítico , Muerte Celular , Femenino , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Células de Purkinje/metabolismo , Serina/metabolismoRESUMEN
In the Fig. 3b western blot of this Article, 'Myc-AlaRS' in row one should have been 'Myc-AAD Aars', 'AlaRS' in row two should have been 'Aars' and 'ANKRD16' in row four should have been 'Ankrd16'. In Fig. 4f, 'ANKRD16' and 'ANKRD16(3xR)' should have been 'Ankrd16' and 'Ankrd163xR; and in Fig. 3c the position of the molecular mass markers had shifted. These figures have been corrected online, and see Supplementary Information to the accompanying Amendment for the original figure.
RESUMEN
Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5-1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.
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Contaminación de Medicamentos , Famotidina , Simulación del Acoplamiento Molecular , Mutágenos , Famotidina/química , Famotidina/análisis , Mutágenos/toxicidad , Mutágenos/análisis , Mutágenos/química , Simulación de Dinámica Molecular , Simulación por Computador , Humanos , Cromatografía Líquida de Alta PresiónRESUMEN
Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.
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Bacteroidetes/genética , Iniciación de la Cadena Peptídica Traduccional , Secuencias Reguladoras de Ácido Ribonucleico , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/genética , Flavobacterium/genética , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Puromicina/farmacología , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , ARN Ribosómico 5S/genética , Ribosomas/ultraestructura , Alineación de Secuencia , Homología de Secuencia , Especificidad de la EspecieRESUMEN
In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved ß-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.
Asunto(s)
Flavobacterium/genética , Regulación Bacteriana de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , Proteínas Bacterianas/biosíntesis , Flavobacterium/metabolismo , Motivos de Nucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30Sâ¢mRNAâ¢tRNAfMet and 30Sâ¢mRNAâ¢tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.
Asunto(s)
Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional , Factor 2 Procariótico de Iniciación/genética , ARN Mensajero/genética , ARN de Transferencia de Metionina/genética , Sistemas de Lectura , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Codón Iniciador , Escherichia coli/metabolismo , Cinética , Mutación , Conformación de Ácido Nucleico , Factor 2 Procariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
In our bodies, a slight pH change causes remarkable activation or serious damage in the biological processes and continuously keeps biological homeostasis. Detection of such a slight pH change has been a constant demand in searching for unusual biological events. In this paper, we demonstrate a novel pH sensory system that has been achieved through a combination of charge neutralization by a slight pH change with aggregation-induced emission (AIE). We selected a cyano-functionalized oligo(phenylene-vinylene) (cyanoOPV) backbone for AIE and introduced ammonium-tethered boronic acid groups as a pH-dependent function. The self-assembling of these dyes (OPV-Cn) was readily achieved by pH-dependent charge neutralization at the neutral pH region. This sensory system showed unusually sensitive pH responsiveness in a narrow pH range. Moreover, this pH change was observed in a biologically important neutral pH region. We therefore believe that this system is broadly applicable to detect the slight pH change occurring in the biological events.
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The control over supramolecular interactions and obtaining information beyond the molecular scale is an extended challenge. The intriguing self-assembly of a perylene-3,4,9,10-tetracarboxylic acid diimide (PDI)-based novel bolaamphiphilic probe is experienced within an artificial environment that is restrained by using supramolecular crystallization and molecular recognition. The bolaamphiphile with a hydrophilic [18]-azacrown ether ring produced nanoaggregates due to differing solubilities in organic and aqueous media. A structural evolution was observed in the presence of alkali metal ions as guests. The metal complexes form a pseudo-cationic structure, which is further involved in an ionic self-assembly with biomolecules, thus resulting new spectroscopic information on the dye self-assembly. The overarching aim of this study is to emphasize the importance of the concept of supramolecular adaptability, which has been used to establish an environment-friendly behavior based on noncovalent forces, thus leading to the evolution of new assembly structures and photophysical properties.
RESUMEN
The newly developed oligophenylenevinylene (OPV)-based fluorescent (FL) chiral chemosensor (OPV-Me) for the representative enantiomeric guest, 1,2-cyclohexanedicarboxylic acid (1,2-CHDA: RR- and SS-form) showed the high chiral discrimination ability, resulting in the different aggregation modes of OPV-Me self-assembly: RR-CHDA directed the fibrous supramolecular aggregate, whereas SS-CHDA directed the finite aggregate. The consequent FL intensity toward RR-CHDA was up to 30 times larger than that toward SS-CHDA. Accordingly, highly enantioselective recognition was achieved. Application to the chirality sensing was also possible: OPV-Me exhibited a linear relationship between the FL intensity and the enantiomeric excess through the morphological development of stereocomplex aggregates. These results clearly show that the chiral recognition ability is manifested by the amplification cascade of the chirality difference through self-assembly.
RESUMEN
Control of higher-order polymer structures attracts a great deal of interest for many researchers when they lead to the development of materials having various advanced functions. Among them, conjugated polymers that are useful as starting materials in the design of molecular wires are particularly attractive. However, an equilibrium existing between isolated chains and bundled aggregates is inevitable and has made their physical properties very complicated. As an attempt to simplify this situation, we previously reported that a polymer chain of a water-soluble polythiophene could be isolated through complexation with a helix-forming polysaccharide. More recently, a covalently self-threading polythiophene was reported, the main chain of which was physically protected from self-folding and chain-chain π-stacking. In this report, we wish to report a new strategy to isolate a water-soluble polythiophene and to control its higher-order structure by a supramolecular approach: that is, among a few bile acids, lithocholate can form stoichiometric complexes with cationic polythiophene to isolate the polymer chain, and the higher-order structure is changeable by the molar ratio. The optical and morphological studies have been thoroughly performed, and the resultant complex has been applied to the selective recognition of two AMP structural isomers.
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Ácidos y Sales Biliares/química , Polímeros/química , Adenosina Monofosfato/química , Dimetilsulfóxido , Ácido Litocólico/química , Microscopía de Fuerza Atómica , Conformación Molecular , Estructura Molecular , Nanocables/química , Nanocables/ultraestructura , Solventes , Espectrofotometría , Tiofenos/química , AguaRESUMEN
Long-term creative approaches have been considered in the design of molecular probes to overcome the quenching effect of important dyes in an aqueous medium. Using the rational donor-acceptor based design principle, we demonstrate herein the different fluorescence states of a non-conjugated symmetrical perylene-azacrown ether system in a solution, from the molecular to the aggregated states. The ethylene-spacer is exceptionally capable of fluorescence enhancement, even in the aggregated state (organic nanoparticle, ONPs, 44 nm), overcoming the quenching effect on changing the solvent from tetrahydrofuran to water. The ONPs with crown ether receptors at the surface show colloidal stability in an aqueous solution. Furthermore, an improved fluorescent state is developed via ONPs-polymer (protamine, Pro) hybridization. Supramolecular interactions between the crown ring and the guanidinium group in Pro play an important role in the ONPs-Pro hybrid formation. The decorated fluorescent hybrid state is finally used as a nano-probe for sensing heparin via the turn-OFF mechanism. The decoration method is further generalized by recognition of the nucleotides. Herein, we detail the bottom-up approach to the molecular design and development of the different fluorescent states of a useful probe. Most excitingly, this new approach is very general and adaptive to facile detection.
RESUMEN
Oligophenylenevinylene (OPV)-based fluorescent (FL) chemosensors exhibiting linear FL responses toward polyanions were designed. Their application to FL sensing of glycosaminoglycans (heparin: HEP, chondroitin 4-sulfate: ChS, and hyaluronic acid: HA) revealed that the charge density encoded as the unit structure directs the mode of OPV self-assembly: H-type aggregate for HEP with 16-times FL increase and J-type aggregate for HA with 93-times FL increase, thus unexpectedly achieving the preferential selectivity for HA in contrast to the conventional HEP selective systems. We have found that the integral magnitude of three factors consisting of binding mechanism, self-assembly, and FL response can amplify the structural information on the target input into the characteristic FL output. This emergent property has been used for a novel molecular recognition system that realizes unconventional FL sensing of HA, potentially applicable to the clinical diagnosis of cancer-related diseases.
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Colorantes Fluorescentes/química , Glicosaminoglicanos/química , Ácido Hialurónico/química , Espectrometría de Fluorescencia , Sulfatos de Condroitina/química , Heparina/química , Concentración de Iones de HidrógenoRESUMEN
Although self-assembly has realized the spontaneous formation of nanoarchitectures, the nanoscopic expression of chemical structural information at the molecular level can alternatively be regarded as a tool to translate molecular structural information with high precision. We have found that a newly developed guanidinium-tethered oligophenylenevinylene exhibits characteristic fluorescence (FL) responses toward L- and meso-tartarate, wherein the different self-assembly modes, termed J- or H-type aggregation, are directed according to the molecular information encoded as the chemical structure. This morphological difference originates from the geometric anti versus gauche conformational difference between L- and meso-tartarate. A similar morphological difference can be reproduced with the geometric C=C bond difference between fumarate and maleate. In the present system, the dicarboxylate structural information is embodied in the inherent threshold concentration of the FL response, the signal-to-noise ratio, and the maximum FL wavelength. These results indicate that self-assembly is meticulous enough to sense subtle differences in molecular information and thus demonstrate the potential ability of self-assembly for the expression of a FL sensory system.
RESUMEN
A novel tetraphenylethene-based fluorescence (FL) chemosensor exhibits nonlinear turn-on FL switching though cooperative binding of L-tartarate, where its convergent binding to form cyclic substructures is responsible for the FL increase. This binding scheme achieves selective detection of dicarboxylates over monocarboxylates, thus is potentially applicable to the preliminary screening for metabolic disorders.
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Aminas/química , Derivados del Benceno/química , Complejos de Coordinación/química , Etilenos/química , Colorantes Fluorescentes/química , Ácidos Picolínicos/química , Tartratos/química , Zinc , Ciclización , Colorantes Fluorescentes/síntesis química , Estereoisomerismo , AguaRESUMEN
5,5'-(1,3,5,7-Tetraoxopyrrolo[3,4-f]isoindole-2,6-diyl)diisophthalic acid (PMDIG) is used to produce a supramolecular hydrogel via acid-base treatment. The field emission scanning electron micrograph and atomic force microscopy micrographs exhibit a fibrillar network structure from intermolecular supramolecular interaction, supported from Fourier transform infrared (FTIR) and UV-vis spectra. The fluorescence intensity of the PMDIG gel is 16 times higher than that of the sodium salt of PMDIG with a 42 nm red shift of the emission peak. Upon addition of an anilinium chloride solution to the PMDIG gel, it transforms into the sol, and when a solid ammonium persulfate is spread over it, a stable hydrogel is produced. The co-assembled PMDIG-polyaniline (PANI) gel exhibits a fibrillar network morphology, and the co-assembly is formed by the supramolecular interaction between the polyaniline (donor) and the PMDIG (acceptor) molecules, which is evident from FTIR spectra and wide angle X-ray scattering results. The UV-vis spectrum of the PMDIG-PANI hydrogel exhibits the characteristic peaks of polaron band transitions of the doped PANI. The PMDIG-PANI co-assembled hydrogel has a 51-fold higher storage modulus, a 52-fold higher elasticity, a 1.4-fold increase in stiffness, and a 5-fold increase of fragility compared to the values of the PMDIG hydrogel. The PMDIG-PANI xerogel exhibits a 4 order of magnitude increase in dc conductivity compared to that of PMDIG, and the I-V characteristic curve exhibits a rectification property under white light illumination showing photocurrent rectification, a new phenomenon reported here for the supramolecular gel systems. A dye-sensitized solar cell fabricated with an ITO/PMDIG-PANI/graphite device shows a power conversion efficiency (η) of 0.1%. A discussion of the mechanism of gel formation and the sol state of the PMDIG-aniline system is included considering the contact angle values of the xerogels.
RESUMEN
An equimolar mixture of riboflavin-5'-phosphate sodium salt (RP) and adenine (AD) dissolved in a phosphate buffer (pH 4.0, 1.0% w/v) produces a red coloured transparent thixotropic hydrogel at 30 °C. The gelation of the RPAD system occurs in the pH range of 25. FTIR spectra and WAXS patterns indicate self-assembly via H-bonding between the >C=O group of RP and the amino/imino group of AD followed by supramolecular organization through a π-stacking process producing a fibrillar network structure. FESEM images clearly indicate that the nanofibres are produced from the intertwining of helical fibrils. The dynamic frequency sweep experiment of the supramolecular gel at a constant strain of 1% exhibits a wide linear viscoelastic region and a considerably higher G' value (460 Pa) than that of G'' (21 Pa) confirming the gel nature of the RPAD system. The hydrogel shows high stiffness (G'/G'' = 3.3), a high yield stress (σ*) (79.5 Pa) and a moderate critical strain (γ = 17.5%). Time sweep experiments at both low (0.1%) and high strain (100%) indicate the thixotropic property of the gel. The RPAD hydrogel shows non-Newtonian viscosity in the shear rate region (0.1158 s(−1)) and after that there is a sudden fall of viscosity. The gel melting point obtained by the falling ball method is 6° higher than that obtained by the DSC method probably due to the presence of the thixotropic property of the gel. The UV-vis spectra indicate a red shift of the ππ* transition band of RP in the RPAD xerogel. On excitation of the RPAD hydrogel at 373 nm it shows twelve times enhancement of emission intensity with a 7 nm red shift of the emission peak. This has been attributed to the enhancement of lifetime from 2.2 ns in RP to 3.4 ns in the RPAD hydrogel. With increase of temperature, the fluorescence intensity of the RPAD hydrogel at first increases till 40 °C, then decreases up to 55 °C and it again increases after 60 °C.
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Mononucleótido de Flavina/química , Fluorescencia , Hidrogeles/química , Adenina/química , Elasticidad , Transición de Fase , Temperatura , ViscosidadRESUMEN
A competitive fluorescence assay of perylene-based molecular receptors has been established, and selective detection of UTP is achieved through improved aggregation arising from the specific interaction of perylene-tethered guanidinium with uridine and phosphate groups in UTP.
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Fluorescencia , Colorantes Fluorescentes/química , Nucleótidos/análisis , Perileno/química , Estructura MolecularRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.