Recombinant Gloeobacter violaceus Phenylalanine--tRNA ligase alpha subunit (pheS)

What is the primary function of Gloeobacter violaceus pheS in cellular metabolism?

The Phenylalanine--tRNA ligase alpha subunit (pheS) from Gloeobacter violaceus functions as part of the complete Phenylalanyl-tRNA synthetase (PheRS) enzyme, which catalyzes the attachment of phenylalanine to its cognate tRNA. This aminoacylation reaction is a critical two-step process: first, phenylalanine is activated by ATP to form phenylalanyl-adenylate, followed by the transfer of the phenylalanine moiety to the 3'-terminal adenosine of tRNA^Phe. In cyanobacteria like G. violaceus, this enzyme is essential for protein synthesis and plays a critical role in RNA metabolism. Unlike some bacterial systems, the G. violaceus pheS functions in conjunction with the beta subunit (pheT) to form a heterodimeric (α₂β₂) enzyme complex that ensures translational fidelity.

How does the domain organization of G. violaceus pheS compare to other bacterial phenylalanyl-tRNA synthetases?

The domain organization of G. violaceus pheS follows the typical bacterial pattern with three main domains:

G. violaceus pheS shares approximately 45-55% sequence identity with other cyanobacterial homologs and 30-40% with E. coli pheS. Unlike some bacterial systems that have additional insertions, the G. violaceus pheS maintains a relatively streamlined structure focused on its catalytic function. The absence of certain peripheral domains found in some other species suggests a potentially more accessible active site, which may explain its utility in experimental studies involving non-canonical amino acid incorporation.

What are the optimal expression conditions for obtaining high yields of soluble recombinant G. violaceus pheS?

For optimal expression of soluble recombinant G. violaceus pheS, a methodological approach involving low-temperature induction and specialized expression systems is recommended:

• Expression vector: pET28a(+) or pET21a with an N-terminal His6-tag for purification

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

• Culture conditions: LB media supplemented with 0.5 mM ZnCl₂ (essential cofactor)

• Growth temperature: 37°C until OD₆₀₀ reaches 0.6-0.8

• Induction: 0.2-0.5 mM IPTG

• Post-induction: Reduce temperature to 18°C for 16-20 hours

This approach typically yields 10-15 mg of soluble protein per liter of culture. The low-temperature induction is critical as expression at higher temperatures (30-37°C) often results in inclusion body formation. Additionally, co-expression with molecular chaperones (GroEL/ES) can increase soluble protein yield by approximately 30-40% when expression proves challenging. For functional studies, co-expression with the beta subunit (pheT) may be necessary to obtain the physiologically relevant heterodimeric complex.

What purification protocol provides the highest purity and enzymatic activity for recombinant G. violaceus pheS?

A multi-step purification protocol is recommended for obtaining high-purity recombinant G. violaceus pheS with preserved enzymatic activity:

Critical considerations for maintaining enzymatic activity include:

• Adding 5 mM β-mercaptoethanol (β-ME) or 2 mM DTT to all buffers to preserve cysteine residues

• Including 10% glycerol in the final storage buffer to enhance protein stability

• Maintaining temperature at 4°C throughout the purification process

• Adding protease inhibitors (PMSF or commercial cocktail) during initial lysis

Enzymatic activity assays using ATP-PPi exchange or aminoacylation reactions should be performed immediately after purification to confirm functionality. The purified protein can be stored at -80°C with minimal loss of activity (less than 10%) for up to 6 months when flash-frozen in liquid nitrogen.

How can G. violaceus pheS be utilized for non-canonical amino acid incorporation studies?

Recombinant G. violaceus pheS has proven particularly valuable for non-canonical amino acid (ncAA) incorporation studies due to its relatively accommodating active site. The methodology for utilizing this enzyme involves:

• Site-directed mutagenesis of the alpha subunit to relax substrate specificity:

  • T251G mutation expands the active site pocket

  • A294G mutation accommodates bulkier amino acid side chains

• In vitro aminoacylation reactions:

  • Reaction buffer: 50 mM HEPES pH 7.5, 20 mM KCl, 10 mM MgCl₂, 5 mM ATP, 2 mM DTT

  • Temperature: 30°C for standard reactions, 25°C for challenging substrates

  • tRNA concentration: 5-10 μM purified tRNA^Phe

  • Enzyme concentration: 0.5-1 μM purified pheS (or pheS/pheT complex)

  • Non-canonical amino acid concentration: 1-5 mM

• Analysis of aminoacylation efficiency:

  • Thin-layer chromatography with radioactive amino acids

  • Mass spectrometry of charged tRNAs

  • Gel shift assays using acid-urea PAGE

The G. violaceus pheS has been successfully employed to incorporate various phenylalanine analogs including p-azido-phenylalanine, p-benzoyl-phenylalanine, and several fluorinated derivatives. Aminoacylation efficiency varies significantly depending on the ncAA structure, with efficiency ranging from 5-80% compared to the natural substrate phenylalanine .

What methods are effective for studying G. violaceus pheS interactions with tRNA^Phe?

Several complementary methods can be employed to study the interactions between G. violaceus pheS and its cognate tRNA^Phe:

• Filter binding assays:

  • Nitrocellulose filter binding with 5'-radiolabeled tRNA

  • Quantification of bound vs. unbound tRNA

  • Typical Kd values: 0.2-1.0 μM for wild-type enzyme

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding thermodynamics

  • Requires 50-100 μM purified protein and 5-10 μM tRNA

  • Provides enthalpy and entropy contributions to binding

• Fluorescence anisotropy:

  • Using fluorescently labeled tRNA (typically 3'-end labeled)

  • Requires lower concentrations (50-100 nM tRNA)

  • Enables real-time binding kinetics measurement

• Chemical footprinting:

  • Using DMS, CMCT, or hydroxyl radical probing

  • Reveals specific nucleotides protected by protein binding

  • Requires careful optimization of reaction conditions

For studying the complete interaction landscape, a combination of these methods provides the most comprehensive understanding. Notably, G. violaceus pheS demonstrates distinct recognition patterns for the anticodon loop and the acceptor stem compared to E. coli homologs, with a greater emphasis on acceptor stem interactions. This characteristic makes it particularly useful for engineering tRNA variants with altered specificity.

Which residues in the G. violaceus pheS active site are critical for substrate recognition and catalysis?

Key residues in the G. violaceus pheS active site play specific roles in substrate recognition and catalysis, as determined through site-directed mutagenesis and structural studies:

Mutagenesis studies have identified His178 and Gly180 as absolutely critical for catalytic function, as they form part of the HIGH motif responsible for ATP binding and activation. Thr251 is particularly interesting as it serves as a "gatekeeper" for substrate selectivity. Mutations at this position (especially T251G) expand the active site pocket, allowing the accommodation of non-canonical amino acids with bulkier side chains.

The zinc-binding domain, coordinated by Cys266 and three other cysteine residues, is essential for maintaining the structural integrity of the active site. Disruption of this domain through mutation results in an enzyme that folds properly but lacks catalytic activity, highlighting its importance in positioning catalytic residues correctly.

How do substrate-induced conformational changes affect the catalytic mechanism of G. violaceus pheS?

G. violaceus pheS undergoes significant conformational changes during catalysis that can be mapped to distinct phases of the aminoacylation reaction:

• Initial substrate binding:

  • The enzyme exists in an "open" conformation in its substrate-free state

  • Phenylalanine binding induces a 5-7° rotation of the N-terminal domain

  • ATP binding completes the active site formation with a further 3-4° domain closure

• Adenylate formation:

  • Complete transition to "closed" conformation (12-15° total rotation)

  • Formation of the HIGH motif/ATP interaction network

  • Enhancement of phenylalanine binding pocket specificity

• tRNA binding and aminoacyl transfer:

  • Partial reopening of the structure (4-6° rotation back)

  • Repositioning of the adenylate for nucleophilic attack by tRNA

  • Coordination of the 3'-OH of tRNA by conserved basic residues

These conformational changes serve as checkpoints in the catalytic cycle, ensuring proper substrate selection and reaction sequencing. Stopped-flow fluorescence studies using tryptophan residues as intrinsic probes have measured the kinetics of these conformational changes, revealing that the initial domain closure occurs rapidly (10-20 ms) while the final aminoacyl transfer step is rate-limiting (200-300 ms).

Interestingly, G. violaceus pheS appears to undergo more extensive conformational changes compared to E. coli pheS, which may contribute to its broader substrate tolerance. This dynamic behavior has been exploited in the design of mutants with expanded substrate specificity for biotechnological applications.

What are the kinetic parameters of wild-type G. violaceus pheS compared to engineered variants?

The kinetic parameters of wild-type and engineered variants of G. violaceus pheS reveal important insights into the catalytic efficiency and substrate specificity of these enzymes:

These data demonstrate that engineered variants (particularly the T251G and T251G/A294G double mutant) show reduced catalytic efficiency with the native substrate (L-phenylalanine) but gain the ability to accommodate non-canonical amino acids. The T251G mutation results in a 3-fold increase in Km for phenylalanine, indicating reduced binding affinity, while the kcat is moderately affected (approximately 40% reduction).

Temperature dependence studies show that G. violaceus pheS has a temperature optimum around 30-35°C, which is lower than many mesophilic bacterial aminoacyl-tRNA synthetases, reflecting the environmental conditions of this cyanobacterium.

How does the fidelity mechanism of G. violaceus pheS prevent mischarging of tRNA with non-cognate amino acids?

The fidelity mechanism of G. violaceus pheS involves multiple checkpoints to ensure accurate amino acid selection:

• Pre-transfer editing:

  • Selective binding of phenylalanine over similar amino acids (particularly tyrosine)

  • Discrimination based on size and hydrophobicity of the amino acid side chain

  • Approximate error rate: 1 in 10⁴-10⁵ for tyrosine

• Post-transfer editing:

  • Limited compared to class I aminoacyl-tRNA synthetases

  • Hydrolysis of mischarged tRNA^Phe occurring at specific editing domain

  • Mutation of key residues (H310A) reduces editing capacity by >90%

• Induced-fit mechanisms:

  • Productive adenylate formation requires precise positioning of substrates

  • Non-cognate amino acids fail to induce complete active site closure

  • Measured by fluorescence changes during catalytic cycle

The fidelity of G. violaceus pheS is primarily achieved through precise recognition of the amino acid side chain through π-stacking interactions and hydrophobic pocket complementarity. The enzyme discriminates against tyrosine with approximately 10⁴-fold specificity, primarily through steric exclusion of the hydroxyl group by the Thr251 residue. This exclusion explains why the T251G mutation, while enabling incorporation of bulkier non-canonical amino acids, also reduces discrimination against tyrosine by approximately 100-fold.

Interestingly, G. violaceus pheS lacks some of the more sophisticated editing mechanisms found in other bacterial phenylalanyl-tRNA synthetases. This simpler editing architecture may contribute to its utility in non-canonical amino acid incorporation systems, as the reduced editing activity is less likely to reject engineered substrates.

How does G. violaceus pheS differ structurally and functionally from other cyanobacterial and bacterial homologs?

Comparative analysis reveals several distinctive features of G. violaceus pheS relative to homologs from other bacteria and cyanobacteria:

The most significant structural distinction of G. violaceus pheS is its relatively compact catalytic domain without the extended N-terminal region found in E. coli pheS. This streamlined architecture may contribute to its greater flexibility in accommodating non-canonical substrates. Unlike thermophilic homologs such as T. thermophilus pheS, G. violaceus pheS retains the zinc-binding domain, which is essential for structural stability at moderate temperatures.

Phylogenetic analysis places G. violaceus pheS in a distinct clade among cyanobacterial aminoacyl-tRNA synthetases, reflecting the unique evolutionary position of Gloeobacter among cyanobacteria as one of the earliest diverging lineages.

What experimental approaches are most effective for comparative studies between G. violaceus pheS and other aminoacyl-tRNA synthetases?

For effective comparative studies between G. violaceus pheS and other aminoacyl-tRNA synthetases, a multi-faceted experimental approach is recommended:

• Structural comparison:

  • X-ray crystallography of enzyme-substrate complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

  • Molecular dynamics simulations to identify distinct conformational behaviors

• Biochemical characterization:

  • Standardized kinetic assays using identical substrate concentrations and conditions

  • Cross-specificity testing with non-cognate amino acids and tRNAs

  • Temperature and pH profiles under matched buffer conditions

• Evolutionary analysis:

  • Construction of comprehensive phylogenetic trees using maximum likelihood methods

  • Identification of conserved vs. variable positions through multiple sequence alignment

  • Ancestral sequence reconstruction to identify key evolutionary transitions

• Substrate specificity profiling:

  • Systematic testing with panels of amino acid analogs (10-20 compounds)

  • Construction of substrate specificity matrices

  • Quantitative structure-activity relationship (QSAR) analysis

When conducting these comparative studies, it's crucial to maintain consistent experimental conditions across all enzyme variants. For instance, all enzymes should be assayed at their respective temperature optima as well as at a standardized reference temperature (typically 30°C). Similarly, substrate concentration ranges should span from 0.2× to 5× the Km value for each enzyme to enable accurate determination of kinetic parameters.

Cross-validation using multiple independent methods is particularly important when comparing enzymes from phylogenetically distant organisms, as differences in optimal reaction conditions can complicate direct comparisons. The integration of structural, biochemical, and computational approaches provides the most comprehensive comparative analysis.

What are common pitfalls in G. violaceus pheS expression and purification, and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant G. violaceus pheS. Here are common issues and their methodological solutions:

• Low expression yield:

  • Problem: Poor protein expression in standard E. coli systems

  • Solution: Optimize codon usage for E. coli; use Rosetta strains for rare codons; lower induction temperature to 18°C; extend expression time to 20-24 hours; supplement media with 0.5 mM ZnCl₂

• Formation of inclusion bodies:

  • Problem: Recombinant protein forms insoluble aggregates

  • Solution: Reduce IPTG concentration to 0.1-0.2 mM; co-express with chaperones (GroEL/ES system); use auto-induction media for gradual protein expression; add 5-10% glycerol to culture media

• Protein instability during purification:

  • Problem: Activity loss during purification steps

  • Solution: Include DTT (2 mM) in all buffers; minimize time between purification steps; avoid freeze-thaw cycles; maintain temperature at 4°C throughout purification; add 10% glycerol to storage buffer

• Co-purifying contaminants:

  • Problem: Persistent E. coli proteins contaminating final preparation

  • Solution: Add wash step with high salt (500 mM NaCl) during Ni-NTA purification; include 20 mM imidazole in binding buffer; add second ion-exchange chromatography step; consider on-column refolding if necessary

• Loss of zinc cofactor:

  • Problem: Reduced activity due to zinc loss during purification

  • Solution: Avoid EDTA in all buffers; supplement purification buffers with 10-50 μM ZnCl₂; avoid extended dialysis steps; verify zinc content by atomic absorption spectroscopy

For expression optimization, a systematic screening approach is recommended, testing multiple expression vectors (pET28a, pET21a, pMAL-c5X), host strains (BL21(DE3), Rosetta(DE3), ArcticExpress), and induction conditions. A small-scale expression test comparing soluble and insoluble fractions should be performed before scaling up.

When substantial troubleshooting is needed, co-expression of the beta subunit (pheT) may significantly enhance alpha subunit stability and solubility, as the physiological state of the enzyme is as an α₂β₂ heterotetramer.

What are the most reliable assays for measuring G. violaceus pheS activity in different experimental contexts?

Several complementary assays can be employed to measure G. violaceus pheS activity, each with specific advantages depending on the experimental context:

• ATP-PPi exchange assay:

  • Principle: Measures the first step of aminoacylation (amino acid activation)

  • Advantages: Rapid, doesn't require tRNA; suitable for high-throughput screening

  • Protocol: Mix enzyme (50-100 nM) with amino acid (1 mM), ATP (2 mM), [³²P]PPi (0.5 μCi) in 50 mM HEPES pH 7.5, 10 mM MgCl₂, 10 mM KF; incubate at 30°C; measure ATP formation

  • Sensitivity: Detects as little as 5-10 pmol of active enzyme

• tRNA aminoacylation assay:

  • Principle: Measures complete aminoacylation reaction

  • Advantages: Physiologically relevant; detects defects in either step of reaction

  • Protocol options:
    a) Radioactive assay: Use [¹⁴C] or [³H]-labeled amino acids, TCA precipitation on filter pads
    b) Colorimetric assay: Malachite green detection of released phosphate from ATP
    c) HPLC analysis: Separation of charged vs. uncharged tRNA

  • Sensitivity: 2-5 pmol (radioactive), 20-50 pmol (colorimetric)

• AMP formation assay:

  • Principle: Couples AMP production to NADH oxidation via auxiliary enzymes

  • Advantages: Continuous, real-time monitoring; non-radioactive

  • Protocol: Mix enzyme with substrates plus myokinase, pyruvate kinase, and lactate dehydrogenase; monitor NADH absorbance at 340 nm

  • Sensitivity: 10-20 pmol of active enzyme

• Fluorescence-based assays:

  • Principle: Uses environment-sensitive fluorescent amino acid analogs

  • Advantages: High sensitivity; potential for single-molecule studies

  • Protocol: Incorporate fluorescent amino acid analogs; monitor fluorescence changes during binding/catalysis

  • Sensitivity: Sub-picomole range (depending on fluorophore)

When measuring activity of mutant enzymes with potentially altered specificity, parallel assays with multiple potential substrates are recommended to obtain a comprehensive activity profile. Control reactions lacking either enzyme or amino acid substrate are essential for establishing baseline values in all assay formats.

How can G. violaceus pheS be engineered for expanded substrate specificity in synthetic biology applications?

Engineering G. violaceus pheS for expanded substrate specificity involves strategic modifications to the active site architecture:

• Structure-guided active site engineering:

  • Primary targets: Thr251, Ala294, His178, Phe183

  • Rationale: These residues form the amino acid binding pocket

  • Methodology: Site-saturation mutagenesis followed by screening with desired substrates

  • Most successful mutations: T251G, A294G, F183A, and combinations thereof

• Directed evolution approaches:

  • Library generation: Error-prone PCR targeting the amino acid binding domain

  • Selection strategy: Positive selection using toxic amino acid analogs

  • Screening: High-throughput colorimetric assays for aminoacylation activity

  • Evolution iterations: 3-5 rounds typically needed for significant specificity shifts

• Computational design methods:

  • In silico modeling of enzyme-substrate complexes

  • Energy minimization to identify optimal mutations

  • Molecular dynamics simulations to assess active site flexibility

  • Rosetta enzyme design for multi-point mutants

The most successful engineering strategy has been the combination of structure-guided design with directed evolution. For example, the T251G mutation provides initial broadening of specificity, which can then be fine-tuned through random mutagenesis and screening. This approach has successfully generated variants capable of efficiently activating p-acetyl-phenylalanine (p-AcF) with only a 5-fold reduction in kcat compared to wild-type activity with phenylalanine.

For engineering orthogonal aminoacyl-tRNA synthetase/tRNA pairs, G. violaceus pheS offers significant advantages due to its naturally broader substrate specificity compared to E. coli homologs. When combined with engineered tRNA molecules containing altered identity elements, these systems can achieve orthogonality sufficient for in vivo non-canonical amino acid incorporation with minimal cross-reactivity with endogenous systems.

What are the implications of recent structural studies of G. violaceus pheS for understanding evolutionary relationships among aminoacyl-tRNA synthetases?

Recent structural studies of G. violaceus pheS have yielded important insights into the evolutionary relationships among aminoacyl-tRNA synthetases:

• Domain architecture conservation:

  • G. violaceus pheS maintains the core catalytic domain with HIGH and KMSKS motifs characteristic of class II aminoacyl-tRNA synthetases

  • The zinc-binding domain represents an ancient feature conserved across diverse bacterial lineages

  • Structural comparisons reveal a closer relationship to ancient aminoacyl-tRNA synthetases than previously recognized

• Active site evolution:

  • Detailed structural analysis shows that key catalytic residues are positioned almost identically to those in distantly related bacteria

  • The substrate binding pocket exhibits intermediate characteristics between highly selective and promiscuous synthetases

  • Conservation of metal coordination geometry suggests early evolutionary fixation of this feature

• Evolutionary implications:

  • G. violaceus represents one of the earliest diverging lineages of cyanobacteria

  • Its aminoacyl-tRNA synthetases retain features from the last universal common ancestor (LUCA)

  • Analysis of G. violaceus pheS structure supports the hypothesis of a simplified genetic code in early life forms

Structural phylogenetic analyses comparing G. violaceus pheS with homologs from archaea, bacteria, and eukaryotes have identified several structural elements that appear to be "molecular fossils" - features preserved from ancient protein synthesis systems. The relatively broad substrate specificity of G. violaceus pheS may reflect an ancestral state where aminoacyl-tRNA synthetases had less stringent recognition properties.

These findings have significant implications for understanding the evolution of the genetic code and translation apparatus. The structural studies suggest that promiscuity was likely an inherent feature of early aminoacyl-tRNA synthetases, with specificity evolving later as the genetic code became more precisely defined. G. violaceus pheS thus serves as an important model for studying the transition from primitive to modern protein synthesis systems.

What emerging technologies might enhance our understanding of G. violaceus pheS function and applications?

Several cutting-edge technologies hold promise for advancing our understanding of G. violaceus pheS:

• Cryo-electron microscopy (Cryo-EM):

  • Application: Visualization of conformational states during catalysis

  • Advantage: Captures dynamic processes not accessible to crystallography

  • Potential insights: Identification of transient intermediates during aminoacylation

  • Technical requirements: Sample preparation optimization for ~150 kDa heterotetramer

• Time-resolved X-ray crystallography:

  • Application: Capturing reaction intermediates at atomic resolution

  • Advantage: Direct visualization of chemical transformations

  • Potential insights: Precise positioning of substrates during adenylate formation

  • Technical challenge: Requires microcrystals and XFEL (X-ray Free Electron Laser) technology

• Single-molecule FRET:

  • Application: Real-time observation of conformational changes

  • Advantage: Reveals heterogeneity in enzyme population behavior

  • Potential insights: Correlation between domain movements and catalytic events

  • Methodology: Strategic placement of fluorophores at domain interfaces

• AlphaFold2 and advanced protein modeling:

  • Application: Prediction of mutant structures and substrate interactions

  • Advantage: Rapid screening of potential mutations without experimental validation

  • Potential insights: Identification of non-obvious residues affecting specificity

  • Methodology: Integration with molecular dynamics simulations

• High-throughput microfluidic screening:

  • Application: Rapid evaluation of thousands of enzyme variants

  • Advantage: Drastically accelerates directed evolution

  • Potential insights: Discovery of unexpected mutations enhancing desired properties

  • Technical requirements: Development of encapsulated activity assays

These emerging technologies, particularly when used in combination, promise to reveal new aspects of G. violaceus pheS function that have remained elusive with conventional approaches. For example, the integration of cryo-EM with single-molecule FRET could provide unprecedented insights into the coupling between structural dynamics and catalytic function, potentially identifying new targetable features for engineering enhanced versions of the enzyme.

How might understanding G. violaceus pheS contribute to broader questions in RNA biology and protein synthesis?

Research on G. violaceus pheS has implications for fundamental questions in RNA biology and protein synthesis:

• Evolution of genetic code fidelity:

  • G. violaceus pheS represents an evolutionary intermediate in translation fidelity

  • Comparative studies may reveal how aminoacyl-tRNA synthetase specificity evolved

  • Potential insights into the origin and expansion of the genetic code

  • Research approach: Ancestral sequence reconstruction and characterization

• RNA-protein recognition mechanisms:

  • G. violaceus pheS-tRNA interactions exemplify specific nucleic acid recognition

  • Identification of molecular determinants for specific vs. promiscuous binding

  • Implications for engineering artificial RNA-protein interfaces

  • Methodology: Comprehensive mutagenesis of both protein and RNA components

• Coordination between translation components:

  • G. violaceus translation system exhibits unique features compared to model organisms

  • Studies may reveal how aminoacyl-tRNA synthetases co-evolved with ribosomes

  • Understanding the integration of protein and RNA components in early life

  • Research approach: Reconstitution of hybrid translation systems

• Non-canonical functions in RNA metabolism:

  • Emerging evidence suggests aminoacyl-tRNA synthetases may have roles beyond aminoacylation

  • G. violaceus pheS might participate in RNA regulatory networks

  • Potential moonlighting functions in RNA metabolism or gene regulation

  • Methodology: RNA-protein interaction studies and functional genomics

• Synthetic genetic codes:

  • G. violaceus pheS engineering provides a foundation for expanding genetic codes

  • Development of orthogonal translation systems with novel chemical capabilities

  • Applications in synthetic biology and biocontainment strategies

  • Research direction: Integration with engineered ribosomes and tRNAs

By bridging ancient and modern features of translation systems, G. violaceus pheS offers a unique window into both the evolutionary past and potential future of protein synthesis. Further investigations may not only answer fundamental questions about how life's translation apparatus evolved but also provide tools for reimagining protein synthesis with expanded chemical capabilities.