Recombinant Acinetobacter sp. Histidine--tRNA ligase (hisS)

What is Histidine--tRNA ligase (HisS) and what is its function in Acinetobacter species?

Histidine--tRNA ligase (HisS) is an aminoacyl-tRNA synthetase that catalyzes the attachment of histidine to its cognate tRNA molecules, a critical step in protein translation. In Acinetobacter species, including the clinically significant A. baumannii, HisS ensures accurate incorporation of histidine during protein synthesis. It belongs to the class II aminoacyl-tRNA synthetases, which are characterized by their structural features and catalytic mechanisms . The enzyme serves as a critical component in maintaining translational fidelity in these bacteria, which is particularly important given their role in healthcare-associated infections and antibiotic resistance .

How does Acinetobacter HisS differ structurally from HisS proteins in other bacterial species?

Acinetobacter HisS belongs to a class of smaller bacterial aminoacyl-tRNA synthetases, compared to some other bacterial species. While the exact sequence characteristics of Acinetobacter HisS have not been extensively studied in comparison to all bacterial species, research on aminoacyl-tRNA synthetases demonstrates significant structural differences between bacterial species. For instance, the histidine sequence of E. coli HisS shows no strong alignment with several other bacterial tRNA synthetases, suggesting it may derive from a different progenitor . Unlike human histidyl-tRNA synthetase, which contains a coiled-coil domain within the first 60 amino acids that is important for both enzymatic activity and antigenicity, bacterial HisS proteins often lack this domain . The structural differences influence substrate interactions and may play roles in species-specific antibiotic resistance mechanisms .

What is the genetic organization of the hisS gene in Acinetobacter species?

The hisS gene in Acinetobacter species encodes the Histidine--tRNA ligase enzyme. While specific details about the genetic organization of hisS in Acinetobacter are not extensively described in the provided search results, comparative studies on aminoacyl-tRNA synthetases suggest that the gene structure includes a coding region and regulatory elements. In E. coli, for example, the hisS locus contains a coding region of 424 codons, with important regulatory elements located in the 5'-noncoding sequence that serve as promoter sites for RNA polymerase . The expression of aminoacyl-tRNA synthetase genes, including hisS, can be affected by various regulatory mechanisms, as observed in studies of bacteria exposed to antimicrobial compounds, where genes controlling protein synthesis accuracy (including hisS) showed activation patterns . Research on A. baumannii genomes has also revealed that metabolic and antibiotic resistance genes can be regulated through complex genetic mechanisms involving secondary metabolite biosynthetic gene clusters and antibiotic resistant genes, which may influence hisS expression .

What are the optimal conditions for recombinant expression of Acinetobacter sp. HisS protein?

The optimal expression of recombinant Acinetobacter sp. HisS protein typically involves bacterial expression systems, particularly E. coli. Based on research methods for similar proteins, the following conditions are recommended:

Expression System Design:

• E. coli BL21(DE3) strain is commonly used as a host for recombinant HisS expression

• Expression vectors containing T7 promoter systems provide controlled induction

• Fusion tags like His6, GST, or MBP can improve solubility and facilitate purification

Culture and Induction Conditions:

• Growth temperature: 30°C pre-induction, reduced to 18-25°C post-induction to maximize soluble protein

• Induction: 0.1-0.5 mM IPTG when culture reaches OD600 of 0.6-0.8

• Post-induction expression time: 4-16 hours (overnight expression at lower temperatures often yields higher amounts of soluble protein)

Media and Supplements:

• Rich media (like LB) with appropriate antibiotics for plasmid maintenance

• Supplementation with glucose (0.5-1%) can reduce basal expression

• Addition of 1-5 mM histidine may improve folding and stability of the synthetase

The expression conditions should be optimized through small-scale tests before proceeding to large-scale production. Protein expression should be monitored by SDS-PAGE and enzymatic activity assays to ensure functional protein production .

What purification strategies are most effective for obtaining high-purity recombinant Acinetobacter HisS?

Effective purification of recombinant Acinetobacter HisS requires a multi-step approach to achieve high purity while maintaining enzymatic activity. Based on successful protocols for similar recombinant proteins, the following methodology is recommended:

Initial Extraction:

• Cell lysis under denaturing conditions can be efficient for HisS, similar to the "rapid histone purification" (RHP) approach that directly solubilizes inclusion bodies without their isolation

• Alternative native extraction using sonication or high-pressure homogenization in buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 300 mM NaCl, 10% glycerol, and 1 mM DTT

Chromatographic Purification Sequence:

• Affinity Chromatography: If His-tagged, use Ni-NTA resin with imidazole gradient elution (20-250 mM)

• Ion Exchange Chromatography: HisS can be purified effectively using ion exchange chromatography similar to methods used for other recombinant proteins

• Size Exclusion Chromatography: Final polishing step using Superdex 200 or similar resin in buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM DTT

Quality Control:

• SDS-PAGE analysis for purity assessment (>95% purity should be achievable)

• Activity assays measuring aminoacylation of tRNA^His

• Mass spectrometry to confirm protein identity and detect potential modifications

The purification protocol should yield protein suitable for enzymatic, structural, and functional studies with minimal batch-to-batch variation .

How can researchers verify the enzymatic activity of purified recombinant HisS?

Verification of enzymatic activity for purified recombinant Acinetobacter HisS requires specific assays that measure its ability to aminoacylate tRNA with histidine. The following methodological approaches can be implemented:

Standard Aminoacylation Assay:

• Prepare reaction mixture containing purified HisS (1-10 µg), tRNA^His (2-5 µM), ATP (2-5 mM), histidine (50-100 µM, including radioactive [³H] or [¹⁴C]-labeled histidine for detection), MgCl₂ (5-10 mM), and buffer (typically 50 mM HEPES pH 7.5, 30 mM KCl)

• Incubate at 37°C for 10-30 minutes

• Precipitate aminoacylated tRNA with trichloroacetic acid (TCA)

• Filter through nitrocellulose membranes and wash

• Measure radioactivity by scintillation counting

ATP-PPi Exchange Assay:

• This alternative assay measures the reverse reaction of aminoacyl-adenylate formation

• Includes [³²P]-PPi in the reaction mixture

• Monitors the incorporation of radioactive phosphate into ATP

• Useful when purified tRNA^His is not available

Enzyme Kinetics Analysis:

• Determine kinetic parameters (Km, kcat) for histidine, tRNA^His, and ATP

• Compare with published values for other bacterial HisS enzymes

• Typical Km values for histidine range from 20-100 µM

Thermal Stability Testing:

• Differential scanning fluorimetry (DSF) can assess protein stability

• Properly folded HisS should show a clear thermal transition

• DSF can also identify ligands that stabilize the enzyme

These assays provide comprehensive verification of enzymatic function and can be complemented with analytical ultracentrifugation (AUC) to confirm proper oligomeric state, typically dimeric for functional HisS .

What domains and motifs are critical for the catalytic activity of Acinetobacter HisS?

The catalytic activity of Acinetobacter HisS depends on several critical domains and motifs that facilitate its aminoacylation function. Based on research on HisS proteins and related aminoacyl-tRNA synthetases, the following structural elements are essential:

Catalytic Domain:

• Contains the active site for histidine and ATP binding

• Features conserved motifs characteristic of class II aminoacyl-tRNA synthetases

• The first 100 amino acids typically contain important catalytic elements, as observed in other bacterial tRNA synthetases

• Mutations in this region, comparable to the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly mutations in human HARS, can significantly impair enzyme function

tRNA Binding Domain:

• Responsible for recognizing and binding the appropriate tRNA^His

• Often contains positively charged residues that interact with the negatively charged tRNA backbone

• May involve recognition of specific identity elements in tRNA^His, particularly the anticodon and acceptor stem

Dimerization Interface:

• HisS typically functions as a dimer

• The dimerization interface is critical for maintaining proper enzyme conformation

• Analytical ultracentrifugation (AUC) studies on similar enzymes confirm the importance of the dimeric structure for function

Conserved Motifs:

• Class II aminoacyl-tRNA synthetases contain three conserved motifs:

  • Motif 1: Involved in dimer formation

  • Motif 2: Forms part of the ATP binding site

  • Motif 3: Contributes to the active site and helps position the amino acid substrate

Bacterial HisS proteins notably lack the coiled-coil domain found in human histidyl-tRNA synthetase (within the first 60 amino acids), which affects their structural organization and potentially their interaction with tRNA . Understanding these structural elements is crucial for interpreting the effects of mutations and designing experiments to probe enzyme function .

How does recombinant HisS contribute to understanding antibiotic resistance mechanisms in Acinetobacter species?

Recombinant HisS serves as a valuable tool for unraveling antibiotic resistance mechanisms in Acinetobacter species through several research approaches:

Protein Synthesis Machinery as Resistance Target:

• Studies show that genes controlling protein synthesis accuracy, including aminoacyl-tRNA synthetases like hisS, are activated in response to antimicrobial treatment

• Recombinant HisS enables investigation of how aminoacylation processes may be altered in resistant strains

• Changes in HisS expression or activity may contribute to translational fidelity shifts that enhance survival under antibiotic pressure

Structural Basis for Resistance:

• Recombinant HisS production permits structural studies to identify potential interaction sites with antibiotics

• Crystallography or cryo-EM analyses using the recombinant protein can reveal how mutations might affect antibiotic binding

• Comparative structural analysis between sensitive and resistant strains may highlight adaptive changes

Functional Genomics Applications:

Metabolic Alterations and Resistance:

• Research indicates that antibiotic resistance in A. baumannii involves metabolic alterations

• Recombinant HisS studies can determine whether changes in histidine incorporation rates affect metabolic pathways linked to resistance

• In vitro translation systems supplemented with recombinant HisS can model these metabolic impacts

Experimental Resistance Development Studies:

• Recombinant HisS can be used in directed evolution experiments to identify mutations conferring resistance

• AFM-based methods similar to those used for detecting A. baumannii antibiotic resistance could incorporate recombinant HisS to study its role in resistance mechanisms

This research is particularly important given that A. baumannii can develop resistance to multiple antibiotics through various mechanisms, including changes in membrane proteins, stress responses, and altered gene expression .

What experimental approaches can detect interactions between antibiotics and Acinetobacter HisS?

Several sophisticated experimental approaches can detect and characterize interactions between antibiotics and Acinetobacter HisS, providing valuable insights into potential inhibition mechanisms:

Biophysical Interaction Assays:

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant HisS on sensor chip

  • Flow antibiotics at various concentrations over the surface

  • Measure binding kinetics (kon, koff) and affinity (KD)

  • Typical expected KD values range from nanomolar to micromolar for specific interactions

• Microscale Thermophoresis (MST):

  • Label recombinant HisS with fluorescent dye

  • Mix with varying concentrations of antibiotics

  • Measure changes in thermophoretic mobility upon binding

  • Provides binding constants in native solution conditions

• Differential Scanning Fluorimetry (DSF):

  • Monitor thermal stability of HisS in presence/absence of antibiotics

  • Binding typically increases melting temperature (Tm)

  • High-throughput method allows screening of multiple compounds

Functional Impact Assays:

• Aminoacylation Inhibition Assays:

  • Measure HisS activity with standard aminoacylation assay

  • Add antibiotics at various concentrations

  • Calculate IC50 values for inhibition

  • Determine inhibition mechanism (competitive, non-competitive, uncompetitive)

• ATP-PPi Exchange Inhibition:

  • Examine effect of antibiotics on adenylate formation step

  • Helps distinguish which reaction step is affected

• Atomic Force Microscopy:

  • Utilize oscillation mode AFM similar to methods developed for detection of A. baumannii antibiotic resistance

  • Detect changes in bacterial nanomotion in response to antibiotics

  • Can provide rapid results in less than an hour compared to traditional methods

Structural Approaches:

• X-ray Crystallography:

  • Co-crystallize HisS with antibiotics

  • Determine atomic-level interaction details

  • Identify specific binding sites and conformational changes

• Nuclear Magnetic Resonance (NMR):

  • Map chemical shift perturbations upon antibiotic binding

  • Identify interaction interfaces

  • Study dynamics of binding-induced conformational changes

Cellular Approaches:

• Gene Expression Analysis:

  • Monitor hisS expression levels in response to antibiotic pressure

  • Correlate with development of resistance phenotypes

  • qRT-PCR validation similar to methods used in proteomic studies of A. baumannii

These complementary approaches provide comprehensive data on antibiotic-HisS interactions, informing both resistance mechanisms and potential new therapeutic strategies .

How can recombinant HisS be used to screen for novel antimicrobial compounds targeting Acinetobacter species?

Recombinant HisS provides an excellent platform for screening novel antimicrobial compounds targeting Acinetobacter species through a multi-tiered approach:

Primary High-Throughput Screening:

• Enzymatic Activity Inhibition Assays:

  • Develop a miniaturized aminoacylation assay using recombinant HisS

  • Screen compound libraries (10,000-100,000 compounds)

  • Identify hit compounds that inhibit >50% activity at 10-20 µM

  • Calculate Z' factor to ensure assay robustness (aim for Z' > 0.5)

• Thermal Shift Assays:

  • Use differential scanning fluorimetry (DSF) as an orthogonal screening method

  • Identify compounds that significantly alter HisS thermal stability

  • Crosscheck with enzymatic assay hits to prioritize candidates

Secondary Screening and Characterization:

• Dose-Response Analysis:

  • Determine IC50 values for hit compounds

  • Establish structure-activity relationships (SAR)

  • Typical minimum criteria: IC50 < 1 µM for further development

• Mechanism of Action Studies:

  • Determine inhibition type (competitive, non-competitive)

  • Identify which substrate interaction is affected (ATP, histidine, tRNA)

  • Use kinetic analysis to calculate Ki values

• Selectivity Profiling:

  • Test activity against human histidyl-tRNA synthetase

  • Aim for >100-fold selectivity for bacterial enzyme

  • Screen against panel of other aminoacyl-tRNA synthetases

Tertiary Cellular Validation:

• Antimicrobial Activity Testing:

  • Determine minimum inhibitory concentration (MIC) against Acinetobacter strains

  • Include clinical isolates with various resistance profiles

  • Compare with standard antibiotics

• Rapid Resistance Detection Methods:

  • Implement AFM-based techniques similar to those developed for A. baumannii antibiotic resistance detection

  • Assess bacterial nanomotion responses to novel compounds

  • Provides rapid validation (within hours) compared to traditional MIC testing (48+ hours)

• Target Validation in Cells:

  • Overexpress HisS in Acinetobacter and test for resistance

  • Use metabolomics to confirm histidine incorporation disruption

  • Monitor changes in protein synthesis rates

Lead Optimization Pipeline:

• Structural Studies with Lead Compounds:

  • Co-crystallize recombinant HisS with promising inhibitors

  • Identify binding modes and key interaction residues

  • Guide medicinal chemistry for improved binding and pharmacokinetics

• Resistance Development Monitoring:

  • Perform serial passage experiments with sub-MIC compound concentrations

  • Sequence hisS gene from resistant mutants

  • Test mutant recombinant proteins to confirm resistance mechanism

This comprehensive screening approach leverages recombinant HisS to identify inhibitors that specifically target Acinetobacter species, potentially addressing the critical need for new antibiotics against multidrug-resistant strains .

How can recombinant HisS be used to investigate translation fidelity mechanisms in Acinetobacter species?

Recombinant HisS provides a powerful tool for investigating translation fidelity mechanisms in Acinetobacter species through several sophisticated experimental approaches:

In Vitro Translation System Development:

• Reconstituted Translation Assays:

  • Establish a cell-free translation system using Acinetobacter components

  • Include purified recombinant HisS alongside other translation factors

  • Measure translation rates and accuracy using reporter constructs

  • Compare wild-type HisS with site-directed mutants affecting catalytic efficiency

• Misacylation Analysis:

  • Assess HisS fidelity by measuring mischarging rates

  • Quantify incorporation of non-cognate amino acids (e.g., glutamine, arginine)

  • Determine error rates using mass spectrometry of translation products

  • Typical experimental setup: incubate HisS with tRNA^His and non-cognate amino acids, detect misacylated products

Molecular Mechanisms of Fidelity Control:

• Structure-Function Analysis:

  • Create recombinant HisS variants with mutations in substrate binding sites

  • Measure changes in aminoacylation kinetics (kcat/Km)

  • Correlate structural changes with fidelity alterations

  • Key targets: conserved motifs in the catalytic domain that influence substrate recognition

• tRNA Recognition Studies:

  • Investigate HisS interactions with tRNA^His identity elements

  • Use modified tRNAs to probe recognition mechanisms

  • Apply methods like SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension) to map interaction sites

Stress Response and Adaptability:

• Environmental Stress Effects:

  • Examine how stress conditions affect HisS fidelity

  • Test recombinant HisS under various pH, ionic strength, and temperature conditions

  • Measure changes in misacylation rates and correlate with growth conditions

  • Relate to studies showing activation of genes controlling protein synthesis accuracy (including hisS) in response to antimicrobial treatments

• Mistranslation as Adaptive Mechanism:

  • Investigate whether controlled mistranslation via HisS contributes to stress adaptation

  • Compare HisS properties from antibiotic-resistant versus sensitive strains

  • Analyze whether changes in translation fidelity correlate with resistance phenotypes

Systems Biology Integration:

• Proteome-Wide Impact Assessment:

  • Combine recombinant HisS studies with proteomics approaches

  • Analyze how HisS activity levels affect the global Acinetobacter proteome

  • Identify proteins particularly sensitive to histidine incorporation accuracy

  • Connect with proteomic analyses that identified differential protein expression between multidrug-resistant and drug-susceptible A. baumannii isolates

These approaches can reveal how translation fidelity contributes to Acinetobacter's remarkable adaptability and antibiotic resistance capabilities, potentially identifying new therapeutic targets .

What are the challenges in using site-directed mutagenesis to study functional residues in Acinetobacter HisS?

Site-directed mutagenesis of Acinetobacter HisS presents several significant challenges that researchers must address to obtain reliable insights into enzyme function:

Technical Challenges:

• Expression and Solubility Issues:

  • Mutations may disrupt protein folding, leading to inclusion body formation

  • Critical residue mutations often reduce expression yields by 50-90%

  • Solutions: Optimize expression conditions (lower temperature, specific media), use solubility tags (MBP, SUMO), explore refolding protocols

• Stability Considerations:

  • Mutations in conserved domains frequently destabilize the protein

  • Mutant proteins may show accelerated degradation during purification

  • Approach: Use differential scanning fluorimetry (DSF) to assess thermal stability of mutants compared to wild-type; implement stabilizing buffers

• Oligomerization Disruption:

  • HisS functions as a dimer; mutations at interfaces may prevent proper assembly

  • Analytical ultracentrifugation (AUC) can confirm proper oligomeric state as demonstrated in similar studies

  • Compensatory mutations may be required to maintain structural integrity

Experimental Design Challenges:

• Selection of Target Residues:

  • Limited structural information specifically for Acinetobacter HisS

  • Need for homology modeling based on related structures

  • Recommendation: Focus on conserved motifs in the catalytic domain first, then expand to species-specific residues

• Distinguishing Direct vs. Indirect Effects:

  • Mutations may impact function through catalytic or structural perturbations

  • Multiple assays needed: aminoacylation kinetics, binding studies, structural analyses

  • Complementary mutations can help distinguish mechanisms

• Quantifying Subtle Effects:

  • Some mutations produce partial rather than complete loss of function

  • Requires precise kinetic analysis (kcat, Km) with multiple substrate concentrations

  • Statistical rigor: minimum triplicate experiments with appropriate controls

Interpretation Challenges:

• Correlation with Physiological Relevance:

  • In vitro behavior may not fully reflect in vivo importance

  • Need for complementary genetic studies (e.g., complementation of hisS mutants)

  • Consider using AFM-based methods similar to those developed for A. baumannii antibiotic resistance detection to link in vitro findings with cellular responses

• Context-Dependent Function:

  • HisS activity may depend on interactions with other cellular components

  • Mutations might affect these interactions without changing core enzymatic function

  • Recommendation: Include protein-protein interaction studies

• Evolutionary Conservation Analysis:

  • Interpreting results requires comparison across bacterial species

  • Some functionally important residues may not be conserved due to coevolution

  • Comparative analysis with multiple bacterial HisS proteins is essential

Solutions Table:

Addressing these challenges requires an integrated approach combining structural, biochemical, and genetic methods to obtain meaningful insights into HisS function .

How can recombinant HisS be utilized in studying evolutionary relationships among Acinetobacter species?

Recombinant HisS provides a valuable molecular tool for investigating evolutionary relationships among Acinetobacter species through several sophisticated approaches:

Comparative Sequence-Structure-Function Analysis:

• Evolutionary Rate Analysis:

  • Express and characterize recombinant HisS from multiple Acinetobacter species

  • Compare sequence conservation patterns in catalytic vs. non-catalytic regions

  • Calculate evolutionary rates (dN/dS ratios) to identify regions under selection

  • Correlate with functional differences in aminoacylation kinetics

• Functional Conservation Testing:

  • Perform cross-species complementation studies

  • Express recombinant HisS from various Acinetobacter species in model organisms

  • Determine which residues are critical for species-specific functions

  • Assess whether HisS from different Acinetobacter species can cross-complement

Phylogenetic Approaches:

• Multi-Gene Phylogeny Integration:

  • Include hisS sequences in phylogenomic analyses of Acinetobacter species

  • Compare HisS-based phylogenies with those derived from whole-genome analyses

  • Identify instances of potential horizontal gene transfer

  • Apply methods similar to the dynamic stochastic block model (DSBM) used for HIV-1 phylogenetic analysis

• Ancestral Sequence Reconstruction:

  • Infer ancestral HisS sequences at key nodes in Acinetobacter phylogeny

  • Express recombinant ancestral HisS proteins

  • Characterize biochemical properties and compare to extant enzymes

  • Track the evolution of substrate specificity and catalytic efficiency

Structural Evolution Studies:

• Comparative Structural Analysis:

  • Determine crystal structures of HisS from diverse Acinetobacter species

  • Identify structural adaptations in different lineages

  • Map sequence variations onto structural models

  • Unlike studies indicating that HisS may derive from a different progenitor than other aminoacyl-tRNA synthetases, focus specifically on intra-genus variations

• Domain Architecture Comparison:

  • Analyze domain organization across the Acinetobacter genus

  • Identify insertions, deletions, or domain shuffling events

  • Express chimeric HisS with domains from different species to test functional implications

Coevolution Analysis:

• HisS-tRNA Coevolution:

  • Compare HisS and tRNA^His sequences from the same species

  • Identify coevolutionary patterns in interacting residues

  • Express recombinant HisS with cognate and non-cognate tRNAs to test specificity

  • Measure aminoacylation efficiency with homologous vs. heterologous components

• Genome Context Analysis:

  • Examine genomic neighborhood of hisS across Acinetobacter species

  • Identify conserved gene clusters and potential operon structures

  • Test whether genomic context correlates with functional properties

  • Similar to approaches used in identifying secondary metabolite biosynthetic gene clusters in Acinetobacter

These approaches can reveal how HisS has evolved within the Acinetobacter genus, potentially shedding light on adaptation mechanisms, speciation events, and the development of species-specific traits including antibiotic resistance .

What are the most common issues in recombinant HisS expression and how can they be resolved?

Researchers frequently encounter several challenges when expressing recombinant Acinetobacter HisS. Here are the most common issues and their methodological solutions:

Low Expression Yields:

• Problem: Poor protein production despite confirmed gene presence
  Solutions:

  • Optimize codon usage for expression host (typically 30-40% increase in yield)

  • Test multiple promoter systems (T7, tac, ara) to identify optimal expression control

  • Adjust induction parameters: IPTG concentration (0.1-1.0 mM), temperature (18-37°C), and duration (4-24 hours)

  • Screen multiple E. coli strains (BL21, Rosetta, Arctic Express) for improved expression

• Problem: Toxicity to host cells
  Solutions:

  • Use tight expression control systems with minimal leakage

  • Lower culture temperature to 18-25°C during expression phase

  • Employ specialized strains with enhanced tolerance (C41/C43)

  • Consider cell-free protein synthesis as an alternative

Solubility and Folding Issues:

• Problem: Formation of inclusion bodies
  Solutions:

  • Apply "rapid histone purification" (RHP)-like approach that directly solubilizes inclusion bodies

  • Express with solubility-enhancing fusion tags (MBP, SUMO, TRX)

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Develop refolding protocol using step-wise dialysis with declining urea/guanidine concentrations

• Problem: Aggregation during purification
  Solutions:

  • Include stabilizing additives: 10% glycerol, 0.1-0.5M arginine, or 0.1% Triton X-100

  • Add catalytic substrates (ATP, histidine) to stabilize native conformation

  • Optimize buffer conditions (pH 7.0-8.0, 150-300 mM NaCl)

  • Use size exclusion chromatography as final purification step

Enzymatic Activity Issues:

• Problem: Low or absent enzymatic activity
  Solutions:

  • Ensure proper folding using circular dichroism spectroscopy

  • Verify structural integrity with limited proteolysis

  • Screen buffer components systematically (pH, salt, metal ions)

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to maintain cysteine residues

• Problem: Batch-to-batch variability
  Solutions:

  • Standardize expression and purification protocols rigorously

  • Implement quality control checkpoints: SDS-PAGE, activity assays, and mass spectrometry

  • Prepare large master batches and store as aliquots at -80°C

  • Include internal standards in activity assays

Stability and Storage Issues:

• Problem: Rapid loss of activity during storage
  Solutions:

  • Identify optimal storage conditions through stability testing

  • Add stabilizers: 10-50% glycerol, 0.1 mg/ml BSA, or 1-5 mM TCEP

  • Flash-freeze aliquots in liquid nitrogen and store at -80°C

  • Avoid repeated freeze-thaw cycles (limit to 1-2 maximum)

By systematically addressing these challenges using the proposed methodological solutions, researchers can significantly improve the yield, quality, and consistency of recombinant Acinetobacter HisS preparations for subsequent functional and structural studies .

How can researchers troubleshoot inconsistent enzyme activity in recombinant HisS preparations?

Inconsistent enzyme activity in recombinant HisS preparations presents a significant challenge for researchers. A methodical troubleshooting approach is essential to identify and resolve the underlying causes:

Systematic Activity Variation Analysis:

• Problem: Variable activity between purification batches
  Diagnostic Tests:

  • Perform side-by-side activity assays with multiple batches

  • Analyze protein purity by SDS-PAGE and mass spectrometry

  • Measure protein concentration using multiple methods (Bradford, BCA, A280)

  Solutions:

  • Standardize every step of the purification protocol

  • Implement specific activity calculations (activity/mg protein)

  • Prepare internal standard (reference batch) for normalization

  • Establish acceptance criteria for batch release

• Problem: Activity decay during storage
  Diagnostic Tests:

  • Monitor activity over time under different storage conditions

  • Analyze for degradation products by SDS-PAGE and Western blotting

  • Assess aggregation by dynamic light scattering

  Solutions:

  • Optimize buffer conditions (pH 7.0-8.0, 150-300 mM NaCl)

  • Add stabilizing agents: 20-50% glycerol, 0.1% BSA, 1 mM DTT

  • Store as single-use aliquots at -80°C to avoid freeze-thaw cycles

  • Consider lyophilization with appropriate cryoprotectants

Enzyme Quality and Integrity Issues:

• Problem: Post-translational modifications affecting activity
  Diagnostic Tests:

  • Mass spectrometry analysis to identify modifications

  • Phosphorylation detection using Pro-Q Diamond staining

  • Western blot with anti-phospho/acetyl antibodies

  Solutions:

  • Express in systems with reduced modification capacity

  • Co-express with phosphatases if phosphorylation is detected

  • Purify under reducing conditions if oxidation is observed

• Problem: Improper folding or oligomerization
  Diagnostic Tests:

  • Circular dichroism spectroscopy to assess secondary structure

  • Analytical ultracentrifugation to determine oligomerization state

  • Differential scanning fluorimetry to evaluate thermal stability

  Solutions:

  • Adjust refolding protocols if using inclusion bodies

  • Include molecular chaperones during expression

  • Optimize purification to maintain native oligomeric state

  • Similar to approaches used in study of CMT2W-associated HARS mutations

Assay and Reaction Condition Optimization:

• Problem: Substrate quality and consistency issues
  Diagnostic Tests:

  • Analyze commercial ATP and amino acid purity

  • Verify tRNA^His integrity by gel electrophoresis

  • Test multiple substrate lots

  Solutions:

  • Source high-quality reagents from reliable suppliers

  • Prepare master mixes for critical components

  • Include internal controls in every assay

  • Vary substrate concentrations to ensure saturation

• Problem: Suboptimal reaction conditions
  Diagnostic Tests:

  • Perform pH optimization (range 6.5-8.5)

  • Titrate divalent cations (Mg²⁺, Mn²⁺) at 1-20 mM

  • Test temperature dependence (25-42°C)

  • Evaluate buffer composition effects

  Solutions:

  • Establish comprehensive condition optimization matrix

  • Determine enzyme kinetic parameters under optimal conditions

  • Standardize reaction time and temperature control

  • Include reaction monitoring time points to ensure linearity

Systematic Troubleshooting Data Table:

By systematically addressing these potential issues, researchers can significantly improve the consistency and reliability of recombinant HisS activity measurements, enabling more reproducible results in functional and inhibitor studies .

What considerations are important when designing kinetic assays for recombinant Acinetobacter HisS?

Designing robust kinetic assays for recombinant Acinetobacter HisS requires careful consideration of multiple factors to ensure accurate, reproducible results. The following methodological guidelines address key aspects of assay design:

Assay Format Selection and Optimization:

• Detection Method Considerations:

  • Radioactive Assays: High sensitivity but requires special handling

    • Use [³H]-histidine (specific activity >20 Ci/mmol) or [¹⁴C]-histidine (50-60 mCi/mmol)

    • TCA precipitation followed by filter binding gives lowest background

    • Typical signal-to-noise ratio should exceed 10:1

  • Colorimetric/Fluorometric Alternatives:

    • Pyrophosphate release detection using coupled enzyme assays

    • BIOMOL Green for phosphate detection (sensitivity ~0.1-2 nmol)

    • Malachite green assay (linear range 0.1-10 nmol phosphate)

  • Coupled Assays:

    • Link aminoacylation to pyrophosphatase and phosphate detection

    • Ensure coupling enzymes are not rate-limiting (use 5-10× excess)

• Reaction Components Optimization:

  • Buffer Selection:

    • HEPES or Tris buffers (50-100 mM, pH 7.0-8.0) minimize interference

    • Include 50-150 mM KCl or NaCl for ionic strength

    • Add 5-10% glycerol to enhance enzyme stability

  • Essential Cofactors:

    • Mg²⁺ (optimally 5-10 mM) as primary metal cofactor

    • Test alternative divalent cations (Mn²⁺, Zn²⁺) at 0.1-5 mM

    • Include reducing agent (1-5 mM DTT or β-mercaptoethanol)

Kinetic Parameter Determination:

• Initial Velocity Measurements:

  • Establish linear range for time course (typically 1-10 minutes)

  • Use enzyme concentrations giving <15% substrate conversion

  • Include multiple time points to confirm linearity

  • Perform reactions at 30-37°C with temperature control (±0.5°C)

• Substrate Concentration Ranges:

  • ATP: 0.01-5 mM (typical Km ~0.1-0.5 mM)

  • Histidine: 1-500 µM (typical Km ~10-50 µM)

  • tRNA^His: 0.1-10 µM (typical Km ~0.5-2 µM)

  • Use minimum 7-8 concentrations spanning 0.2-5× Km values

• Data Analysis Approaches:

  • Apply appropriate kinetic models (Michaelis-Menten, Hill, etc.)

  • Use non-linear regression rather than linearization methods

  • Calculate standard errors for all parameters

  • Validate with Eadie-Hofstee or Hanes-Woolf plots for linearity

Controls and Validation:

• Critical Control Reactions:

  • No-enzyme controls to establish background

  • Heat-inactivated enzyme controls (95°C, 10 min)

  • Substrate omission controls for each component

  • Positive control with commercial aminoacyl-tRNA synthetase

• Assay Validation Parameters:

  • Reproducibility: CV <15% between replicates

  • Robustness: Evaluate influence of minor parameter changes

  • Z'-factor: Calculate for high-throughput applications (aim for >0.5)

  • Stability: Test reagent stability over typical assay duration

Special Considerations for Inhibitor Studies:

• Inhibition Kinetics Design:

  • Pre-incubate enzyme with inhibitor (5-15 min)

  • Use multiple substrate and inhibitor concentrations

  • Include appropriate solvent controls (DMSO typically <2%)

  • Determine inhibition mechanism (competitive, non-competitive, etc.)

• Artifact Recognition and Elimination:

  • Test for inhibitor aggregation via dynamic light scattering

  • Evaluate inhibitor specificity against related enzymes

  • Include detergent controls (0.01% Triton X-100) to identify promiscuous inhibitors

  • Consider time-dependent inhibition effects