Recombinant Chicken Tyrosine--tRNA ligase, cytoplasmic (YARS), partial

What is the function of Tyrosine--tRNA ligase in cellular metabolism?

Tyrosine--tRNA ligase (YARS) catalyzes the attachment of tyrosine to tRNA(Tyr) in a two-step reaction essential for protein synthesis. First, tyrosine is activated by ATP to form Tyr-AMP, and then the aminoacyl group is transferred to the acceptor end of tRNA(Tyr) . This aminoacylation reaction is critical for maintaining translational fidelity during protein synthesis by ensuring the correct amino acid is incorporated into growing polypeptide chains. Beyond its canonical role in translation, YARS may possess additional non-canonical functions in cellular signaling pathways, as seen in mammalian systems .

How does chicken YARS compare structurally to YARS from other species?

Chicken YARS shares significant structural homology with YARS from other vertebrates, maintaining the characteristic class I aminoacyl-tRNA synthetase architecture. Like other YARS proteins, it likely functions as a homodimer . While specific structural data for chicken YARS is limited, comparative studies with other species suggest conservation of key catalytic residues involved in substrate binding and catalysis, such as the threonine residue that stabilizes negatively charged reaction intermediates . The catalytic domain contains the HIGH and KMSKS motifs typical of class I synthetases, while the highly conserved CP1 domain is involved in tRNA recognition.

What expression systems are commonly used for recombinant YARS production?

Recombinant YARS proteins are typically expressed in prokaryotic systems such as E. coli, which allows for high yield and relatively straightforward purification protocols . For expression of partial chicken YARS, bacterial expression systems utilizing histidine tags facilitate efficient purification through immobilized metal affinity chromatography (IMAC). Alternative systems including yeast, baculovirus, or mammalian cell lines may be employed when post-translational modifications or specific folding environments are required . The choice of expression system should be guided by experimental requirements, with E. coli being preferred for structural and biochemical studies where glycosylation is not essential.

What are recommended storage conditions for recombinant chicken YARS?

For optimal stability and activity retention, recombinant chicken YARS should be stored at -20°C for long-term storage or at -80°C for extended preservation . For working solutions, storage at 4°C is suitable for up to one week. The protein is typically maintained in a buffer containing glycerol (20%) to prevent freeze-thaw damage, along with reducing agents such as DTT (1mM) to protect cysteine residues from oxidation . To minimize activity loss, repeated freeze-thaw cycles should be avoided, and the addition of carrier proteins (0.1% HSA or BSA) is recommended for dilute solutions . Proper aliquoting upon initial thawing can help maintain enzyme integrity throughout your research project.

How can researcher-optimized aminoacylation assays distinguish between canonical and non-canonical activities of chicken YARS?

Distinguishing between canonical aminoacylation and non-canonical functions of chicken YARS requires carefully designed biochemical assays that can separately measure these activities. For canonical aminoacylation activity, researchers should employ a tRNA charging assay using radiolabeled tyrosine ([³H] or [¹⁴C]tyrosine) or ATP ([γ-³²P]ATP), followed by acid precipitation and scintillation counting to quantify charged tRNA(Tyr). The reaction mixture should contain:

To investigate potential non-canonical activities, researchers should design assays based on mammalian YARS studies, which have demonstrated cytokine-like activities and interactions with PARP1 . Pull-down assays, co-immunoprecipitation, or yeast two-hybrid systems can identify novel protein interaction partners specific to chicken YARS. Comparative assays using truncated versions or site-directed mutants with compromised aminoacylation activity but intact non-canonical domains will help distinguish these functions.

What are the critical considerations for generating site-directed mutants of chicken YARS for structure-function studies?

When designing site-directed mutants of chicken YARS for structure-function studies, researchers should prioritize several key considerations:

• Catalytic residue identification: Based on homology with bacterial TyrRS, mutations of putative active site residues such as the threonine equivalent to Thr234 in bacterial TyrRS should be prioritized . Conservative mutations (Thr→Ser) versus non-conservative mutations (Thr→Ala) can provide insights into the precise role of these residues.

• Domain interface residues: Since YARS functions as a homodimer , mutations at the dimer interface can elucidate the importance of dimerization for catalytic activity.

• tRNA recognition elements: Mutations in the CP1 domain and the C-terminal domain can help map the tRNA binding surface.

• Expression validation: Western blotting using anti-His tag antibodies or specific anti-YARS antibodies should confirm proper expression of mutant proteins .

• Activity assays: Steady-state kinetic parameters (kcat, KM) should be determined for each mutant and compared with wild-type enzyme to quantify the impact of mutations.

Researchers should also consider control mutations at non-conserved, surface-exposed residues distant from the active site to confirm that changes in activity are specific to the mutated functional residues rather than due to global structural perturbations.

How can researchers effectively differentiate between YARS1 (cytoplasmic) and YARS2 (mitochondrial) activities in chicken tissues?

Differentiating between YARS1 (cytoplasmic) and YARS2 (mitochondrial) activities in chicken tissues requires specialized approaches that exploit their distinct subcellular localization, substrate preferences, and biochemical properties:

• Subcellular fractionation: Isolate cytoplasmic and mitochondrial fractions from chicken tissues using differential centrifugation. The purity of fractions should be validated using markers such as GAPDH (cytoplasmic) and cytochrome c oxidase (mitochondrial).

• Immunological differentiation: Generate or obtain antibodies specific to unique epitopes in chicken YARS1 and YARS2. Western blotting and immunohistochemistry can then be used to distinguish between these isoforms .

• Activity assays with selective inhibitors: Resveratrol has been shown to inhibit human YARS1 and promote its nuclear localization . Researchers can test whether this compound similarly affects chicken YARS1 but not YARS2.

• Substrate specificity: While both enzymes charge tRNA(Tyr) with tyrosine, they may exhibit differences in kinetic parameters or preferences for specific tRNA isoacceptors. Assays using purified mitochondrial versus cytoplasmic tRNA populations can help differentiate their activities.

• pH and salt sensitivity profiles: Systematically characterize the activity of both enzymes across different pH values and salt concentrations to identify conditions where one isoform remains active while the other is inhibited.

These approaches, used in combination, provide a comprehensive strategy for distinguishing between the two YARS isoforms in chicken tissues.

What are the methodological challenges in investigating potential moonlighting functions of chicken YARS?

Investigating moonlighting functions of chicken YARS presents several methodological challenges that researchers must address:

• Separating enzymatic and non-enzymatic activities: Creating variants with mutations that specifically abolish aminoacylation activity while preserving potential secondary functions is essential. This requires detailed structure-function knowledge of chicken YARS.

• Identifying relevant interaction partners: Techniques such as BioID, proximity labeling, or cross-linking mass spectrometry should be employed to identify proteins that interact with chicken YARS in different cellular compartments.

• Confirming physiological relevance: Demonstrating that observed moonlighting functions occur at physiologically relevant concentrations and conditions is crucial. Cell-based assays using chicken cell lines with YARS knockdown/knockout followed by complementation with wild-type or function-specific mutants can help establish relevance.

• Tissue-specific expression patterns: Comprehensive expression profiling across chicken tissues may reveal enrichment patterns suggesting tissue-specific moonlighting functions.

• Evolutionary conservation assessment: Comparative studies with YARS from other species can determine whether potential moonlighting functions are conserved or chicken-specific, providing insights into their evolutionary significance.

Based on studies of mammalian YARS1, researchers should investigate potential roles in nuclear poly-ADP-ribosylation, cytokine-like activities, or immune signaling pathways , while recognizing that chicken YARS may have evolved distinct moonlighting functions.

How should researchers design kinetic assays to determine the catalytic efficiency of chicken YARS?

Designing rigorous kinetic assays for chicken YARS requires careful consideration of reaction conditions, substrate concentrations, and analytical methods:

• Steady-state kinetic analysis: To determine kinetic parameters (KM, kcat, kcat/KM), researchers should use a coupled enzyme assay monitoring ATP consumption or pyrophosphate release. The standard reaction mixture should include:

• Pre-steady-state kinetics: Rapid quench-flow techniques can investigate individual steps in the aminoacylation reaction, particularly the rate-limiting step.

• Substrate variation: Systematically vary one substrate while keeping others at saturating concentrations to determine individual Michaelis constants.

• Temperature and pH optimization: Characterize activity across temperatures (25-42°C) and pH values (6.5-8.5) to determine optimal conditions and physiological relevance.

• Data analysis: Apply appropriate kinetic models (Michaelis-Menten, substrate inhibition, etc.) using non-linear regression. For complex kinetic behaviors, global fitting of multiple datasets may be necessary.

These approaches provide a comprehensive kinetic characterization of chicken YARS and facilitate comparisons with YARS from other species.

What strategies can researchers employ to improve the solubility and stability of recombinant chicken YARS?

Researchers can employ several strategies to enhance the solubility and stability of recombinant chicken YARS:

• Optimization of expression conditions:

  • Lower induction temperature (16-20°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Use of specialized E. coli strains (Rosetta, Origami, SHuffle)

• Buffer optimization:

  • Include stabilizing agents: glycerol (10-20%), sucrose (5-10%)

  • Add reducing agents: DTT (1-5 mM) or TCEP (0.5-2 mM)

  • Test various salts (NaCl, KCl) at different concentrations (50-500 mM)

  • Evaluate buffering agents (HEPES, Tris, phosphate) at various pH values

• Protein engineering approaches:

  • Removal of hydrophobic surface patches

  • Introduction of surface-exposed charged residues

  • Deletion of flexible/disordered regions

  • Creation of fusion proteins with solubility enhancers (MBP, SUMO, thioredoxin)

• Storage condition optimization:

  • Flash-freezing in liquid nitrogen

  • Addition of protease inhibitors

  • Inclusion of carrier proteins (BSA, 0.1%)

  • Lyophilization with appropriate cryoprotectants

• High-throughput screening:

  • Thermal shift assays to identify stabilizing buffer conditions

  • Limited proteolysis to identify stable domains

  • Dynamic light scattering to monitor aggregation propensity

These strategies should be systematically tested to identify optimal conditions for maintaining the activity and structural integrity of recombinant chicken YARS during purification, storage, and experimental procedures.

What are the most effective methods for assessing the fidelity of chicken YARS in tRNA charging reactions?

Assessing the fidelity of chicken YARS in tRNA charging requires specialized methods that can detect misaminoacylation events:

• Acid gel electrophoresis: This technique separates charged and uncharged tRNAs based on their mobility differences. Researchers should use:

  • 6.5% polyacrylamide gels (pH 5.0)

  • Northern blotting with tRNA(Tyr)-specific probes

  • Quantification of aminoacylation levels using phosphorimaging

• Mass spectrometry-based approaches:

  • LC-MS/MS analysis of digested aminoacyl-tRNAs

  • Determination of aminoacylation products with a mass accuracy of <5 ppm

  • Heavy isotope-labeled amino acids as internal standards

• Hydrolysis protection assays:

  • Treatment of aminoacylated tRNA with mild alkali

  • Differential hydrolysis rates between correctly and incorrectly charged tRNAs

  • Analysis by denaturing PAGE or HPLC

• In vitro translation fidelity assays:

  • Reporter constructs with tyrosine codons in critical positions

  • Analysis of translation products by mass spectrometry

  • Quantification of amino acid substitution rates

• Competition assays with near-cognate amino acids:

  • Inclusion of phenylalanine as competitor (structurally similar to tyrosine)

  • Determination of discrimination factors (ratio of kcat/KM values)

  • Analysis under varying conditions (temperature, pH, salt)

These methods provide comprehensive insights into the aminoacylation fidelity of chicken YARS and can identify conditions that may compromise translational accuracy in research applications.

How can researchers validate the activity and specificity of recombinant chicken YARS preparations?

Validating the activity and specificity of recombinant chicken YARS preparations requires a multi-faceted approach:

• Aminoacylation activity assays:

  • Measurement of initial velocities using radiolabeled substrates

  • Determination of specific activity (nmol/min/mg)

  • Comparison with reference standards or literature values

• Purity assessment:

  • SDS-PAGE with Coomassie staining (>90% purity expected)

  • Western blotting with anti-YARS or anti-tag antibodies

  • Mass spectrometry to identify contaminants

• Substrate specificity tests:

  • Aminoacylation assays with non-cognate tRNAs

  • Competition experiments with near-cognate amino acids

  • Determination of discrimination factors

• Structural integrity validation:

  • Circular dichroism spectroscopy

  • Thermal denaturation profiles

  • Size-exclusion chromatography to confirm oligomeric state

• Functional comparison with mammalian YARS:

  • Side-by-side activity assays

  • Cross-species tRNA charging efficiency

  • Inhibition profiles with known inhibitors

Researchers should establish quality control criteria based on these parameters to ensure batch-to-batch consistency and reliable experimental outcomes when working with recombinant chicken YARS.

What are common pitfalls in experimental design when studying chicken YARS, and how can they be avoided?

Researchers should be aware of several common pitfalls when designing experiments with chicken YARS:

• Inadequate enzyme stability monitoring:

  • Pitfall: Activity loss during storage or throughout experiments

  • Solution: Regular activity checks using standardized assays; inclusion of proper controls; optimal storage conditions with glycerol and reducing agents

• Neglecting metal ion requirements:

  • Pitfall: Inconsistent activity due to variable metal ion concentrations

  • Solution: Careful titration of Mg²⁺ (primary cofactor) ; testing for effects of other divalent cations; inclusion of EDTA controls

• Improper tRNA preparation:

  • Pitfall: Partially degraded or misfolded tRNA substrates

  • Solution: Rigorous quality control of tRNA preparations; proper refolding protocols; verification of charging competence

• Oversight of post-translational modifications:

  • Pitfall: Different activity profiles between recombinant and native enzyme

  • Solution: Characterization of potential PTMs in native chicken YARS; selection of appropriate expression systems if PTMs are critical

• Incomplete kinetic analysis:

  • Pitfall: Simplified kinetic models that miss complex mechanisms

  • Solution: Comprehensive initial velocity studies; product inhibition analysis; consideration of ordered binding mechanisms

• Ignoring potential moonlighting functions:

  • Pitfall: Missing physiologically relevant non-canonical activities

  • Solution: Design experiments that can detect both canonical and non-canonical functions; consider cellular context

By anticipating these pitfalls, researchers can design more robust experiments that yield reliable and physiologically relevant data about chicken YARS function.

How can researchers distinguish between enzymatic and non-enzymatic properties of chicken YARS in cellular contexts?

Distinguishing enzymatic from non-enzymatic properties of chicken YARS in cellular contexts requires strategic experimental approaches:

• Catalytically inactive mutants:

  • Generate point mutations in catalytic residues that abolish aminoacylation activity

  • Verify loss of enzymatic function in vitro

  • Express these mutants in chicken cell lines to assess non-canonical functions

• Domain-specific analysis:

  • Create domain deletion/truncation variants

  • Express individual domains to identify which mediate non-canonical interactions

  • Use domain swapping with other synthetases to create chimeric proteins

• Spatiotemporal localization studies:

  • Perform immunofluorescence microscopy or live-cell imaging with fluorescently tagged YARS

  • Monitor subcellular redistribution under stress conditions

  • Correlate localization patterns with potential moonlighting functions

• Interaction proteomics:

  • Conduct immunoprecipitation followed by mass spectrometry

  • Compare interaction partners of wild-type versus catalytically inactive YARS

  • Validate key interactions using techniques like FRET or BiFC

• Pharmacological approaches:

  • Test compounds that specifically inhibit the catalytic activity (e.g., analogs of tyrosyl-adenylate)

  • Assess whether non-canonical functions persist during enzymatic inhibition

  • Utilize resveratrol, which has been shown to inhibit human YARS1 catalytic activity while promoting nuclear functions

These approaches provide complementary evidence to delineate the diverse functions of chicken YARS beyond its canonical role in protein synthesis.

How does chicken YARS compare functionally to YARS from other avian and non-avian vertebrates?

Comparative analysis of YARS across species reveals important evolutionary insights:

• Sequence conservation patterns:

  • Core catalytic domains show high conservation (>80% identity) across vertebrates

  • C-terminal domains exhibit greater divergence, suggesting species-specific regulatory mechanisms

  • Key catalytic residues (equivalent to Thr234 in bacterial YARS) are invariant across species

• Kinetic parameter comparisons:

  • Avian YARS typically exhibits kinetic parameters similar to other vertebrate orthologs:

(Note: These values represent typical ranges based on related synthetases; specific values for chicken YARS may vary)

• tRNA recognition elements:

  • Avian YARS recognizes similar identity elements in tRNA(Tyr) as mammalian orthologs

  • Species-specific differences in anticodon recognition may exist, reflecting codon usage biases

• Moonlighting functions:

  • Cytokine-like functions observed in mammalian YARS require investigation in chicken YARS

  • The interaction with PARP1 seen in human YARS1 may be conserved in chicken, suggesting ancient origin

• Subcellular localization patterns:

  • Nuclear localization signals and export sequences show varying degrees of conservation

  • Stimuli triggering subcellular redistribution may differ between avian and mammalian systems

These comparisons provide context for understanding chicken YARS function within the broader evolutionary landscape of aminoacyl-tRNA synthetases.

What insights can structural biology approaches provide about chicken YARS function and evolution?

Structural biology approaches offer valuable insights into chicken YARS function and evolution:

• Homology modeling and structural prediction:

  • Models based on crystal structures from related species can predict:

    • Active site architecture and substrate binding modes

    • Conformational changes during catalysis

    • Potential allosteric regulation sites

  • Molecular dynamics simulations can reveal species-specific dynamic properties

• X-ray crystallography targets:

  • Crystal structures of chicken YARS in different functional states could reveal:

    • Apo enzyme conformation

    • Tyrosine-bound state

    • ATP-bound state

    • Tyrosyl-adenylate intermediate complex

    • tRNA-bound state

• Comparative structural analysis:

  • Structural alignment with bacterial TyrRS (e.g., from Geobacillus stearothermophilus) can identify:

    • Conserved catalytic machinery

    • Vertebrate-specific structural elements

    • Potential sites for non-canonical functions

• Structure-guided functional studies:

  • Identification of surface patches unique to avian YARS

  • Design of chimeric proteins to test function of specific structural elements

  • Rational design of mutations to test mechanistic hypotheses

• Evolutionary analysis based on structure:

  • Mapping sequence conservation onto structural models

  • Identifying regions under positive or purifying selection

  • Correlating structural features with species-specific functional adaptations

These structural approaches complement biochemical and cellular studies, providing a mechanistic understanding of chicken YARS function that can guide further experimental investigations.