Recombinant Borrelia burgdorferi Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the functional role of Phenylalanine-tRNA ligase in Borrelia burgdorferi pathogenesis?

Phenylalanine-tRNA ligase (PheRS) in Borrelia burgdorferi, composed of alpha and beta (pheT) subunits, plays a critical role in protein synthesis by catalyzing the attachment of phenylalanine to its cognate tRNA. Unlike many bacterial aminoacyl-tRNA synthetases that are adapted to specific environmental niches, B. burgdorferi's PheRS must function effectively across varying conditions experienced during the bacterium's complex life cycle, which alternates between tick vectors and mammalian hosts. The enzyme maintains translation fidelity in dramatically different temperature and pH environments, making it essential for survival and pathogenesis .

The beta subunit (pheT) specifically contains the catalytic domain responsible for aminoacylation activity. Research indicates that inhibition of aminoacyl-tRNA synthetases disrupts protein synthesis and bacterial growth, positioning pheT as a potential therapeutic target for Lyme disease treatment. The enzyme's catalytic activity is particularly critical during the rapid adaptive response required when transitioning between host environments .

What expression systems yield optimal production of functional recombinant B. burgdorferi pheT?

E. coli-based expression systems have demonstrated superior results for recombinant production of B. burgdorferi proteins. For pheT specifically, the following methodological approach has proven effective:

• Vector selection: pET expression systems with T7 promoters provide strong, controlled expression

• Host strain: BL21(DE3) or Rosetta(DE3) for rare codon optimization

• Fusion tags: MBP (maltose-binding protein) fusion significantly enhances solubility and proper folding of B. burgdorferi proteins, as evidenced by successful expression of other B. burgdorferi proteins

• Induction conditions: 0.5mM IPTG at 18°C for 16-20 hours minimizes inclusion body formation

A comparative analysis of expression systems for recombinant B. burgdorferi proteins demonstrated that MBP-fusion proteins consistently achieve >90% purity following affinity chromatography, similar to what has been achieved with VlsE proteins . Codon optimization based on B. burgdorferi's AT-rich genome (average GC content 28.6%) is essential, as the bacterium shows marked bias toward AU-rich codons in a ratio of 2:1 up to 20:1 depending on the amino acid .

How should researchers validate the structural integrity of recombinant pheT?

Multiple complementary techniques should be employed to confirm proper folding and functionality of recombinant pheT:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure content

• Thermal Shift Assays: To evaluate thermal stability and proper folding

• Size Exclusion Chromatography: To confirm monomeric/oligomeric state

• Enzymatic Activity Assessment: Aminoacylation assays measuring attachment of phenylalanine to tRNA^Phe

• Binding Assays: Surface Plasmon Resonance (SPR) to quantify interactions with substrates

Activity validation requires comparison with established parameters for aminoacyl-tRNA synthetases. A properly folded pheT should demonstrate Michaelis-Menten kinetics with catalytic efficiency (kcat/KM) values in the range of 10^5-10^6 M^-1s^-1 for cognate tRNA aminoacylation.

What techniques enable structural characterization of B. burgdorferi pheT and how do they inform drug design?

Structural characterization of B. burgdorferi pheT requires an integrated approach:

• X-ray Crystallography: The gold standard for high-resolution structures, though crystallization of B. burgdorferi pheT presents challenges due to conformational flexibility. Preliminary crystallization trials should employ sparse matrix screens with protein concentrations of 5-15 mg/mL in low-salt buffers (20-50 mM) at pH 7.0-8.0.

• Cryo-Electron Microscopy: Particularly valuable for visualizing pheT in complex with tRNA substrates or potential inhibitors. Sample preparation should utilize graphene oxide grids to minimize preferred orientation issues.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): For mapping dynamic regions and conformational changes upon substrate binding.

• Molecular Dynamics Simulations: To model conformational flexibility and identify potential allosteric sites.

The active site architecture of aminoacyl-tRNA synthetases reveals multiple druggable pockets. The ATP binding site is highly conserved while the amino acid binding pocket offers greater potential for selectivity. A structure-guided approach focuses on compounds that exploit species-specific features of the enzyme's catalytic or editing domains .

How can researchers effectively design aminoacyl-tRNA synthetase inhibitors specific to B. burgdorferi pheT?

Rational design of B. burgdorferi pheT inhibitors should follow this methodological framework:

• Comparative Structural Analysis: Identify unique features of B. burgdorferi pheT versus human orthologs to maximize selectivity

• Fragment-Based Screening: Utilize thermal shift assays to identify initial binding fragments

• Structure-Activity Relationship Development: Systematically modify hit compounds to improve potency and selectivity

• Binding Pocket Targeting Strategy: Focus on three potential sites:

  • ATP binding pocket (more conserved)

  • Phenylalanine binding pocket (moderately specific)

  • tRNA interaction domain (most species-specific)

The design approach leverages knowledge gained from other aminoacyl-tRNA synthetase studies, where binding site mutations have successfully altered specificity for substrates. Similar to the approach used for incorporating p-acetyl-L-phenylalanine via engineered synthetases, researchers can identify key residues in B. burgdorferi pheT that differ from human orthologs .

What approaches are effective for studying potential synergistic effects between pheT inhibitors and existing antibiotics?

A systematic investigation of potential synergies requires:

• Checkerboard Assays: Determine Fractional Inhibitory Concentration (FIC) indices across concentration matrices

• Time-Kill Studies: Establish bactericidal versus bacteriostatic effects of combination treatments

• Efflux Pump Interaction Analysis: Evaluate whether pheT inhibitors affect antibiotic accumulation

• Resistance Development Monitoring: Serial passage experiments comparing resistance development rates

Synergy assessment should examine combinations with antibiotics from different classes:

• β-lactams (cell wall synthesis inhibitors)

• Macrolides (alternative protein synthesis inhibitors)

• Tetracyclines (commonly used for Lyme disease)

Research on other aminoacyl-tRNA synthetase inhibitors suggests that targeting multiple steps in bacterial protein synthesis can produce synergistic effects while reducing the likelihood of resistance development.

What are the essential controls and validation steps for aminoacylation assays with recombinant pheT?

A rigorous aminoacylation assay requires the following controls and validation steps:

• Positive Controls:

  • Commercial E. coli PheRS with verified activity

  • Native B. burgdorferi extract (if available)

• Negative Controls:

  • Heat-inactivated pheT (95°C for 10 minutes)

  • Assays lacking essential components (ATP, tRNA, or phenylalanine)

  • Assays with non-cognate amino acids (e.g., tyrosine)

• Validation Parameters:

  • Linear reaction range determination

  • Enzyme concentration optimization

  • Time-course analysis to establish steady-state conditions

  • pH and temperature optimization reflective of physiological conditions

• Data Quality Assessment:

  • Technical replicates (minimum n=3)

  • Biological replicates (minimum n=3 independent protein preparations)

  • Positive control recovery (80-120% of expected activity)

The aminoacylation reaction can be monitored through various methods, including:

• Radioactive assays using [³H]-phenylalanine

• Colorimetric pyrophosphate detection

• MALDI-TOF mass spectrometry to detect charged tRNA species

Each method has specific technical considerations that must be addressed to ensure reliable data interpretation.

How should differential scanning fluorimetry (DSF) be optimized for studying pheT-ligand interactions?

DSF optimization for pheT requires methodical parameter refinement:

• Buffer Optimization:

  • Screen multiple buffers (HEPES, Tris, Phosphate) at pH 6.5-8.0

  • Test salt concentrations (50-200 mM NaCl)

  • Evaluate stabilizing additives (5-10% glycerol, 1-5 mM MgCl₂)

• Protein Concentration:

  • Titrate across 0.1-1.0 mg/mL to determine optimal signal-to-noise ratio

  • Verify linear range between protein concentration and fluorescence signal

• Dye Selection and Optimization:

  • Compare SYPRO Orange, SYPRO Red, and ANS for compatibility

  • Determine optimal dye:protein ratio (typically 5-10:1)

• Thermal Parameters:

  • Rate of temperature increase (0.5-1.0°C/min)

  • Temperature range (25-95°C)

  • Equilibration time at each temperature point

• Ligand Evaluation:

  • Include substrate controls (ATP, phenylalanine, tRNA)

  • Establish minimum significant ΔTm shift (typically >1.5°C)

  • Develop concentration-response curves for promising ligands

DSF data analysis should include both Tm shifts and curve shape analysis, as some ligands may stabilize specific protein conformations without significantly altering the melting temperature.

What strategies effectively address the challenges of preparing tRNA substrates for pheT activity studies?

Preparation of suitable tRNA substrates presents several technical challenges that can be addressed through these methodological approaches:

• Source Options for tRNA^Phe:

  Source Method	Advantages	Disadvantages	Typical Yields
  Commercial yeast tRNA^Phe	High purity, immediate availability	May not perfectly match bacterial specificity	N/A (purchased)
  In vitro transcription	Sequence control, high uniformity	Lacks post-transcriptional modifications	2-5 mg/L reaction
  Recombinant expression in E. coli	Better mimics natural tRNA structure	Complex purification, heterogeneity	1-3 mg/L culture
  Direct purification from B. burgdorferi	Native structure including modifications	Extremely low yields, technically challenging	0.1-0.5 mg/L culture

• Quality Control Measures:

  • Denaturing PAGE analysis (>90% purity)

  • A260/A280 ratio assessment (ideal: 1.8-2.0)

  • Aminoacylation efficiency with control PheRS enzyme

  • Thermal denaturation profile to confirm proper folding

• Special Considerations for B. burgdorferi tRNA Studies:

  • Account for B. burgdorferi's unusual AT-rich codon usage

  • Consider potential unique post-transcriptional modifications

  • Evaluate the need for specific ions (Mg²⁺, NH₄⁺) in reaction buffers

For researchers without specialized RNA expertise, commercial sources followed by validation of aminoacylation competence represent the most reliable approach for initial studies.

How should kinetic data from pheT aminoacylation assays be analyzed to extract mechanistic insights?

Comprehensive kinetic analysis of pheT requires multiple analytical approaches:

• Steady-State Kinetic Parameters Determination:

  • Initial velocity measurements across substrate concentration ranges

  • Non-linear regression fitting to Michaelis-Menten equation

  • Analysis of kcat, KM, and kcat/KM for each substrate

  • Calculation of specificity constants for different amino acid/tRNA combinations

• Reaction Mechanism Investigation:

  • Product inhibition studies to distinguish ordered vs. random mechanisms

  • Isotope exchange experiments to identify rate-limiting steps

  • Pre-steady-state kinetics to capture transient intermediates

• Data Transformation Approaches:

  • Lineweaver-Burk plots for mechanism visualization (though not for parameter determination)

  • Eadie-Hofstee and Hanes-Woolf plots as complementary analyses

  • Global fitting to comprehensive models using specialized software

• Inhibition Analysis:

  • Determination of inhibition constants (Ki) and inhibition modalities

  • IC50 to Ki conversion using the Cheng-Prusoff equation when appropriate

  • Time-dependent inhibition analysis to identify potential covalent inhibitors

Proper interpretation requires consideration of B. burgdorferi's unusual environmental adaptations. The enzyme likely exhibits temperature-dependent kinetic parameters that may differ significantly from model organisms, necessitating analysis across temperature ranges relevant to both tick vector (23-25°C) and mammalian host (37°C) environments .

What approaches can differentiate between specific and non-specific binding in pheT-ligand interaction studies?

Distinguishing specific from non-specific binding requires a multi-faceted approach:

• Competition Assays:

  • Displacement studies with known substrates (ATP, phenylalanine)

  • IC50 determination with proper controls for compound interference

• Binding Selectivity Assessment:

  • Comparison with related aminoacyl-tRNA synthetases

  • Evaluation against denatured protein controls

  • Testing against non-catalytic protein controls (BSA, ovalbumin)

• Biophysical Characterization:

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Surface Plasmon Resonance (SPR) for binding kinetics

  • Microscale Thermophoresis (MST) for solution-phase interactions

  • NMR for binding site identification

• Structural Validation:

  • X-ray co-crystallization or soaking experiments

  • Hydrogen-deuterium exchange mass spectrometry

  • Site-directed mutagenesis of predicted binding residues

Specific binding typically demonstrates:

• Saturable binding with defined stoichiometry

• Temperature and pH dependence consistent with physiological function

• Competition with natural substrates

• Binding energetics dominated by enthalpy rather than entropy

How can comparative genomics inform the specificity analysis of B. burgdorferi pheT across different strains and related species?

Comparative genomics offers valuable insights into pheT function and specificity:

• Sequence Conservation Analysis:

  • Multiple sequence alignment of pheT across Borrelia species

  • Identification of highly conserved catalytic residues

  • Mapping strain-specific variations to functional domains

• Structural Homology Modeling:

  • Template-based modeling using known aminoacyl-tRNA synthetase structures

  • Analysis of active site conservation and divergence

  • Prediction of species-specific binding pocket features

• Evolutionary Analysis:

  • Selection pressure analysis (dN/dS ratios) across gene regions

  • Identification of positively selected sites indicating adaptation

  • Phylogenetic reconstruction of enzyme evolution within the genus

• Cross-Species Functional Comparison:

  • Recombinant expression of pheT from related species

  • Comparative kinetic analysis across Borrelia species

  • Cross-species substrate utilization profiles

The AT-rich genome of B. burgdorferi (28.6% GC content) creates unique codon usage patterns that may influence tRNA recognition properties of pheT . Comparative analysis across B. burgdorferi strains (such as type strain B31) and other Borrelia species causing tick-borne relapsing fever (TBRF) can reveal adaptations specific to their distinct lifestyles and host environments .

How can recombinant pheT be utilized in developing new diagnostic approaches for Lyme disease?

Recombinant pheT offers several avenues for improving Lyme disease diagnostics:

• Serological Assay Development:

  • Evaluation as a potential antigen in multiplex serological arrays

  • Assessment of immunoreactivity in patient sera during different disease stages

  • Incorporation into modified immunoblot assays alongside other recombinant antigens

• Epitope Mapping Strategies:

  • Identification of B-cell epitopes unique to B. burgdorferi

  • Development of peptide-based diagnostic assays with increased specificity

  • Cross-reactivity assessment with other Borrelia species

• Specificity Enhancement:

  • Comparative analysis against TBRF Borrelia species antigens

  • Identification of unique immunogenic regions

  • Development of differential diagnostic panels

The current diagnostic approach for Lyme disease relies on a two-tier testing method involving an initial screening test followed by Western blot confirmation, but this approach has limitations in sensitivity and specificity . Immunoblots using recombinant B. burgdorferi antigens have demonstrated superior performance compared to whole-cell lysate Western blots, suggesting that carefully selected recombinant proteins like pheT could potentially improve diagnostic accuracy .

Comparative studies with sera from patients with and without Lyme disease, as well as those with TBRF and other conditions that may cause false positives, are essential for establishing diagnostic utility. The sensitivity, specificity, positive and negative predictive values must be rigorously determined through blinded assessments with reference sera collections .

What methodological approaches enable the use of pheT as a potential therapeutic target for Lyme disease?

A comprehensive drug discovery campaign targeting pheT would follow this methodological framework:

• Target Validation:

  • Genetic knockdown studies to confirm essentiality

  • Chemical biology approaches with tool compounds

  • In vitro growth inhibition correlation with enzyme inhibition

• High-Throughput Screening (HTS) Strategy:

  • Development of aminoacylation assays adaptable to 384-well format

  • Fluorescence-based readouts for increased throughput

  • Primary screen at single concentration (10-20 μM) with Z' > 0.5

  • Dose-response confirmation of primary hits

• Medicinal Chemistry Optimization:

  • Structure-activity relationship studies

  • Improvement of physiochemical properties

  • ADME profile optimization

  • Selectivity enhancement versus human PheRS

• Preclinical Evaluation:

  • In vitro efficacy against multiple B. burgdorferi strains

  • Determination of bactericidal vs. bacteriostatic effects

  • Assessment in relevant infection models

  • Pharmacokinetic/pharmacodynamic relationship establishment

The design of aminoacyl-tRNA synthetase inhibitors benefits from rational approaches similar to those used for incorporating unnatural amino acids, where specific binding pocket modifications dramatically alter substrate specificity . Structure-guided design can exploit differences between bacterial and mammalian synthetases to achieve selective inhibition.

How can researchers address solubility and stability challenges with recombinant B. burgdorferi pheT?

Successful production of soluble, stable recombinant pheT requires systematic optimization:

• Solubility Enhancement Strategies:

  • Fusion Partners: MBP tag has demonstrated >90% purity and excellent solubility with other B. burgdorferi proteins

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

  • Addition of solubility-enhancing additives (0.5M arginine, 5-10% glycerol)

  • Expression temperature optimization (16-18°C typically optimal)

• Stability Optimization:

  • Buffer screening matrix (pH 6.5-8.0, salt 50-500 mM)

  • Stabilizing additives identification (glycerol, trehalose, sucrose)

  • Thermal stability assessment via DSF across conditions

  • Long-term storage condition optimization (-80°C vs. liquid nitrogen)

• Problem-Solving Decision Tree:

  Problem	First Approach	Alternative Strategy	Advanced Solution
  Inclusion bodies	Lower temperature (16°C)	MBP fusion	Refolding from inclusion bodies
  Proteolytic degradation	Add protease inhibitors	Optimize purification speed	Engineer out proteolytic sites
  Aggregation	Add 5-10% glycerol	Reduce protein concentration	Add non-ionic detergents (0.01% Triton X-100)
  Activity loss	Add reducing agents	Test different buffer systems	Co-purify with substrate

• Quality Control Metrics:

  • Size exclusion chromatography to verify monodispersity

  • Dynamic light scattering to assess aggregation propensity

  • Thermal shift assays to determine stability under various conditions

  • Functional activity retention tracking during storage

Experience with other B. burgdorferi proteins suggests that MBP fusion tags significantly enhance solubility while maintaining >90% purity through affinity chromatography steps .

What strategies are most effective for developing specific antibodies against B. burgdorferi pheT for research applications?

Development of specific anti-pheT antibodies requires careful planning and validation:

• Antigen Design Considerations:

  • Full-length protein vs. unique peptide epitopes

  • Consideration of surface accessibility based on structural models

  • Selection of regions with low homology to human PheRS

  • Avoidance of cross-reactive epitopes with other Borrelia species

• Production Options:

  • Polyclonal antibodies: Higher sensitivity, multiple epitope recognition

  • Monoclonal antibodies: Higher specificity, renewable resource

  • Recombinant antibodies: Customizable specificity, consistent production

• Validation Essentials:

  • Western blot against recombinant pheT and B. burgdorferi lysates

  • Immunoprecipitation efficiency assessment

  • Cross-reactivity testing against related Borrelia species

  • Immunohistochemistry in infected tissues

• Application-Specific Optimization:

  • Western blot: Determine optimal antibody dilution and blocking conditions

  • Immunofluorescence: Fixation method optimization (paraformaldehyde vs. methanol)

  • ELISA: Coating buffer and detection system optimization

  • Flow cytometry: Permeabilization protocol development

Proper validation requires demonstration of specificity against both recombinant protein and native B. burgdorferi lysates. Additionally, any antibodies developed should be tested against related Borrelia species causing TBRF to ensure they don't cross-react with homologous proteins, which is particularly important given the antigenic similarities between Lyme disease and TBRF Borrelia species .

How might structural and functional studies of pheT inform our understanding of B. burgdorferi's adaptation to different host environments?

Investigating pheT's role in environmental adaptation offers insights into B. burgdorferi pathogenesis:

• Temperature-Adaptive Mechanisms:

  • Comparative enzymatic activity at tick (23°C) vs. mammalian (37°C) temperatures

  • Structural dynamics changes across temperature ranges

  • Temperature-dependent substrate binding and catalytic efficiency

• Nutrient Limitation Responses:

  • pheT function under amino acid limitation conditions

  • Adaptive responses to tRNA availability fluctuations

  • Integration with stringent response pathways

• Host Immune Evasion Connections:

  • Potential interactions with VlsE and other antigenic variation systems

  • Role in stress responses during immune challenge

  • Contribution to persister cell formation

• Methodological Approaches:

  • Temperature-controlled enzymatic assays

  • Differential gene expression analysis across host conditions

  • Protein-protein interaction studies under varying environmental conditions

B. burgdorferi's complex life cycle requires adaptation to dramatically different environments. While VlsE is well-characterized as contributing to immune evasion , the potential role of essential housekeeping enzymes like pheT in environmental adaptation remains an emerging area of investigation. The bacterium's limited biosynthetic capabilities suggest that protein synthesis machinery must be highly optimized to function across diverse conditions.

What are the potential applications of engineered B. burgdorferi pheT variants in biotechnology?

Engineered pheT variants offer several biotechnological applications:

• Expanded Genetic Code Applications:

  • Development of orthogonal aminoacyl-tRNA synthetases based on pheT

  • Incorporation of unnatural amino acids into proteins

  • Creation of novel protein functionalities through chemical biology

• Biosensor Development:

  • Engineering pheT-based sensors for detecting environmental phenylalanine

  • Development of high-throughput screening platforms for aminoacyl-tRNA synthetase inhibitors

  • Creation of cellular reporters for monitoring protein synthesis

• Methodology for pheT Engineering:

  • Structure-guided mutagenesis of binding pocket residues

  • Directed evolution approaches for altered specificity

  • Computational design of protein interfaces

• Implementation Strategy:

  • Initial target selection (e.g., p-acetyl-L-phenylalanine incorporation)

  • Mutation selection based on molecular docking and energy calculations

  • Screening for desired activity using reporter systems

Similar engineering approaches have been successfully applied to other aminoacyl-tRNA synthetases, such as the M. jannaschii tyrosyl-tRNA synthetase, where mutations in key residues (Tyr32, Asp158, Ile159) altered specificity to accept unnatural amino acids like p-acetyl-L-phenylalanine . These approaches could be adapted to B. burgdorferi pheT to develop novel biotechnological tools.

What unexplored research questions about B. burgdorferi pheT would advance our understanding of Lyme disease pathogenesis?

Several critical knowledge gaps remain unexplored:

• Persistence Mechanisms:

  • Role of pheT in bacterial persistence during antibiotic treatment

  • Potential alterations in tRNA aminoacylation during dormancy

  • Contribution to post-treatment Lyme disease syndrome pathophysiology

• Host-Pathogen Interactions:

  • Potential extracellular functions of pheT beyond protein synthesis

  • Immunomodulatory properties of pheT or its fragments

  • Role in adaptation to specific host microenvironments

• Regulatory Networks:

  • Integration of pheT function with other adaptive responses

  • Transcriptional and post-translational regulation across the infectious cycle

  • Connection to the limited metabolic capabilities of B. burgdorferi

• Research Methodologies:

  • Conditional knockdown systems to study essentiality in vivo

  • Single-cell approaches to examine heterogeneity in pheT expression

  • Systems biology integration of aminoacyl-tRNA synthetase functions

The genomic analysis of B. burgdorferi revealed its limited metabolic capabilities , suggesting that protein synthesis machinery like pheT may play especially critical roles in pathogen survival and adaptation. Understanding how these essential functions intersect with pathogenesis mechanisms could reveal new therapeutic approaches for Lyme disease.

How might advances in structural biology techniques enable new insights into B. burgdorferi pheT function and inhibition?

Emerging structural biology approaches offer unprecedented opportunities:

• Cryo-Electron Microscopy Advances:

  • Direct visualization of pheT-tRNA complexes at near-atomic resolution

  • Structural determination of conformationally heterogeneous states

  • Integration with in situ cellular tomography for native context

• Time-Resolved Structural Studies:

  • X-ray free-electron laser (XFEL) applications for capturing reaction intermediates

  • Time-resolved crystallography to visualize catalytic mechanism

  • Temperature-jump studies to examine conformational dynamics

• Integrative Structural Biology:

  • Combining multiple techniques (crystallography, cryo-EM, SAXS, NMR)

  • Computational integration of sparse experimental data

  • Molecular dynamics simulations informed by experimental constraints

• Methodological Implementation Strategy:

  • Sample preparation optimization for specific techniques

  • Data collection and processing pipelines development

  • Computational framework for integrating multiple data types

These approaches could reveal the detailed molecular mechanisms underlying pheT function in B. burgdorferi, potentially identifying unique structural features that could be exploited for therapeutic development. The VlsE protein has been successfully produced as a recombinant protein with >90% purity , suggesting that similar approaches could be applied to obtain sufficient quantities of pheT for advanced structural studies.