Recombinant Methanocaldococcus jannaschii UPF0333 protein MJ1469 (MJ1469)

What is Methanocaldococcus jannaschii and why is it significant in research?

Methanocaldococcus jannaschii is a hyperthermophilic, strictly hydrogenotrophic, methanogenic archaeon that was isolated from a deep-sea hydrothermal vent. It represents an evolutionary ancient lineage and has significant importance in understanding extremophile biology and early evolution of life . M. jannaschii grows optimally at high temperatures and requires sulfide for growth, making it an important model organism for studying adaptations to extreme environments. Its complete genome was one of the first archaeal genomes to be sequenced, providing valuable insights into the unique biochemistry and molecular biology of Archaea. The study of its proteins, including uncharacterized ones like MJ1469, contributes to our understanding of archaeal metabolism and adaptation strategies.

What expression systems are typically used for recombinant production of M. jannaschii proteins?

For more authentic expression, researchers have developed genetic systems for M. jannaschii itself, which allow for homologous expression of proteins. For instance, a genetic system has been developed that uses mevinolin resistance as a selectable marker and homologous recombination to integrate modified genes into the M. jannaschii chromosome . This approach was successfully used to express FprA with affinity tags, demonstrating that M. jannaschii can be engineered for homologous protein expression .

What are the optimal conditions for handling and storing recombinant MJ1469 protein?

Due to the thermophilic origin of MJ1469, standard handling protocols for mesophilic proteins may need modification. The protein is likely to exhibit high thermal stability, which can be beneficial for long-term storage but may require special considerations during purification and activity assays.

For storage, recombinant MJ1469 protein should generally be kept at -80°C in buffer conditions that maintain stability. Since the protein originates from a hyperthermophile, addition of glycerol (typically 10-20%) can help prevent denaturation during freeze-thaw cycles. Buffers containing reducing agents may help maintain any potential disulfide bonds in their native state.

What purification strategies are most effective for recombinant His-tagged MJ1469?

Purification of His-tagged MJ1469 typically involves immobilized metal affinity chromatography (IMAC) using Ni-NTA or cobalt-based resins. Based on information about similarly expressed M. jannaschii proteins, the following protocol is recommended:

• Cell lysis under native or denaturing conditions, depending on protein solubility

• IMAC purification using a linear or step imidazole gradient (typically 20-250 mM)

• Size exclusion chromatography to remove aggregates and achieve higher purity

• Optional: ion exchange chromatography to remove any remaining contaminants

For proteins from hyperthermophiles like M. jannaschii, a heat treatment step (70-80°C for 15-30 minutes) before chromatography can be highly effective, as it denatures most E. coli proteins while leaving the thermostable target protein intact. This heat treatment can significantly improve purity and simplify subsequent purification steps.

Similar purification approaches have been successfully used for other M. jannaschii proteins. For example, the Mj-FprA protein from M. jannaschii BM31 strain was purified using a Streptactin XT superflow column with 10 mM D-biotin elution, yielding 0.26 mg purified protein per liter of culture .

What analytical methods are recommended for assessing the structural integrity of purified MJ1469?

To ensure the structural integrity of purified MJ1469, a combination of analytical methods is recommended:

• SDS-PAGE: To assess purity and approximate molecular weight (expected around 154 amino acids plus the tag)

• Western blot: Using anti-His antibodies to confirm the presence of the His-tagged protein

• Mass spectrometry: For accurate molecular weight determination and peptide mapping (similar to the approach used for Mj-FprA, which identified 41 peptides covering 55% of the protein's primary structure)

• Circular dichroism (CD): To evaluate secondary structure elements and thermal stability

• Dynamic light scattering (DLS): To assess homogeneity and detect aggregation

• Differential scanning calorimetry (DSC): To determine thermal transition temperatures

For MJ1469 specifically, comparing the thermal stability profile with that of mesophilic homologs (if available) can provide insights into its thermoadaptation mechanisms.

What approaches are recommended for determining the function of uncharacterized proteins like MJ1469?

For uncharacterized proteins like MJ1469, a multi-faceted approach to functional determination is recommended:

• Bioinformatic analysis: Use sequence similarity, structural prediction, and phylogenetic analysis to identify potential functions. Compare with characterized proteins from related archaeal species.

• Structural studies: X-ray crystallography or cryo-EM to determine the three-dimensional structure, which may reveal functional domains or active sites.

• Protein-protein interaction studies: Pull-down assays, yeast two-hybrid, or co-immunoprecipitation to identify interaction partners that might suggest functional pathways.

• Genetic approaches: Gene knockout or knockdown in M. jannaschii using the established genetic system , followed by phenotypic analysis. The genetic system developed for M. jannaschii allows for homologous recombination and has been successfully used to manipulate genes in this organism.

• Enzymatic activity screening: Systematic testing of potential substrates based on structural similarity to known enzymes or predicted functions.

• Transcriptomic analysis: Identify conditions under which MJ1469 is upregulated, which may provide clues about its function. Previous global transcriptional analyses have been conducted for M. jannaschii and could serve as a reference .

How can I test if MJ1469 is involved in sulfur metabolism or oxidative stress responses?

Given that M. jannaschii requires sulfide for growth and possesses unique adaptations for sulfur metabolism , testing MJ1469's potential involvement in these pathways is reasonable:

• Enzyme activity assays: Test if MJ1469 exhibits any activity related to sulfur metabolism, such as sulfite reduction or sulfide oxidation. For comparison, the F420-dependent sulfite reductase (Fsr) in M. jannaschii has been characterized with a specific activity of 0.57 μmol sulfite reduced min⁻¹ mg⁻¹ protein .

• Substrate binding assays: Use isothermal titration calorimetry or surface plasmon resonance to test binding of sulfur compounds.

• Gene expression analysis: Determine if expression of MJ1469 changes in response to different sulfur sources or oxidative stress conditions.

• Complementation studies: Test if MJ1469 can complement mutants of related organisms defective in sulfur metabolism genes.

• Co-expression analysis: Identify if MJ1469 is co-expressed with known sulfur metabolism genes such as the coenzyme F420-dependent sulfite reductase (Fsr), which allows M. jannaschii to grow with sulfite as the sole sulfur source .

For oxidative stress response testing, compare the activity to known oxidative stress proteins like FprA from M. jannaschii, which has F420H2 oxidase activity and reduces O2 to H2O .

What methods are available for studying the potential interaction of MJ1469 with coenzyme F420?

Coenzyme F420 is a key electron carrier in methanogens, involved in various redox reactions . To investigate potential interactions between MJ1469 and F420:

• Spectroscopic assays: Monitor changes in F420 fluorescence or absorbance in the presence of MJ1469 and potential substrates. F420 has characteristic absorbance at 420 nm that changes upon reduction.

• Enzyme activity assays: Test if MJ1469 exhibits F420-dependent enzymatic activities, similar to those described for F420-dependent sulfite reductase (Fsr) or F420H2 oxidase (FprA) .

• Binding studies: Use fluorescence quenching or isothermal titration calorimetry to directly measure binding between MJ1469 and F420.

• Structural studies: Co-crystallize MJ1469 with F420 to determine binding sites and interaction modes.

• Comparative analysis: Compare with known F420-dependent enzymes from M. jannaschii, such as FprA which shows high activity (2,100 μmole/min/mg) with oxygen and F420H2 , or Fsr which reduces sulfite to sulfide using H2F420 as electron source (Km: sulfite, 12 μM; H2F420, 21 μM) .

What are the challenges in developing a reliable in vitro activity assay for MJ1469?

Developing reliable activity assays for uncharacterized proteins from hyperthermophiles presents several challenges:

• Temperature requirements: Optimal enzyme activity for M. jannaschii proteins typically occurs at elevated temperatures (70-85°C). Maintaining assay components stability at these temperatures can be challenging.

• Unknown substrates: Without functional annotation, identifying potential substrates requires systematic screening approaches.

• Cofactor requirements: M. jannaschii enzymes often require unique cofactors such as coenzyme F420, which may not be commercially available and might need to be purified from methanogen cultures.

• Anaerobic conditions: As M. jannaschii is a strict anaerobe, the protein may require oxygen-free conditions for proper activity, necessitating specialized equipment for anaerobic work.

• Protein stability issues: Recombinantly expressed archaeal proteins may not fold correctly in E. coli, potentially requiring refolding or expression optimizations.

For developing a MJ1469 assay, researchers should consider multiple detection methods (spectrophotometric, fluorometric, coupled enzyme assays) and test various environmental conditions (pH, salt concentration, temperature) based on M. jannaschii's native environment.

How can I establish structure-function relationships for MJ1469?

To establish structure-function relationships for MJ1469, a comprehensive approach combining structural analysis with functional studies is recommended:

• Structural determination: Obtain high-resolution structures through X-ray crystallography or cryo-EM. If these are challenging, homology modeling based on structurally characterized members of the UPF0333 family can provide initial insights.

• Domain analysis: Identify conserved domains and predict functional sites using bioinformatic tools and structural comparison with characterized proteins.

• Site-directed mutagenesis: Based on structural insights, create targeted mutations of predicted functional residues and assess their impact on any identified activities.

• Truncation analysis: Generate truncated versions of MJ1469 to identify minimal functional domains.

• Thermal stability analysis: Compare wild-type and mutant proteins using differential scanning fluorimetry to correlate structural features with thermostability.

• Molecular dynamics simulations: Simulate protein behavior at different temperatures to understand thermoadaptation mechanisms and flexibility of potential active sites.

This approach has been successful with other M. jannaschii proteins. For example, structural and functional analysis of F420-dependent enzymes like FprA revealed domains responsible for F420 binding and catalytic activity .

What considerations are important when comparing MJ1469 with homologs from mesophilic organisms?

When comparing MJ1469 with mesophilic homologs, several important considerations should be taken into account:

• Thermoadaptation features: Hyperthermophilic proteins typically have increased surface charge, more ionic interactions, shorter loops, and higher hydrophobic core packing. These features should be analyzed to understand thermostability mechanisms.

• Kinetic parameters: Enzymes from hyperthermophiles often display lower activity at mesophilic temperatures but higher stability. Comparative kinetic analysis should be performed at both standard and elevated temperatures.

• Substrate specificity: Substrate preferences may differ between mesophilic and thermophilic homologs, requiring broader substrate screening.

• Cofactor requirements: Binding affinity for cofactors like F420 may differ between homologs, affecting activity measurements.

• Evolutionary context: Phylogenetic analysis can help determine if MJ1469 represents an ancestral form or a specialized adaptation.

• Protein flexibility: Thermophilic proteins often maintain similar flexibility at their physiological temperatures compared to mesophilic homologs at lower temperatures. This "corresponding states" concept should be considered when comparing functional properties.

Similar comparative approaches have been used for other M. jannaschii proteins, such as FprA, which showed substantially higher specific activity (2,100 μmole/min/mg) compared to homologs from mesophilic Methanobrevibacter arboriphilus (55 μmole/min/mg) and thermophilic Methanothermobacter marburgensis (110 μmole/min/mg) .

What strategies can address poor solubility of recombinant MJ1469 expressed in E. coli?

Poor solubility is a common challenge when expressing archaeal proteins in E. coli. For MJ1469, consider the following strategies:

• Optimization of expression conditions:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Use specialized E. coli strains designed for expressing difficult proteins (e.g., ArcticExpress, Rosetta)

• Fusion tags to enhance solubility:

  • MBP (maltose-binding protein) tag in addition to the His tag

  • SUMO or thioredoxin fusion systems

  • NusA tag for highly insoluble proteins

• Co-expression with chaperones:

  • GroEL/GroES system

  • DnaK/DnaJ/GrpE system

  • Specialized archaeal chaperones if available

• Buffer optimization during purification:

  • Include stabilizing additives (glycerol, arginine, trehalose)

  • Test different pH conditions and salt concentrations

  • Add reducing agents if cysteine residues are present

• Refolding from inclusion bodies:

  • Solubilize inclusion bodies with urea or guanidinium chloride

  • Perform gradual dialysis or on-column refolding

  • Add molecular chaperones during refolding

These approaches have been successful with other challenging archaeal proteins and could be adapted for MJ1469 based on its specific properties.

How can I validate that recombinantly produced MJ1469 is properly folded and functional?

Validating proper folding and functionality of recombinant MJ1469 requires multiple approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

  • Fluorescence spectroscopy to assess tertiary structure through intrinsic tryptophan fluorescence

  • Size exclusion chromatography to confirm monomeric state or expected oligomerization

• Thermal stability analysis:

  • Differential scanning fluorimetry to determine melting temperature

  • CD thermal melting curves

  • Activity retention after heat treatment (expected high thermostability for M. jannaschii proteins)

• Functional validation:

  • Activity assays if function is known or predicted

  • Ligand binding studies using isothermal titration calorimetry or microscale thermophoresis

  • Comparison with native protein if available

• Conformational dynamics:

  • Hydrogen-deuterium exchange mass spectrometry to assess protein dynamics

  • Limited proteolysis to identify flexible regions

  • Comparison with in silico predictions of structure

For a protein from a hyperthermophile like M. jannaschii, demonstrating thermostability (e.g., resistance to denaturation at 70-80°C) would provide strong evidence of proper folding, as incorrectly folded proteins typically lose this characteristic thermostability.

What are the key considerations for designing experiments to study protein-protein interactions involving MJ1469?

When investigating protein-protein interactions involving MJ1469, several important considerations should guide experimental design:

• Temperature considerations:

  • Interactions may only occur at temperatures relevant to M. jannaschii's growth (65-85°C)

  • Experimental methods must be adapted for high-temperature conditions

  • Consider performing experiments at multiple temperatures to compare interaction dynamics

• Partner identification strategies:

  • Pull-down assays using tagged MJ1469 with M. jannaschii cell lysates

  • Bacterial or yeast two-hybrid systems modified for thermophilic proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • In silico prediction of interaction partners based on genomic context or co-expression data

• Interaction characterization methods:

  • Surface plasmon resonance (SPR) adapted for high temperature

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Microscale thermophoresis for interaction studies in complex solutions

  • Förster resonance energy transfer (FRET) for in vitro or in vivo interaction studies

• Validation approaches:

  • Mutagenesis of predicted interaction interfaces

  • Competition assays with peptides derived from interaction interfaces

  • Co-crystallization of MJ1469 with interaction partners

  • Functional assays to determine biological relevance of interactions

• Special considerations for archaeal proteins:

  • Post-translational modifications present in archaea but not in recombinant systems

  • Influence of extreme conditions (temperature, pH, salt) on interaction dynamics

  • Potential involvement of archaeal-specific cofactors in mediating interactions

Understanding protein-protein interactions is crucial for elucidating the biological function of uncharacterized proteins like MJ1469 within the context of M. jannaschii's cellular networks.