Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ0904 (MJ0904)

What is Methanocaldococcus jannaschii protein MJ0904 and why study it?

MJ0904 is an uncharacterized protein from the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii. The interest in studying this protein stems from M. jannaschii's unique metabolic pathways that differ significantly from bacterial and eukaryotic systems. Similar to the investigation of MJ1447 (described in search result ), studying uncharacterized proteins like MJ0904 can provide insights into novel biochemical pathways specific to archaeal metabolism. The protein may be involved in unique stress response mechanisms that allow M. jannaschii to thrive in extreme environments, including high temperatures and pressures found in deep-sea hydrothermal vents.

Methodologically, researchers typically begin characterization by conducting bioinformatic analyses of the protein sequence to identify potential functional domains, followed by recombinant expression and preliminary biochemical characterization. Comparative genomic approaches with other archaea can provide initial clues about potential functions based on genomic context and conservation patterns.

What expression systems are most effective for recombinant MJ0904 production?

For archaeal proteins like MJ0904, several expression systems have proven effective, with each offering distinct advantages:

When expressing archaeal proteins like MJ0904, researchers should consider using approaches similar to those employed for other M. jannaschii proteins. The gene can be amplified by PCR from genomic DNA using specific primers that introduce appropriate restriction sites, as demonstrated in the protocol for MJ1447 gene amplification . The amplified product should be purified, digested with appropriate restriction enzymes, and ligated into a suitable expression vector, followed by transformation into the chosen expression host.

What purification strategies are most suitable for MJ0904?

The purification strategy for MJ0904 should be designed based on the protein's predicted properties and the addition of affinity tags during recombinant expression:

• Initial clarification: Cell lysis using sonication on ice with multiple rounds, similar to the protocol described in search result , in a buffer containing 6 M urea, 2 M thiourea, and 50 mM ammonium bicarbonate with protease inhibitors.

• Heat treatment: Leveraging the thermostability of archaeal proteins, crude extracts can be heated (70-80°C for 15-20 minutes) to precipitate most host proteins while MJ0904 remains soluble.

• Chromatography: A typical purification scheme would include:

  • Affinity chromatography (if His-tagged)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Quality control: Assess purity by SDS-PAGE and verify protein identity through mass spectrometry using approaches similar to those described for cuproproteome analysis .

For proteins that prove difficult to purify in native conditions, denaturing purification followed by refolding can be attempted, though this often results in lower yields of active protein.

How can I verify the structural integrity of recombinant MJ0904?

Verifying the structural integrity of recombinant MJ0904 is essential to ensure that functional studies are conducted with properly folded protein. Multiple complementary approaches are recommended:

• Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure content (α-helices, β-sheets). For archaeal proteins, comparing the CD spectrum at different temperatures (25°C vs. 80°C) can indicate thermal stability.

• Fluorescence Spectroscopy: Intrinsic tryptophan fluorescence can indicate tertiary structure integrity. Native folding typically results in blue-shifted emission maxima compared to denatured states.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): Determines the oligomeric state and homogeneity of the purified protein.

• Thermal Shift Assay: Measures protein stability through the change in fluorescence of a hydrophobic dye as the protein unfolds with increasing temperature. As MJ0904 comes from a hyperthermophile, it should display high thermal stability (Tm likely >80°C).

• Limited Proteolysis: Properly folded proteins typically show resistance to proteolytic digestion compared to misfolded variants. Results can be analyzed by SDS-PAGE or mass spectrometry using methods similar to those described in the protein analysis protocols .

What bioinformatic approaches can predict MJ0904 function?

Predicting the function of uncharacterized proteins like MJ0904 requires a multi-faceted bioinformatic approach:

• Sequence-Based Analysis:

  • PSI-BLAST searches against protein databases to identify remote homologs

  • Multiple sequence alignment with detected homologs

  • Motif searching using PROSITE, PFAM, and other domain databases

• Structural Prediction:

  • Secondary structure prediction (PSIPRED, JPred)

  • Tertiary structure prediction using AlphaFold2 or RoseTTAFold

  • Ligand binding site prediction (COACH, COFACTOR)

• Genomic Context Analysis:

  • Examination of neighboring genes in the M. jannaschii genome

  • Identification of conserved gene clusters across archaeal species

  • Protein-protein interaction network prediction

• Phylogenetic Analysis:

  • Construction of phylogenetic trees with homologs

  • Analysis of evolutionary conservation patterns

  • Investigation of selective pressure (dN/dS ratio)

When analyzing results, researchers should integrate multiple lines of evidence rather than relying on any single prediction. For instance, if structural prediction indicates a potential binding site for a specific cofactor, this hypothesis should be tested experimentally through binding assays or activity tests with that cofactor.

How can I design experiments to determine the biochemical function of MJ0904?

Determining the biochemical function of an uncharacterized protein requires a systematic experimental approach based on preliminary bioinformatic analyses:

• Substrate Screening:

  • Develop a panel of potential substrates based on bioinformatic predictions

  • Test enzymatic activity using spectrophotometric, fluorometric, or coupled enzyme assays

  • Conduct metabolite profiling using mass spectrometry to identify reaction products

• Cofactor Requirements:

  • Systematically test common cofactors (metals, nucleotides, vitamins)

  • Use ICP-MS to identify bound metals in the purified protein

  • Perform activity restoration experiments with purified protein after chelation treatment

• Structure-Function Analysis:

  • Identify conserved residues through multiple sequence alignment

  • Generate point mutations of these residues

  • Assess the impact on activity, stability, and binding properties

• Protein-Protein Interaction Studies:

  • Pull-down assays with M. jannaschii lysate

  • Bacterial two-hybrid or yeast two-hybrid screening

  • Crosslinking mass spectrometry to identify interaction partners

Data from these experiments should be presented in clear, informative tables following the principles outlined in search result , with careful attention to statistical analysis and proper controls. The experimental plan should incorporate both positive controls (known enzymes from related pathways) and negative controls (reaction mixtures lacking MJ0904) to validate results.

What proteomics approaches are most effective for studying MJ0904 interactions?

For studying protein interactions of MJ0904, several complementary proteomics approaches can be employed, similar to those used in the cuproproteome study :

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged MJ0904 (His-tag or FLAG-tag)

  • Perform pull-down experiments under native conditions

  • Identify co-purifying proteins by LC-MS/MS

  • Use label-free quantitative proteomics to distinguish specific from non-specific interactions

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers (BS3, DSS, or formaldehyde) to stabilize transient interactions

  • Digest crosslinked samples following protocols similar to those described in search result

  • Analyze by specialized XL-MS workflows to identify crosslinked peptides

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Compare deuterium uptake of MJ0904 alone versus in complex with potential partners

  • Map interaction surfaces based on protection patterns

• Thermal Proteome Profiling (TPP):

  • Monitor thermal stability changes of MJ0904 upon ligand binding

  • Identify cellular targets through whole-proteome thermal shift assays

Sample preparation for these experiments requires careful optimization, particularly for a protein from an extremophile. Extraction buffers should be designed to maintain protein-protein interactions while effectively solubilizing membrane-associated complexes. The urea/thiourea extraction protocol described in search result provides a good starting point, though lower concentrations may be needed to preserve interactions.

How does temperature affect the activity and stability of recombinant MJ0904?

Understanding the temperature dependence of MJ0904 activity and stability is crucial given its origin from a hyperthermophilic organism. Researchers should systematically evaluate:

• Temperature Optima:

  • Measure activity across a wide temperature range (30-95°C)

  • Determine temperature optimum and compare with growth temperature of M. jannaschii (85°C)

  • Assess activity at mesophilic temperatures to evaluate functional conservation

• Thermal Stability Profile:

  • Monitor unfolding using CD spectroscopy at increasing temperatures

  • Perform differential scanning calorimetry (DSC) to determine transition temperatures

  • Compare stability in different buffer conditions and with potential stabilizing factors

• Long-term Stability:

  • Measure activity retention after prolonged incubation at different temperatures

  • Compare stability of recombinant protein with native protein (if available)

The above table represents expected patterns based on typical hyperthermophilic proteins, but actual values must be determined experimentally. Researchers should present their data in clear, comparative tables following the guidelines in search result , separating dependent variables in columns for easier comparison.

What are the challenges in crystallizing MJ0904 for structural studies?

Crystallizing proteins from hyperthermophiles like M. jannaschii presents unique challenges and opportunities:

• Sample Preparation Challenges:

  • Ensuring high purity (>95% by SDS-PAGE) and homogeneity (monodisperse by DLS)

  • Removing bound nucleic acids that may copurify (check A260/A280 ratio)

  • Determining optimal buffer conditions that maintain stability while promoting crystal contacts

• Crystallization Strategies:

  • Screening at elevated temperatures (room temperature to 60°C)

  • Utilizing high-salt conditions that mimic the native environment

  • Testing both vapor diffusion and microbatch methods

  • Considering crystallization with potential ligands or cofactors to stabilize the protein

• Data Collection Considerations:

  • Crystals from thermophilic proteins often diffract to higher resolution due to structural rigidity

  • Radiation damage may be less severe compared to mesophilic proteins

  • Cryoprotection protocols need optimization to prevent ice formation

• Alternative Approaches:

  • If crystallization proves challenging, consider cryo-electron microscopy (cryo-EM)

  • NMR spectroscopy for structural determination of domains or smaller constructs

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope determination

Researchers should systematically document crystallization trials, following proper data presentation methods outlined in search result , including all relevant parameters (protein concentration, buffer composition, temperature, precipitants) to facilitate reproducibility.

How can I analyze contradictory data regarding MJ0904 function?

When faced with contradictory data about MJ0904 function, researchers should apply a systematic approach to resolve inconsistencies:

The resolution of contradictory data often leads to new insights about protein function, potentially revealing context-dependent activities or regulatory mechanisms not previously appreciated.

What are the best approaches for studying post-translational modifications of MJ0904?

Studying post-translational modifications (PTMs) of archaeal proteins requires specialized approaches due to their unique modifications:

• Identification of PTMs:

  • Use high-resolution mass spectrometry with multiple fragmentation methods (CID, HCD, ETD)

  • Employ enrichment strategies for specific modifications (e.g., TiO2 for phosphopeptides)

  • Process samples following protocols similar to those in search result , with adaptations for specific PTM analysis

• Mapping Modification Sites:

  • Perform bottom-up proteomics with careful consideration of digestion enzymes

  • Consider using multiple proteases beyond trypsin (Lys-C, Glu-C, chymotrypsin) to improve sequence coverage

  • Implement top-down proteomics to analyze intact protein forms

• Functional Significance:

  • Generate site-directed mutants of modified residues

  • Compare activity, stability, and localization of wild-type and mutant forms

  • Analyze the conservation of modification sites across related species

• Archaeal-Specific Considerations:

  • Look for unique archaeal modifications not common in bacteria or eukaryotes

  • Consider the effect of extreme growth conditions on modification patterns

  • Investigate if modifications affect thermostability

The sample preparation protocol outlined in search result , which includes reduction with dithiothreitol, alkylation with iodoacetamide, and sequential digestion with Lys-C and trypsin, provides a solid foundation for PTM analysis, though modifications may be needed based on the specific PTMs targeted.

How should I optimize protein extraction from expression systems for maximum MJ0904 yield?

Optimizing protein extraction is critical for maximizing yield and maintaining the native structure of MJ0904:

• Lysis Buffer Optimization:

  • Test different buffer compositions (varying salt concentrations, pH values)

  • Evaluate the necessity of denaturants (urea/thiourea as in search result ) versus native conditions

  • Include appropriate protease inhibitors to prevent degradation

• Physical Disruption Methods:

  • Compare sonication (as described in ), French press, and bead beating

  • Optimize sonication parameters (power, pulse duration, intervals) to minimize protein denaturation

  • For thermostable proteins, consider thermal lysis as an initial purification step

• Solubility Enhancement:

  • Test solubility-enhancing additives (glycerol, arginine, non-detergent sulfobetaines)

  • Evaluate mild detergents for membrane-associated fractions

  • Consider fusion partners known to enhance solubility (MBP, SUMO, thioredoxin)

• Extraction Yield Measurement:

  • Quantify protein in each fraction (soluble, insoluble, flow-through)

  • Use Western blotting to track the target protein throughout the extraction process

  • Assess activity recovery to ensure functional protein extraction

Data presentation should follow the guidelines in search result , keeping tables simple with clear comparisons between methods and avoiding repetition of information between text, tables, and figures.

What is the best way to present comparative data on MJ0904 and its homologs?

Presenting comparative data on MJ0904 and its homologs requires careful organization to highlight meaningful patterns:

• Sequence Comparison:

  • Present sequence identity/similarity in a matrix format

  • Highlight conserved domains and critical residues

  • Use multiple sequence alignments for detailed analysis of conservation patterns

• Biochemical Properties:

  • Organize data with dependent variables in columns for clearer comparison, as recommended in search result

  • Compare kinetic parameters, stability metrics, and reaction specificities

  • Include statistical analyses to validate significant differences

• Structural Comparison:

  • Present RMSD values for structural alignments

  • Highlight differences in active sites and binding pockets

  • Use structural overlays to visualize key differences

• Evolutionary Analysis:

  • Include phylogenetic trees with bootstrap values

  • Map functional differences onto evolutionary relationships

  • Analyze rates of evolution for different protein domains

Following the principles outlined in search result , data should be organized to facilitate comparison, with similar data presented in columns rather than rows. The first column typically lists independent variables (e.g., different homologs), while subsequent columns contain dependent variables (e.g., kinetic parameters, thermal stability).

How can I effectively communicate MJ0904 experimental results in scientific publications?

Effective communication of MJ0904 research findings requires careful attention to data presentation:

• Organization of Results:

  • Follow the "first general, then specific" principle described in search result

  • Begin with response rates and research participant description (if applicable)

  • Present key findings followed by relevant statistical analyses

  • Ensure all data directly address the research questions

• Selection of Presentation Format:

  • Use text for simple data with few categories

  • Employ tables for precise numerical values and large amounts of related data

  • Utilize graphics for highlighting trends and proportions

  • Avoid repeating the same information in multiple formats

• Table Construction:

  • Create self-explanatory tables with clear titles, as outlined in search result

  • Organize columns for comparison of dependent variables

  • Use footnotes for definitions and statistical significance indicators

  • Limit tables to essential information that supports the narrative

• Writing Style:

  • Use past tense for describing results

  • Keep explanations simple yet comprehensive

  • Present general findings before specific details

  • Avoid including methods or discussion in the results section

When preparing manuscripts, researchers should consult the "Uniform Requirements for Manuscripts Submitted to Medical Journals" or equivalent guidelines for their field, as mentioned in search result , to ensure proper formatting and content organization.

How might understanding MJ0904 advance archaeal systems biology?

The characterization of uncharacterized proteins like MJ0904 has significant implications for advancing archaeal systems biology:

• Metabolic Network Reconstruction:

  • Filling gaps in existing metabolic models of M. jannaschii

  • Identifying novel pathways similar to the ribose-5-phosphate biosynthesis pathway described in search result

  • Enabling more accurate flux balance analysis predictions

• Evolutionary Insights:

  • Understanding archaeal-specific adaptations to extreme environments

  • Clarifying the evolutionary relationship between archaea and other domains of life

  • Identifying novel protein families unique to archaeal lineages

• Biotechnological Applications:

  • Discovering enzymes with unique properties for industrial applications

  • Developing thermostable proteins for high-temperature bioprocesses

  • Creating new tools for synthetic biology based on archaeal components

• Fundamental Biology:

  • Revealing novel mechanisms of protein stabilization at extreme conditions

  • Understanding archaeal-specific regulatory networks

  • Providing insights into the diversity of life's biochemical solutions

Research on proteins like MJ0904 contributes to a more complete understanding of archaeal biology, similar to how the study of pentose phosphate pathway alternatives in M. jannaschii revealed the functioning of the ribulose monophosphate pathway in this organism . Such discoveries often have implications beyond archaea, potentially informing our understanding of early life on Earth.

What are promising future research directions for MJ0904 characterization?

Based on current trends in archaeal protein research, several promising directions for MJ0904 characterization emerge:

• Integrative Structural Biology:

  • Combining X-ray crystallography, cryo-EM, and computational modeling

  • Using hydrogen-deuterium exchange mass spectrometry to map dynamics

  • Applying in-cell NMR to study the protein in a native-like environment

• Systems-Level Analysis:

  • CRISPR-based gene editing in M. jannaschii or model archaea

  • Global interactome mapping to position MJ0904 in cellular networks

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to understand context-dependent function

• Synthetic Biology Applications:

  • Engineering MJ0904 for enhanced stability or altered specificity

  • Developing MJ0904-based biosensors for extreme environments

  • Creating chimeric proteins with mesophilic homologs to understand thermostability determinants

• Comparative Studies:

  • Characterizing homologs across the archaeal domain

  • Investigating potential horizontal gene transfer events involving MJ0904

  • Examining functional conservation and divergence across evolutionary lineages

These research directions should be pursued using methodological approaches similar to those described in the search results, including protein isolation and characterization techniques , careful experimental design, and proper data presentation methods .