Recombinant Methanococcus maripaludis Methionine--tRNA ligase (metG), partial

Basic Research Questions

• What is the function of Methanococcus maripaludis Methionine--tRNA ligase (metG) in archaeal translation?

Methanococcus maripaludis Methionine--tRNA ligase (metG), also known as methionyl-tRNA synthetase (MetRS), catalyzes the attachment of methionine to its cognate tRNA (tRNAMet) in an ATP-dependent reaction as part of the translation process. This aminoacylation reaction is crucial for protein synthesis as it ensures the correct incorporation of methionine into growing polypeptide chains.

In M. maripaludis, metG plays a particularly important role in the organism's unique metabolic adaptations as a hydrogenotrophic methanogen. The enzyme belongs to the class I aminoacyl-tRNA synthetase family, characterized by its Rossmann fold catalytic domain and HIGH/KMSKS signature sequences for ATP binding.

Methodologically, researchers studying metG function should employ aminoacylation assays that measure the enzyme's activity by quantifying the attachment of radiolabeled methionine to tRNAMet, followed by acid precipitation and scintillation counting. For kinetic analyses, steady-state measurements using varying concentrations of ATP, methionine, and tRNAMet are recommended.

• What expression systems are available for producing recombinant M. maripaludis metG?

Multiple expression systems have been developed for recombinant production of M. maripaludis metG:

For archaeal proteins like metG, the pst promoter system represents a significant advancement for expression within M. maripaludis itself. This phosphate-responsive promoter increases expression 4- to 6-fold when medium phosphate drops to growth-limiting concentrations, decoupling heterologous gene expression from growth without requiring inducers .

For heterologous expression in E. coli, the protein can be biotinylated in vivo using AviTag-BirA technology, where BirA catalyzes amide linkage between biotin and a specific lysine of the AviTag .

Methodologically, researchers should consider using the pMEV5mT vector with the pst promoter, which has demonstrated success with other archaeal proteins that are potentially toxic when overexpressed .

• How should researchers optimize storage and handling of recombinant M. maripaludis metG preparations?

Proper storage and handling of recombinant M. maripaludis metG is critical for maintaining its enzymatic activity:

The protein is typically supplied as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For optimal stability, 5-50% glycerol should be added to the final preparation .

Storage recommendations:

• Long-term storage: -20°C or -80°C

• Working aliquots: 4°C for up to one week

• Avoid repeated freeze-thaw cycles, which can significantly reduce activity

When handling the reconstituted protein:

• Briefly centrifuge vials before opening to bring contents to the bottom

• Use anaerobic techniques when possible, as M. maripaludis proteins evolved in strictly anaerobic environments

• Include reducing agents (e.g., DTT or β-mercaptoethanol) in buffers to maintain cysteine residues in reduced states

• Consider adding metal cofactors that may be essential for activity

For activity assays, researchers should verify protein purity (typically >85% by SDS-PAGE) and determine optimal buffer conditions, as archaeal enzymes often have different pH and salt optima compared to their bacterial or eukaryotic counterparts.

Advanced Research Questions

• How does the metG enzyme integrate into M. maripaludis' unique methionine metabolism, and what methodologies best study this relationship?

M. maripaludis exhibits distinctive methionine metabolism that differs from other domains of life, with metG playing a crucial role in this network. Unlike many organisms, M. maripaludis possesses a methionine biosynthesis pathway where the sulfur for methionine is acquired from inorganic sulfide rather than cysteine .

The integration of metG within this metabolism can be studied through several methodological approaches:

• Isotopic tracing experiments: Using 35S-labeled sulfide to track sulfur incorporation into methionine and subsequently into charged tRNAMet. This approach has confirmed that M. maripaludis acquires sulfur for methionine from inorganic sulfide, not cysteine .

• Metabolic flux analysis: Quantifying the flow of metabolites through the methionine biosynthesis pathway using LC-MS/MS to measure intermediates before and after metG inhibition or knockdown.

• Genetic complementation studies: Similar to the approaches used with metH (methionine synthase), researchers can study the conditional essentiality of metG in different growth conditions . This may reveal previously unknown regulatory mechanisms controlling metG expression and activity.

• Cobalamin-related interactions: Given the importance of cobalamin (vitamin B12) in M. maripaludis metabolism and its relationship with methionine biosynthesis, researchers should investigate potential regulatory links between cobalamin availability and metG activity . Methodologically, this could involve:

  • Growth experiments with varying cobalamin concentrations

  • Quantitative PCR to measure metG expression under different cobalamin conditions

  • Riboswitch analyses to identify potential cobalamin-responsive elements controlling metG expression

These approaches would provide insight into how metG functions within the broader metabolic network of M. maripaludis and its adaptation to anaerobic environments.

• What challenges exist in expressing functional metG in heterologous systems, and how can they be overcome?

Expression of functional archaeal metG in heterologous systems presents several challenges due to the unique biology of archaea:

The pst promoter system represents a significant advancement for expressing potentially toxic proteins in M. maripaludis. This system responds to inorganic phosphate concentration in the growth medium, with expression increasing 4- to 6-fold when medium phosphate drops to growth-limiting concentrations. This regulated system effectively decouples growth from heterologous gene expression without adding inducers .

For proteins similar to metG, expression under the pst promoter has shown significant improvements compared to the constitutive hmvA promoter. For example, when expressing the potentially toxic arginine methyltransferase MmpX, the pst promoter enabled high expression levels that weren't possible with constitutive promoters, presumably because toxic effects were mitigated when expression occurred between mid-log and early stationary phase .

When expressing metG in E. coli, researchers should consider:

• Using specialized strains like Rosetta or Arctic Express for archaeal proteins

• Lowering induction temperature to 18-20°C

• Adding rare tRNAs to overcome codon bias issues

• Incorporating specialized tags that can be later removed by specific proteases

• How can researchers design experiments to elucidate the structural and functional domains of M. maripaludis metG?

Designing experiments to elucidate the structural and functional domains of M. maripaludis metG requires a multi-faceted approach:

• Domain prediction and truncation analysis:

  • Perform bioinformatic analysis to predict distinct domains

  • Create a series of truncated metG constructs (N-terminal, C-terminal, and internal domain deletions)

  • Express each truncation using the pst promoter system

  • Assess aminoacylation activity of each truncation

  • This approach can identify which regions are essential for catalysis, tRNA binding, and structural integrity

• Site-directed mutagenesis strategies:

  • Target conserved residues in the predicted catalytic site (HIGH/KMSKS motifs)

  • Create alanine-scanning mutants across putative functional regions

  • Test activity of each mutant using aminoacylation assays

  • Quantify the kinetic parameters (kcat, KM) for substrate binding and catalysis

• Structural analysis methods:

  • X-ray crystallography of metG alone and in complex with substrates

  • Cryo-EM analysis for visualizing metG-tRNA interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify flexible regions and binding interfaces

  • Small-angle X-ray scattering (SAXS) for solution structure determination

• Cross-linking and interaction studies:

  • Use chemical cross-linking coupled with mass spectrometry to identify interaction interfaces

  • Apply fluorescence resonance energy transfer (FRET) to measure distances between domains during catalysis

  • Employ native mass spectrometry to determine oligomeric states

• Comparative analysis with related enzymes:

  • Compare with MetRS from Methanococcus jannaschii and other related archaea

  • Identify unique features of M. maripaludis metG that may relate to its specialized metabolism

  • Construct chimeric enzymes to test domain functionality across species

These approaches would provide comprehensive insight into the structure-function relationships of metG and help understand how it has adapted to the unique metabolic requirements of M. maripaludis.

• How does metG expression regulation integrate with the broader metabolic network of M. maripaludis?

Understanding how metG expression regulation integrates with M. maripaludis' broader metabolic network requires examining several interconnected systems:

• Phosphate regulation and energy metabolism:
  The pst promoter system, which responds to phosphate limitation, may link metG expression to the organism's energy status . This connection is significant because:

  • Phosphate availability affects ATP production

  • ATP is a direct substrate for the aminoacylation reaction

  • Under phosphate limitation, organisms must carefully regulate ATP-consuming processes

  Methodological approach: Monitor metG expression levels using RT-qPCR under varying phosphate concentrations while simultaneously measuring intracellular ATP levels using luciferase-based assays.

• Integration with methanogenesis:
  M. maripaludis generates energy primarily through methanogenesis, converting CO₂ to methane using H₂ as an electron donor . This process involves:

  • Coenzyme F420 as an electron carrier

  • Na⁺ gradient formation for ATP synthesis

  • Cobalamin-dependent enzymes

  Methodological approach: Compare metG expression and activity during growth on different substrates (H₂/CO₂ vs. formate) to determine how energy source affects aminoacyl-tRNA synthetase function.

• Sulfur metabolism connections:
  M. maripaludis acquires sulfur for methionine from inorganic sulfide rather than cysteine , suggesting a unique regulatory network that may include metG.

  Methodological approach: Track metG expression under various sulfur sources (sulfide, elemental sulfur, cysteine) using proteomics and transcriptomics to identify co-regulated genes.

• Nitrogen regulation interplay:
  The TCA cycle regulation in M. maripaludis is poorly understood but appears to involve 2-oxoglutarate levels as a nitrogen status indicator . Since protein synthesis (and thus metG function) represents a major nitrogen sink, there may be coordinated regulation.

  Methodological approach: Analyze metG expression under nitrogen limitation and excess, particularly in relation to NrpR-regulated genes.

• Data integration framework:
  To fully understand these connections, researchers should:

  • Perform RNA-seq under various nutrient limitations

  • Conduct ChIP-seq to identify transcription factors binding near metG

  • Use metabolic flux analysis to quantify changes in methionine metabolism

  • Apply systems biology approaches to model how metG regulation responds to environmental changes

This integrated approach would reveal how metG expression is coordinated with the organism's broader metabolic demands and adaptations to its ecological niche.

• What novel methodologies can be developed to study the role of metG in archaeal translation fidelity?

Developing novel methodologies to study metG's role in archaeal translation fidelity requires innovative approaches addressing the unique aspects of archaeal biology:

• In vivo misincorporation detection systems:

  • Engineer a reporter system containing strategically placed methionine codons

  • Mutate key residues in metG that may affect tRNA recognition

  • Quantify mistranslation events using mass spectrometry

  • Develop fluorescent reporters that activate upon methionine misincorporation

• Ribosome profiling tailored for archaea:

  • Adapt ribosome profiling techniques for M. maripaludis growth conditions

  • Compare translational efficiency and pausing at methionine codons in wild-type vs. metG mutants

  • Analyze how different environmental conditions affect translation fidelity

• tRNA modification analysis platform:

  • Develop a high-throughput method to analyze modifications on archaeal tRNAMet

  • Investigate how these modifications affect metG recognition and charging

  • Create a comprehensive database of M. maripaludis tRNA modifications

• Real-time single-molecule charging assays:

  • Apply FRET-based approaches to monitor metG-tRNA interactions in real-time

  • Develop a microfluidic platform for single-molecule studies under anaerobic conditions

  • Integrate with total internal reflection fluorescence (TIRF) microscopy

• Genetic suppressor screens:

  • Develop a selection system where survival depends on accurate methionine incorporation

  • Identify suppressors that restore function to compromised metG variants

  • Map genetic interactions between metG and other translation components

• Isotopic pulse-chase in archaea:

  • Adapt pulse-chase methods for archaeal culture conditions

  • Use 35S-methionine to track charged tRNA pools and incorporation rates

  • Develop computational models to interpret kinetic data

• Archaeal cell-free translation systems:

  • Engineer a complete M. maripaludis-derived cell-free translation system

  • Systematically vary metG concentrations and mutant forms

  • Measure translation rates and error frequencies with defined templates

• Cryo-electron tomography of archaeal polysomes:

  • Visualize translating ribosomes in archaeal cells

  • Track metG association with ribosomes under different conditions

  • Compare wild-type and metG variant effects on polysome formation