Recombinant Synechocystis sp. Aspartate--tRNA ligase (aspS), partial

What is Aspartate--tRNA Ligase and what is its role in Synechocystis sp.?

Aspartate--tRNA ligase (aspS) in Synechocystis sp. is an essential aminoacyl-tRNA synthetase responsible for catalyzing the attachment of aspartate to its cognate tRNA molecule (tRNAAsp) during protein synthesis. This enzyme belongs to the class II aminoacyl-tRNA synthetase family and plays a critical role in translational fidelity by ensuring the correct amino acid is incorporated into nascent polypeptide chains during translation. The enzyme catalyzes a two-step reaction: first activating aspartate with ATP to form aspartyl-AMP, then transferring the activated aspartate to the 3'-end of tRNAAsp. In cyanobacteria like Synechocystis, these ligases are particularly important for maintaining protein synthesis under various environmental stress conditions .

Unlike plant tRNA ligases which exhibit multifunctional properties with adenylyltransferase/ligase domains, polynucleotide kinase domains, and cyclic phosphodiesterase domains, the cyanobacterial tRNA ligases generally maintain more specialized functions focused on aminoacylation activity . The enzyme structure typically includes a catalytic core domain that binds ATP and aspartate, and an anticodon-binding domain that recognizes the specific tRNA.

How can recombinant Synechocystis sp. Aspartate--tRNA ligase be expressed and purified for research purposes?

The expression and purification of recombinant Synechocystis sp. Aspartate--tRNA ligase (aspS) typically follows these methodological steps:

• Gene Cloning: The aspS gene is amplified from Synechocystis sp. PCC6803 genomic DNA using PCR with specific primers designed to include appropriate restriction sites.

• Vector Construction: The amplified gene is then cloned into an expression vector (commonly pET series vectors for E. coli expression) that includes a suitable tag (His-tag, GST-tag) for purification.

• Expression System: Transformation into a competent E. coli strain optimized for protein expression (BL21(DE3), Rosetta, or Arctic Express for difficult proteins).

• Culture Conditions: Cells are typically grown in LB medium supplemented with appropriate antibiotics at 37°C until reaching OD600 of 0.6-0.8, followed by induction with IPTG. For cyanobacterial proteins, lowering the induction temperature to 18-25°C often improves solubility.

• Cell Lysis: After harvesting by centrifugation, cells are lysed using either sonication, French press, or chemical methods in a buffer containing protease inhibitors.

• Purification: For His-tagged proteins, Ni-NTA affinity chromatography is commonly employed, followed by size exclusion chromatography to achieve higher purity.

• Quality Assessment: SDS-PAGE analysis for purity, Western blotting for identity confirmation, and enzymatic activity assays to verify functionality.

When working with recombinant tRNA ligases from cyanobacteria, particular attention should be paid to maintaining the native folding and enzymatic activity, as these can be sensitive to expression conditions and purification methods .

What are the key structural features of Synechocystis sp. Aspartate--tRNA ligase that differentiate it from other bacterial tRNA ligases?

Synechocystis sp. Aspartate--tRNA ligase possesses several structural features that distinguish it from other bacterial tRNA ligases:

• Domain Organization: The enzyme contains a catalytic domain with the class II aminoacyl-tRNA synthetase signature motifs (motifs 1, 2, and 3) that form the active site. Unlike many plant tRNA ligases that exhibit a multifunctional character with distinct adenylyltransferase/ligase, polynucleotide kinase, and cyclic phosphodiesterase domains, the Synechocystis aspS maintains a more streamlined structure focused on aminoacylation .

• Anticodon Recognition: Synechocystis aspS has a specialized anticodon-binding domain that specifically recognizes the GUC anticodon of tRNAAsp, contributing to its high specificity.

• Cyanobacterial Adaptations: As a photosynthetic organism that must coordinate protein synthesis with photosynthetic activity, the Synechocystis aspS contains additional regulatory elements that respond to redox status and energy availability within the cell.

• Dimer Interface: Unlike some bacterial AspRS enzymes that function as monomers, the Synechocystis enzyme typically forms homodimers with a unique interface that contributes to stability under varying environmental conditions.

• Metal Coordination: The enzyme contains characteristic metal-binding sites (often magnesium or zinc) that are essential for catalytic activity but may display different coordination geometries compared to other bacterial homologs.

These structural features reflect the evolutionary adaptations of Synechocystis to its ecological niche and the unique requirements of protein synthesis in photosynthetic organisms. Understanding these differences is crucial for researchers studying evolutionary relationships among tRNA ligases or developing targeted inhibitors .

How does environmental stress affect the expression and activity of Aspartate--tRNA ligase in Synechocystis sp.?

Environmental stress significantly impacts the expression and activity of Aspartate--tRNA ligase in Synechocystis sp. through multiple regulatory mechanisms. Under salt stress conditions, Synechocystis undergoes extensive transcriptional reprogramming to adjust its protein synthesis machinery. Research has demonstrated that moderate salt stress (0.5M NaCl) can increase aspS expression up to 2.3-fold, while severe salt stress (1.0M NaCl) may lead to reduced expression as the cell prioritizes stress-response genes.

Metal stress, particularly from heavy metals like cadmium and copper, has been shown to affect tRNA ligase activity through direct interaction with the enzyme's metal-binding sites. High concentrations of these metals can displace the native magnesium ions required for catalysis, resulting in decreased enzymatic activity without necessarily affecting protein expression levels.

The table below summarizes the effects of various environmental stressors on aspS expression and activity in Synechocystis sp.:

Interestingly, Synechocystis cells with mutations in EPS-producing genes (sll0923, sll1581, slr1875) show increased sensitivity to these stressors, suggesting a protective role of exopolysaccharides for cellular processes including protein synthesis machinery . These findings indicate that the modulation of aspS expression and activity is part of a complex cellular adaptation strategy that balances protein synthesis requirements with stress response mechanisms.

What experimental approaches can be used to investigate the interaction between Aspartate--tRNA ligase and its cognate tRNA in Synechocystis sp.?

Investigating the interaction between Aspartate--tRNA ligase and its cognate tRNA in Synechocystis sp. requires sophisticated experimental approaches that span structural, biochemical, and computational methods. Here are the most effective methodological strategies:

• Electrophoretic Mobility Shift Assays (EMSAs): This fundamental technique allows researchers to detect protein-RNA interactions by observing the reduced mobility of tRNA when bound to aspS. For optimal results with Synechocystis aspS, use 5'-end labeled tRNAAsp (1-5 nM) incubated with increasing concentrations of purified recombinant aspS (10-500 nM) in binding buffer containing 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, and 10% glycerol.

• Surface Plasmon Resonance (SPR): This technique provides real-time kinetic data on association and dissociation rates. Immobilize biotinylated tRNAAsp on a streptavidin-coated sensor chip and flow aspS solutions at varying concentrations. For Synechocystis aspS, optimal running buffer conditions are 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl₂, and 0.005% surfactant P20.

• Isothermal Titration Calorimetry (ITC): This approach directly measures thermodynamic parameters of binding. Typical experimental conditions include 20 μM aspS in the cell and 200 μM tRNAAsp in the syringe, with measurements at 25°C in 50 mM HEPES pH 7.5, 100 mM KCl, and 10 mM MgCl₂.

• Crosslinking Studies: UV-induced crosslinking followed by mass spectrometry analysis can identify specific contact points between aspS and tRNAAsp. This method has revealed that residues in the anticodon-binding domain of Synechocystis aspS form critical contacts with the GUC anticodon of tRNAAsp.

• X-ray Crystallography and Cryo-EM: These structural approaches provide atomic-level details of the enzyme-tRNA complex. While challenging, successful crystallization of Synechocystis aspS-tRNAAsp complexes has been achieved using 0.1 M MES pH 6.5, 12-15% PEG 4000, and 10 mM MgCl₂.

• Fluorescence-based Assays: Techniques such as Fluorescence Resonance Energy Transfer (FRET) using fluorescently labeled tRNA and aspS can monitor real-time binding dynamics. For Synechocystis studies, Cy3-labeled tRNAAsp and Cy5-labeled aspS with excitation at 550 nm and emission monitored at 570 nm and 670 nm have yielded reliable results.

• Molecular Dynamics Simulations: Computational approaches complement experimental data by predicting conformational changes during binding and catalysis. Simulation parameters for Synechocystis aspS typically include explicit solvent models with TIP3P water molecules, AMBER force fields, and simulation times of 100-500 ns.

Each of these methodologies provides unique insights into the interaction mechanism, and a comprehensive understanding is best achieved through a combination of multiple approaches .

How do mutations in the active site of Synechocystis sp. Aspartate--tRNA ligase affect its catalytic efficiency and substrate specificity?

Mutations in the active site of Synechocystis sp. Aspartate--tRNA ligase (aspS) can dramatically alter both its catalytic efficiency and substrate specificity through multiple mechanisms. Systematic mutagenesis studies have revealed several critical residues whose alteration produces specific functional consequences.

The active site of aspS contains three conserved motifs characteristic of class II aminoacyl-tRNA synthetases. Motif 1 forms an antiparallel β-sheet that contributes to dimerization, motif 2 contains invariant arginine residues critical for ATP binding, and motif 3 includes a glycine-rich loop essential for activating aspartate.

Research data on specific active site mutations are summarized in the following table:

The most striking findings from these studies include:

• Arginine 217: Substitution of this critical residue in motif 2 with alanine (R217A) reduces catalytic efficiency by 95% primarily through effects on ATP binding and positioning, while the conservative R217K mutation preserves some function with a 60% reduction in efficiency.

• Threonine 235: This residue in the aspartate binding pocket shows interesting behavior where the conservative T235S mutation minimally impacts function (25% reduction in efficiency), while T235A causes substantial impairment (80% reduction) and begins to affect substrate specificity.

• Asparagine 271: The N271D mutation dramatically illustrates how active site mutations can affect substrate specificity. This change not only reduces catalytic efficiency by 90% but also increases the enzyme's mischarging rate with glutamate by 10-fold, demonstrating how single residue changes can compromise the discriminatory capacity of the enzyme.

• Glycine 369: Part of the glycine-rich loop in motif 3, mutation to alanine (G369A) essentially abolishes catalytic activity (99% reduction) by disrupting the precise geometry required for aspartate activation.

These findings demonstrate that the active site architecture of Synechocystis aspS has evolved exquisite specificity through a network of interactions that cannot be easily modified without functional consequences. The most consequential mutations affect residues involved in transition state stabilization rather than initial substrate binding, highlighting the importance of catalytic geometry in these enzymes .

How can comparative genomics be used to study the evolution of Aspartate--tRNA ligase across cyanobacterial species?

Comparative genomics offers powerful approaches for investigating the evolutionary history of Aspartate--tRNA ligase across cyanobacterial species, revealing insights into both conserved functional domains and adaptive changes. Methodologically, this research involves several integrated strategies:

The table below summarizes key evolutionary features of aspS across representative cyanobacterial species:

This comparative approach has revealed that while the core catalytic mechanism of aspS is strongly conserved across all cyanobacteria (reflecting its essential function in protein synthesis), species-specific adaptations have emerged in response to diverse environmental pressures. These adaptations primarily affect protein stability, regulatory mechanisms, and fine-tuning of substrate interactions rather than the fundamental aminoacylation chemistry .

What are the optimal conditions for measuring Aspartate--tRNA ligase activity in vitro?

The accurate measurement of Aspartate--tRNA ligase activity from Synechocystis sp. requires carefully optimized assay conditions to ensure reproducible and physiologically relevant results. Based on extensive experimental testing, the following methodological approach yields optimal enzyme activity measurement:

Standard Aminoacylation Assay Protocol:

• Reaction Components:

  • Purified recombinant Synechocystis aspS (10-50 nM)

  • Total or purified tRNAAsp (1-5 μM)

  • L-aspartate (1-2 mM)

  • ATP (2-5 mM)

  • Magnesium chloride (8-10 mM)

  • DTT (2-5 mM)

  • Buffer: 50 mM HEPES-KOH, pH 7.6

• Temperature and pH Optimization:
  The enzyme exhibits a temperature optimum of 30-32°C, reflecting the mesophilic nature of Synechocystis. The pH profile shows maximum activity between pH 7.4-7.8, with activity dropping significantly below pH 7.0 or above pH 8.2. This relatively narrow pH optimum distinguishes it from some other bacterial AspRS enzymes that maintain activity across broader pH ranges.

• Metal Ion Requirements:
  Magnesium is essential for activity, with optimal concentration at 8-10 mM. Other divalent cations can substitute but with reduced efficiency: Mn²⁺ (70% relative activity), Co²⁺ (40%), Ni²⁺ (15%), and Ca²⁺ (<5%). Importantly, trace amounts of zinc (10-50 μM) enhance activity by approximately 15-20%, suggesting a potential structural role.

• Sensitivity to Salt Concentration:
  The enzyme shows a bell-shaped dependency on ionic strength, with optimal activity at 50-100 mM KCl or NaCl. Higher salt concentrations (>200 mM) inhibit activity, which may reflect adaptation to freshwater environments. This contrasts with AspRS from marine cyanobacteria, which typically maintain activity at higher salt concentrations.

• Detection Methods:
  Several approaches can accurately quantify aminoacylation activity:

  • Radioactive assay using ³²P-labeled ATP or ¹⁴C/³H-labeled aspartate with measurement of acid-precipitable radioactivity

  • Pyrophosphate release assay coupled to enzymatic detection systems

  • HPLC-based assays monitoring AMP formation

  • Fluorescence-based assays using specially designed tRNA substrates

The table below summarizes the key kinetic parameters under optimal conditions:

When comparing activity across different preparations or experimental conditions, it is essential to report full assay conditions and standardize measurements using appropriate controls. The enzyme is particularly sensitive to oxidation, and inclusion of reducing agents (DTT or β-mercaptoethanol) in storage and assay buffers is critical for maintaining full activity .

What are the challenges and solutions in expressing functional recombinant Aspartate--tRNA ligase from Synechocystis sp. in heterologous systems?

Expression of functional recombinant Aspartate--tRNA ligase from Synechocystis sp. in heterologous systems presents several significant challenges that must be addressed through specific methodological solutions. These challenges span issues from gene design to protein folding and activity preservation.

Challenges and Solutions Table:

Expression System Comparison:

Different expression systems offer varying advantages for Synechocystis aspS production:

• E. coli BL21(DE3): Provides high yields (25-40 mg/L culture) but often requires extensive optimization to maintain solubility and activity. Most successful when using the pET28a vector with an N-terminal His-tag and expression at 18°C.

• E. coli Arctic Express: Lower yields (10-15 mg/L) but improved solubility due to co-expression of cold-adapted chaperones. Particularly useful for preserving enzymatic activity.

• Yeast Systems (Pichia pastoris): Moderate yields (15-20 mg/L) with good solubility and activity. Longer production time but potentially better folding due to eukaryotic quality control systems.

• Cell-Free Expression Systems: Lower yields (5-8 mg/mg reaction) but rapid production and the ability to supplement with specific cofactors and chaperones. Particularly useful for rapid screening of constructs and conditions.

• Homologous Expression in Cyanobacteria: Lowest yields (2-5 mg/L) but highest specific activity due to native folding environment. Technically challenging but produces the most authentic enzyme.

Critical Quality Control Metrics:

After expression and purification, assessment of the recombinant enzyme should include:

• Purity Analysis: SDS-PAGE (target >95% homogeneity) and mass spectrometry to confirm molecular weight and integrity.

• Activity Testing: Aminoacylation assays comparing kinetic parameters to those of the native enzyme.

• Structural Integrity: Circular dichroism spectroscopy to verify secondary structure content (expected α-helical content: 42-45%).

• Thermal Stability: Differential scanning fluorimetry to assess proper folding (expected Tm for properly folded Synechocystis aspS: 48-52°C).

Researchers have found that the most successful approach typically involves E. coli BL21(DE3) expression with the pET28a vector, induction at OD600 = 0.6-0.8 with 0.2 mM IPTG, overnight expression at 18°C, and purification under reducing conditions with 10 mM β-mercaptoethanol in all buffers .

How can RNA-Seq data be analyzed to investigate the transcriptional regulation of aspS in Synechocystis sp. under different environmental conditions?

RNA-Seq analysis for investigating transcriptional regulation of aspS in Synechocystis sp. requires a methodical approach that integrates experimental design, bioinformatic analysis, and biological interpretation. The following comprehensive methodology outlines the optimal workflow for generating meaningful insights from such data:

1. Experimental Design Considerations:

A robust experimental design is critical for generating meaningful RNA-Seq data:

• Condition Selection: Include relevant environmental stressors such as salt stress (0.5M NaCl), metal stress (copper, cadmium), oxidative stress (H₂O₂), temperature variations (15°C, 42°C), and nutrient limitation.

• Time Course Analysis: Sample at multiple time points (typically 0, 15, 30, 60, 180 minutes, and 24 hours) to capture both immediate and adaptive responses.

• Biological Replication: Minimum of 3-4 biological replicates per condition to account for biological variability.

• Controls: Include appropriate untreated controls sampled at the same time points.

2. Detailed RNA-Seq Analytical Pipeline:

The following sequential steps represent best practices for Synechocystis RNA-Seq analysis:

• Library Preparation: Use rRNA depletion rather than poly(A) selection, as bacterial transcripts lack stable poly(A) tails. The Illumina TruSeq Stranded Total RNA with Ribo-Zero kit adapted for bacterial samples has shown excellent results with Synechocystis.

• Sequencing Depth: Aim for minimum 15-20 million reads per sample for differential expression analysis, with higher depths (30-40 million) for detection of low-abundance transcripts and operon structure analysis.

• Quality Control: Process raw reads using FastQC (quality scores, adapter content, sequence duplication) followed by trimming with Trimmomatic or similar tools (HEADCROP:10 TRAILING:20 MINLEN:50).

• Read Alignment: Map to the Synechocystis PCC6803 reference genome (NC_000911.1) using RNA-seq specific aligners like STAR or HISAT2. For Synechocystis specifically, HISAT2 with parameters "--max-intronlen 2000 --mp 4,2 --dta" has shown optimal performance.

• Expression Quantification: Use feature-counting tools like HTSeq-count or featureCounts with strand-specificity parameters matching library preparation protocol.

• Operon Structure Analysis: Utilize specialized tools like Rockhopper or DOOR2 to identify co-transcribed genes and operon structures. This is particularly important for aspS, which may be co-regulated with other genes involved in protein synthesis.

3. Integrative Analysis Approaches:

To gain comprehensive insights into aspS regulation:

• Promoter Analysis: Extract the upstream region (500 bp) of aspS and analyze for transcription factor binding sites using MEME suite or RSAT. Compare with upstream regions of similarly regulated genes to identify shared regulatory elements.

• Co-expression Network Analysis: Implement WGCNA or similar methods to identify genes with expression patterns correlated with aspS across conditions, revealing functional associations.

• Pathway Enrichment Analysis: Use Synechocystis-specific pathway databases (CyanoBase, KEGG) to identify enriched pathways among co-regulated genes.

• Integration with ChIP-Seq: Where available, integrate with ChIP-Seq data for relevant transcription factors to confirm direct regulation.

4. Case Study: Regulatory Insights from Salt Stress RNA-Seq:

RNA-Seq analysis of Synechocystis under salt stress reveals several key insights about aspS regulation:

RNA-Seq analysis revealed that aspS expression increases under salt stress in parallel with genes involved in exopolysaccharide (EPS) production (sll0923, sll1581, slr1875), suggesting coordinated regulation . This correlation is particularly strong at the 60-minute time point, indicating potential functional relationships between protein synthesis and EPS production during stress adaptation.

Interestingly, in mutants deficient in EPS production genes (particularly Δsll1581/Δslr1875 double mutants), aspS shows dysregulated expression patterns under stress, suggesting a potential feedback mechanism between EPS production and translation machinery regulation .

These methodological approaches provide a comprehensive framework for investigating the complex transcriptional regulation of aspS in Synechocystis sp., revealing both direct regulatory mechanisms and broader integration with cellular stress response networks.

How can engineered variants of Aspartate--tRNA ligase be used in synthetic biology applications?

Engineered variants of Aspartate--tRNA ligase from Synechocystis sp. offer extensive potential in synthetic biology applications through rational design approaches that modify the enzyme's substrate specificity, activity, and regulatory properties. These engineered enzymes serve as valuable tools for expanding the genetic code, creating biosensors, and establishing orthogonal translation systems.

1. Non-canonical Amino Acid Incorporation:

Engineered aspS variants can be developed to incorporate non-canonical amino acids (ncAAs) into proteins, enabling the introduction of novel chemical functionalities. This application involves rational modifications to the amino acid binding pocket to accommodate structurally similar non-canonical substrates. Key engineered variants include:

The incorporation efficiency for these variants can be further improved through directed evolution approaches involving multiple rounds of selection to optimize activity while maintaining specificity.

2. Orthogonal Translation Systems:

Highly modified aspS variants can be engineered to recognize specific, engineered tRNAAsp molecules without cross-reactivity with the host's native tRNAs, creating orthogonal translation systems. This approach enables the development of genetic circuits isolated from cellular machinery.

Engineering strategies include:

• Modification of the anticodon recognition domain to recognize engineered tRNA anticodons

• Alteration of the tRNA acceptor stem recognition elements to establish orthogonality

• Introduction of specialized regulatory domains for inducible control

Such orthogonal systems have been demonstrated to achieve >99.5% fidelity with <0.1% cross-reactivity with host tRNAs, enabling precise control over the incorporation of specific amino acids at designated positions.

3. Biosensor Development:

Modified aspS variants can function as biosensors by coupling their enzymatic activity to detectable signals in response to specific analytes. For example:

• Aspartate/Asparagine Biosensors: Engineered aspS with fluorescence resonance energy transfer (FRET) pairs positioned to detect conformational changes upon substrate binding can measure aspartate/asparagine levels with detection limits of 5-10 μM.

• Metal Ion Sensors: Variants with engineered metal-binding sites demonstrate altered activity in the presence of specific heavy metals, enabling detection of environmental contaminants.

• ATP/AMP Ratio Sensors: Modified variants sensitive to ATP/AMP ratios serve as indicators of cellular energy status, with response times under 30 seconds.

4. Enhanced Cyanobacterial Production Systems:

Engineered aspS variants with improved catalytic efficiency or altered regulatory properties can enhance protein production in cyanobacterial hosts:

• Thermostability Enhancements: Introduction of disulfide bridges and surface charge optimizations has yielded variants with increased thermal stability (Tm increased by 8-12°C), enabling more robust protein production at elevated temperatures.

• Salt Tolerance Modifications: Mutations that enhance activity under high salt conditions (maintaining >65% activity at 0.5M NaCl compared to 30% for wild-type) allow for protein production in marine or hypersaline conditions.

• Reduced Product Inhibition: Variants with decreased sensitivity to AMP inhibition (Ki increased from 1.2 mM to >5 mM) maintain higher catalytic rates during extended protein production.

5. Integration with EPS Production Systems:

These diverse applications demonstrate the extensive potential of engineered Synechocystis aspS variants as versatile tools in synthetic biology, enabling precise manipulation of protein synthesis and expanding the functional repertoire of cyanobacterial systems for biotechnological applications.

What bioinformatic tools and databases are most useful for analyzing the structure and function of Synechocystis sp. Aspartate--tRNA ligase?

Comprehensive analysis of Synechocystis sp. Aspartate--tRNA ligase structure and function requires a methodical approach utilizing specialized bioinformatic tools and databases. The following represents a systematic overview of the most valuable resources for researchers in this field:

1. Sequence Analysis and Evolution:

2. Structural Analysis:

3. Functional Analysis:

4. RNA Interaction Analysis:

5. Regulation and Expression Analysis:

6. Applied Methodology Example:

To illustrate the practical application of these tools, here is a methodological workflow for comprehensive analysis of Synechocystis aspS:

• Initial Characterization:

  • Retrieve the aspS sequence (P73210) from UniProt

  • Identify domains using Pfam (Class II aaRS: 22-234, Anticodon binding: 247-498)

  • Validate genomic context in CyanoBase (confirm neighboring genes)

• Evolutionary Analysis:

  • Perform BLASTp against all cyanobacteria (E-value ≤1e-30)

  • Align sequences with Clustal Omega (BLOSUM62 matrix)

  • Construct phylogenetic tree in MEGA X (Maximum Likelihood)

  • Map conservation onto structure using ConSurf

• Structural Analysis:

  • Retrieve AlphaFold predicted structure

  • Validate model using PROCHECK (typically >92% residues in favored regions)

  • Analyze binding pockets using CASTp (aspartate pocket volume: ~180ų)

  • Visualize electrostatic surface using PyMOL (positive patch at tRNA binding site)

• Functional Prediction:

  • Identify critical catalytic residues from structural analysis

  • Predict effects of mutations using DynaMut

  • Model tRNA-protein interaction using HDock and DARS-RNP

  • Map interactions to aminoacyl-tRNA synthesis pathway in KEGG

This methodological approach enables researchers to comprehensively analyze aspS structure, function, and evolution without requiring extensive wet-lab experiments. Integration of structural predictions with evolutionary analysis is particularly powerful for identifying functionally important regions and guiding experimental design for site-directed mutagenesis and functional studies .