Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ0184 (MJ0184)

What is Methanocaldococcus jannaschii uncharacterized protein MJ0184?

MJ0184 is a relatively small protein (77 amino acids) found in the thermophilic archaeon Methanocaldococcus jannaschii, with the UniProt ID Q57643. The protein has the amino acid sequence MNIMITKQFDRHLKYYTTIVKVFANGIILITAYYLVFELPVGYLIGLYIIMFVVWLLVSMFFLGRLLDFMAKMDLKK, suggesting potential membrane association based on its hydrophobic regions. The protein was identified during the complete genome sequencing of M. jannaschii but remains functionally uncharacterized, making it an important target for fundamental research into archaeal biology .

How is recombinant MJ0184 typically produced for research purposes?

Recombinant MJ0184 is commonly produced using E. coli expression systems. The gene encoding MJ0184 is cloned into an expression vector containing an N-terminal His-tag, which facilitates purification. The protein is expressed in E. coli strains optimized for heterologous protein expression and subsequently purified using affinity chromatography, typically with Ni-NTA resin. According to product specifications, the recombinant protein is provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For research applications, the protein must be reconstituted in appropriate buffer solutions, with recommendations to avoid repeated freeze-thaw cycles and to store working aliquots at 4°C for up to one week .

What evolutionary significance does MJ0184 potentially have in archaea?

The evolutionary significance of MJ0184 lies in its potential to provide insights into archaeal-specific biology. M. jannaschii was the first archaeon to have its complete genome sequenced, which identified many genes unique to the archaeal domain of life . As an uncharacterized protein, MJ0184 may represent novel biological functions specific to methanogenic archaea or adaptations to extreme environments. Comparative genomic analyses across archaeal species could reveal whether MJ0184 represents a conserved archaeal protein or a specialized adaptation in Methanocaldococcus species. Such evolutionary analyses would contribute to our understanding of archaeal diversity and the molecular mechanisms underlying their unique ecological niches.

What expression systems optimize the production of functional recombinant MJ0184?

For optimal expression of recombinant MJ0184, researchers should consider:

• Vector selection: Vectors with strong, inducible promoters (T7, tac) with an N-terminal His-tag have proven successful .

• Host strain optimization: E. coli BL21(DE3) or Rosetta strains can address codon bias issues between archaeal and bacterial genes.

• Temperature modulation: Despite M. jannaschii being thermophilic, expression in E. coli should be conducted at lower temperatures (16-25°C) after induction to improve proper folding.

• Induction protocol: Use IPTG at concentrations of 0.1-0.5 mM, with induction at mid-log phase (OD600 ~0.6-0.8).

• Growth media: LB media supplemented with glycerol can improve protein yield and stability.

Expression should be monitored via SDS-PAGE analysis at different time points post-induction to determine the optimal harvest time .

What are the recommended protocols for reconstitution and storage of MJ0184?

For proper reconstitution and storage of MJ0184:

• Initial reconstitution: Briefly centrifuge the vial of lyophilized protein before opening. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage buffer optimization: Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage .

• Aliquoting: Prepare small working aliquots to avoid repeated freeze-thaw cycles.

• Storage conditions: Store working aliquots at 4°C for up to one week. For long-term storage, keep at -20°C or -80°C .

• Thawing protocol: Thaw frozen aliquots quickly at room temperature, followed by brief centrifugation before use.

The storage buffer (Tris/PBS-based, 6% Trehalose, pH 8.0) is designed to maintain protein stability . Researchers should verify protein integrity after reconstitution using methods like gel electrophoresis or activity assays.

What analytical techniques are most suitable for studying the structural properties of MJ0184?

Due to the small size (77 amino acids) and potential membrane association of MJ0184, complementary analytical techniques are recommended:

• Spectroscopic methods:

  • Circular dichroism (CD) for secondary structure determination

  • Fluorescence spectroscopy to probe tertiary structure and ligand binding

  • Nuclear magnetic resonance (NMR) for solution structure determination

• Crystallographic approaches:

  • X-ray crystallography with appropriate detergents or lipidic cubic phase methods

  • Electron crystallography for membrane-embedded forms

• Biophysical characterization:

  • Size-exclusion chromatography to assess oligomeric state

  • Analytical ultracentrifugation for homogeneity and assembly analysis

  • Differential scanning calorimetry for thermal stability assessment

• Computational approaches:

  • Molecular dynamics simulations in membrane environments

  • Homology modeling and ab initio structure prediction

• Membrane interaction studies:

  • Lipid binding assays using archaeal-like lipid compositions

  • Fluorescence anisotropy measurements in reconstituted systems

These approaches should consider the thermophilic nature of M. jannaschii and potentially include analyses at elevated temperatures .

How might the high-resolution structure of MJ0184 be determined?

Determining the high-resolution structure of MJ0184 presents unique challenges due to its small size and potential membrane association. A multi-faceted approach is recommended:

• Solution NMR spectroscopy:

  • Particularly suitable for small proteins like MJ0184 (77 amino acids)

  • Requires isotope labeling (15N, 13C) of recombinant protein

  • Can be performed in detergent micelles or lipid nanodiscs

  • Enables study of dynamics and conformational changes

• X-ray crystallography:

  • Requires high-purity protein preparations (>95%)

  • May need fusion partners (T4 lysozyme, BRIL) to aid crystallization

  • Screening various detergents and precipitants is critical

  • Lipidic cubic phase methods may be suitable for membrane proteins

• Cryo-electron microscopy:

  • Challenging for small proteins, but feasible with recent advances

  • May require incorporation into larger complexes or nanodiscs

  • Provides native-like structural information

• Integrative approaches:

  • Combining low-resolution experimental data with computational modeling

  • Molecular dynamics simulations to refine structures in membrane environments

  • Cross-validation using orthogonal structural methods

For each approach, protein stability in the experimental conditions must be verified, and the thermophilic nature of M. jannaschii should be considered when interpreting structural data .

What computational approaches can predict potential functions of MJ0184?

Given the uncharacterized nature of MJ0184, computational approaches offer valuable insights into potential functions:

• Sequence-based analysis:

  • Profile-based searches (PSI-BLAST, HMMer) against diverse databases

  • Identification of conserved motifs or domains

  • Remote homology detection using sensitive methods like HHpred

• Structure-based prediction:

  • Protein threading against structural databases

  • Binding site prediction and molecular docking

  • Electrostatic surface analysis for interaction interfaces

• Genomic context analysis:

  • Examination of neighboring genes and operonic structures

  • Phylogenetic profiling to identify co-evolving genes

  • Gene fusion events that might suggest functional relationships

• Network-based approaches:

  • Protein-protein interaction prediction

  • Metabolic network analysis for potential pathway involvement

  • Co-expression network integration

• Machine learning methods:

  • Function prediction using integrated features

  • Deep learning approaches trained on multi-omics data

Each prediction should be evaluated based on statistical significance and validated experimentally where possible. The unique biology of archaea should be considered when interpreting computational predictions .

How does MJ0184 potentially contribute to the thermostability of M. jannaschii?

As a protein from a hyperthermophilic archaeon that grows optimally at around 85°C, MJ0184 likely incorporates adaptations for thermostability:

• Amino acid composition analysis:

  • Increased proportion of charged residues that can form stabilizing salt bridges

  • Higher hydrophobicity in the core regions

  • Reduced occurrence of thermolabile residues (Asn, Gln, Cys)

• Structural features:

  • Predicted compact folding with minimal surface loops

  • Potential disulfide bonds or metal coordination sites

  • Increased secondary structure elements for rigidity

• Membrane interaction dynamics:

  • Adaptation to the unique archaeal membrane lipids (ether-linked isoprenoids)

  • Potential role in maintaining membrane integrity at high temperatures

  • Specialized lipid-protein interactions

• Comparative analysis:

  • Comparison with homologs from mesophilic organisms

  • Identification of thermostability-conferring residues

  • Evolutionary analysis of thermoadaptation mechanisms

Experimental validation could include thermal stability assays, comparative structural analysis, and mutagenesis studies targeting predicted thermostabilizing features .

What experimental approaches can elucidate the function of MJ0184?

To systematically investigate the function of this uncharacterized protein, a multi-level experimental approach is recommended:

• Localization studies:

  • Immunolocalization in native M. jannaschii (if antibodies available)

  • Fluorescent protein fusions in heterologous systems

  • Subcellular fractionation and membrane association analysis

• Interaction partner identification:

  • Pull-down assays using His-tagged MJ0184

  • Crosslinking followed by mass spectrometry

  • Yeast two-hybrid or bacterial two-hybrid screening

• Phenotypic analysis:

  • Gene knockout/knockdown in closely related archaeal species

  • Overexpression phenotypes in heterologous hosts

  • Complementation studies in model organisms

• Biochemical characterization:

  • Systematic enzymatic activity screening

  • Binding assays with potential ligands

  • Membrane perturbation or transport assays

• Stress response correlation:

  • Expression analysis under various stress conditions

  • Protein level analysis during different growth phases

  • Response to specific environmental challenges

These approaches should be integrated to build a comprehensive functional model, with particular attention to the thermophilic and anaerobic lifestyle of M. jannaschii .

How can researchers assess potential protein-protein interactions involving MJ0184?

Investigating protein-protein interactions for MJ0184 requires approaches adapted to archaeal membrane proteins:

• In vitro interaction studies:

  • Surface plasmon resonance with immobilized MJ0184

  • Microscale thermophoresis for solution-based interaction analysis

  • Isothermal titration calorimetry for thermodynamic characterization

  • Pull-down assays with His-tagged MJ0184

• In vivo/ex vivo approaches:

  • Co-immunoprecipitation from M. jannaschii cell extracts

  • Bacterial/archaeal two-hybrid systems

  • Proximity labeling methods (BioID, APEX) in heterologous systems

  • Fluorescence resonance energy transfer (FRET) for direct interaction visualization

• Computational prediction validation:

  • Testing interactions predicted by genomic context analysis

  • Validating co-evolution-based interaction predictions

  • Structure-based interaction predictions followed by mutagenesis

• High-throughput screening:

  • Protein microarray analysis

  • Phage display screening

  • Ribosome display for peptide interaction partners

For each method, appropriate controls must be included, and the unique properties of archaeal proteins (thermostability, different codon usage) should be considered when designing experiments .

What considerations are important when designing site-directed mutagenesis studies for MJ0184?

Site-directed mutagenesis of MJ0184 requires careful planning to maximize informational output:

• Target residue selection strategy:

  • Conserved residues identified through multiple sequence alignment

  • Charged or polar residues in hydrophobic regions

  • Residues predicted to be at interaction interfaces

  • Potential catalytic residues based on structural modeling

• Mutation design principles:

  • Conservative substitutions to assess structural roles

  • Charge reversals to probe electrostatic interactions

  • Alanine scanning for systematic functional mapping

  • Introduction of reporter groups (Cys for labeling, Trp for fluorescence)

• Expression and functional analysis:

  • Verification of proper folding after mutation

  • Thermal stability comparison with wild-type protein

  • Functional assays (if known function) or interaction studies

  • Structural analysis of significant mutants

• Interpretation framework:

  • Statistical analysis of multiple mutants

  • Structure-function correlation

  • Evolutionary context of critical residues

  • Integration with other experimental data

Special consideration should be given to the thermophilic nature of M. jannaschii, as mutations may have different effects at high temperatures compared to standard laboratory conditions .

How should researchers interpret inconsistent experimental results when studying MJ0184?

When faced with inconsistent experimental results for MJ0184, researchers should implement a systematic troubleshooting approach:

• Sample quality assessment:

  • Verify protein purity using multiple methods (SDS-PAGE, mass spectrometry)

  • Check for proper folding using spectroscopic techniques

  • Assess aggregation state using size-exclusion chromatography

  • Confirm identity via peptide mapping or sequencing

• Experimental condition evaluation:

  • Consider temperature effects (M. jannaschii is thermophilic)

  • Evaluate buffer composition and pH effects

  • Assess the impact of reducing agents and metal ions

  • Examine detergent effects for membrane-associated studies

• Methodological cross-validation:

  • Apply orthogonal techniques to verify results

  • Introduce positive and negative controls specific to each assay

  • Perform spike-in experiments to assess matrix effects

  • Blind analysis to reduce experimenter bias

• Data integration framework:

  • Weigh evidence based on methodological robustness

  • Consider biological plausibility of each result

  • Develop multiple working hypotheses consistent with subsets of data

  • Design discriminating experiments to resolve contradictions

Inconsistencies may reveal important biological properties, such as temperature-dependent functions or context-specific interactions, particularly relevant for thermophilic archaeal proteins .

What statistical approaches are appropriate for analyzing high-throughput data related to MJ0184?

High-throughput experiments investigating MJ0184 require robust statistical analysis:

• Differential expression analysis:

  • Appropriate normalization for platform-specific biases

  • Multiple testing correction (FDR, Bonferroni) for large datasets

  • Power analysis to ensure sufficient sample size

  • Sensitivity analysis to assess robustness of findings

• Interaction network analysis:

  • Enrichment analysis for functional categories

  • Topological analysis of network properties

  • Module detection algorithms to identify functional clusters

  • Permutation tests to assess significance of network features

• Structure-function relationships:

  • Multiple sequence alignment statistical analysis

  • Coevolution analysis using mutual information or direct coupling analysis

  • Regression models for structure-activity relationships

  • Machine learning approaches for integrating multiple data types

• Experimental design considerations:

  • Factorial designs to assess interaction effects

  • Time-series analysis for dynamic processes

  • Dose-response modeling for binding or activity studies

  • Bayesian approaches for incorporating prior knowledge

Particular attention should be paid to the unique properties of archaeal systems when interpreting statistical results, as standard assumptions based on bacterial or eukaryotic systems may not apply .

How can researchers integrate structural and functional data to develop hypotheses about MJ0184?

Integrating structural and functional data requires a multi-dimensional approach:

• Structure-guided functional mapping:

  • Identify potential binding pockets or catalytic sites

  • Map conservation patterns onto structural models

  • Analyze electrostatic and hydrophobic surface properties

  • Predict membrane interaction interfaces

• Data visualization and integration:

  • Structural visualization with mapped functional data

  • Network representations of interaction data

  • Heat maps for condition-dependent activities

  • Principal component analysis for multi-parameter data reduction

• Computational hypothesis generation:

  • Molecular docking with potential ligands

  • Molecular dynamics simulations under varying conditions

  • Virtual screening approaches for function prediction

  • Machine learning integration of heterogeneous data types

• Iterative hypothesis testing framework:

  • Develop multiple competing hypotheses consistent with data

  • Design experiments specifically to discriminate between hypotheses

  • Prioritize experiments based on information gain potential

  • Update structural and functional models based on new data

This integrated approach can lead to testable hypotheses about the role of MJ0184 in archaeal biology, particularly in the context of membrane processes and thermoadaptation .