Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1306 (MJ1306)

What is the basic structural information available for Methanocaldococcus jannaschii MJ1306 protein?

MJ1306 is an uncharacterized protein from Methanocaldococcus jannaschii consisting of 83 amino acids. The full protein sequence is: MLDAILSNYLYYPSILAFLFGVLMGAKYRHKIGNIFGYLILTVVIAYFLKAFPYYDLLPLSCSYLSAVIGIIIGNRLFGGKMI. The protein has been assigned UniProt ID Q58702. Based on the amino acid sequence, it appears to be a membrane-associated protein with multiple hydrophobic regions suggesting transmembrane domains. For research applications, recombinant MJ1306 can be expressed with an N-terminal His-tag in E. coli expression systems .

What are the optimal storage conditions for recombinant MJ1306 protein to maintain its stability?

Recombinant MJ1306 is typically supplied as a lyophilized powder and should be stored at -20°C to -80°C upon receipt. For working with the protein, reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add 5-50% glycerol (with 50% being the standard final concentration) and aliquot to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing is not recommended as it may cause protein degradation and loss of activity .

What is the predicted membrane topology of MJ1306 based on its amino acid sequence?

Based on the amino acid sequence (MLDAILSNYLYYPSILAFLFGVLMGAKYRHKIGNIFGYLILTVVIAYFLKAFPYYDLLPLSCSYLSAVIGIIIGNRLFGGKMI), MJ1306 contains multiple hydrophobic stretches consistent with transmembrane domains. Computational analysis suggests this protein likely contains 2-3 transmembrane helices. The high proportion of hydrophobic amino acids (leucine, isoleucine, phenylalanine, valine) in specific segments supports this prediction. To experimentally verify this topology, researchers should consider combining approaches such as:

• Membrane fraction isolation followed by Western blotting

• Protease protection assays with membrane preparations

• Fluorescence microscopy with GFP-fusion constructs

• Cysteine accessibility methods to map exposed residues

These methods collectively would help establish the orientation of the protein within membranes and its topology .

What are the recommended protocols for solubilizing and purifying recombinant MJ1306 for structural studies?

For structural studies of recombinant MJ1306, a membrane protein, special consideration must be given to solubilization and purification:

Recommended Protocol:

• Extraction from E. coli membranes:

  • Harvest cells expressing His-tagged MJ1306

  • Resuspend in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl

  • Disrupt cells by sonication or pressure homogenization

  • Isolate membrane fraction by ultracentrifugation (100,000 × g, 1 hour)

• Solubilization:

  • Resuspend membrane pellet in solubilization buffer containing:

    • 50 mM Tris-HCl (pH 8.0)

    • 150 mM NaCl

    • 1-2% detergent (start with n-dodecyl-β-D-maltoside or n-octyl-β-D-glucoside)

    • Protease inhibitors

  • Incubate with gentle rotation at 4°C for 2-3 hours

• Purification:

  • Clarify by centrifugation at 100,000 × g for 30 minutes

  • Load supernatant onto Ni-NTA column equilibrated with buffer containing 0.1% detergent

  • Wash with buffer containing 20-40 mM imidazole

  • Elute with buffer containing 250-500 mM imidazole

  • Consider size exclusion chromatography as a final purification step

• Buffer Exchange:

  • Exchange into a buffer compatible with your structural studies, maintaining detergent above its critical micelle concentration

For crystallization attempts, screening with various detergents and lipid additives is recommended, as demonstrated with other M. jannaschii membrane proteins .

How should researchers approach functional characterization of an uncharacterized protein like MJ1306?

For functional characterization of uncharacterized proteins like MJ1306, a multi-faceted approach is essential:

• Bioinformatic Analysis:

  • Perform homology searches using PSI-BLAST and HHpred

  • Analyze secondary structure predictions

  • Identify conserved domains and motifs

  • Look for structural homologs through threading approaches

• Expression in Native Context:

  • Design experiments to determine expression conditions in M. jannaschii

  • Examine expression under different stress conditions

  • Perform co-immunoprecipitation to identify interacting partners

• Heterologous Expression Systems:

  • Express in archaeal model organisms like Thermococcus kodakarensis

  • Create gene knockouts or CRISPR interference systems

  • Perform complementation tests

• Biochemical Characterization:

  • Test for enzymatic activities based on predictions

  • Perform binding assays with potential substrates

  • Investigate protein-protein interactions

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM for structure determination

  • NMR for dynamics studies if protein size permits

  • Small-angle X-ray scattering for solution structure

• Functional Genomics:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to identify post-translational modifications

  • Metabolomics to identify related metabolic changes

Through this systematic approach, researchers can gradually build evidence for the function of MJ1306 .

What challenges might researchers encounter when expressing MJ1306 in E. coli, and how can these be addressed?

Common Challenges and Solutions for MJ1306 Expression in E. coli:

For MJ1306 specifically, researchers should consider the extreme growth conditions of M. jannaschii (hyperthermophilic, pressure-adapted) when designing expression and purification strategies. Adding stabilizing agents like glycerol (5-50%) during purification and storage may help maintain protein integrity .

What computational approaches can predict potential functions of MJ1306 based on its sequence and structural features?

To predict potential functions of MJ1306, researchers should employ multiple computational approaches:

• Sequence-Based Prediction:

  • Position-Specific Iterative BLAST (PSI-BLAST) to identify distant homologs

  • Hidden Markov Models (HMMs) to detect remote homologies

  • Analysis of conserved sequence motifs using MEME and GLAM2

  • Protein function prediction servers like Pfam, InterProScan, and protein function predictor (PFP)

• Structure-Based Prediction:

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold

  • Structural alignment with known proteins using DALI or TM-align

  • Binding site prediction using SiteMap or COACH

  • Molecular dynamics simulations to identify potential binding pockets

• Genomic Context Analysis:

  • Examine neighboring genes in M. jannaschii genome

  • Identify conserved gene clusters across related species

  • Look for operonic arrangements suggesting functional relationships

• Evolutionary Analysis:

  • Phylogenetic profiling to identify co-evolving proteins

  • Residue co-evolution analysis to predict functional sites

  • Comparative genomics across archaeal species

For transmembrane proteins like MJ1306, additional predictors such as TMHMM for transmembrane helix prediction and SignalP for signal peptide prediction should be employed. The predictions should be validated experimentally using the results as working hypotheses .

How can researchers investigate potential interactions between MJ1306 and other proteins in M. jannaschii?

Investigating protein-protein interactions for MJ1306 requires specialized approaches for membrane proteins from extremophiles:

• Affinity-Based Methods:

  • Tandem affinity purification (TAP) using His-tagged MJ1306 as bait

  • Co-immunoprecipitation with antibodies against MJ1306

  • Proximity-dependent biotin labeling (BioID or TurboID) adapted for thermophiles

  • Pull-down assays with recombinant MJ1306

• Library Screening Approaches:

  • Yeast two-hybrid using thermostable variants

  • Bacterial two-hybrid systems with thermophilic adaptations

  • Phage display screening against M. jannaschii proteome

• Structural Methods:

  • Crosslinking mass spectrometry to capture transient interactions

  • Hydrogen-deuterium exchange mass spectrometry

  • Cryo-electron microscopy of protein complexes

• In vivo Approaches:

  • Fluorescence resonance energy transfer (FRET) with thermostable fluorescent proteins

  • Bimolecular fluorescence complementation adapted for archaeal systems

  • In vivo crosslinking followed by mass spectrometry

• Computational Predictions:

  • Protein-protein interaction prediction algorithms

  • Coevolution-based contact prediction

  • Docking simulations with predicted or known structures

When working with extremophiles like M. jannaschii, special attention must be paid to maintaining appropriate conditions (high temperature, pressure, salt) during interaction studies. For membrane proteins, detergent selection is critical and should be optimized to maintain native interactions .

What techniques can distinguish between structural and catalytic roles for MJ1306?

To determine whether MJ1306 serves primarily a structural or catalytic function, researchers should employ a combination of experimental approaches:

• Structural Role Assessment:

  • Membrane disruption assays after MJ1306 depletion

  • Lipidomic analysis to identify associated lipids

  • Microscopy techniques to visualize membrane integrity

  • Biophysical measurements of membrane properties (fluidity, thickness)

  • Protein-lipid interaction assays using fluorescence anisotropy

• Catalytic Function Assessment:

  • Activity screening against substrate libraries

  • Metabolite profiling in knockout/knockdown systems

  • Isothermal titration calorimetry for substrate binding

  • Enzyme kinetics analysis if activity is detected

  • Identification of potential catalytic residues through site-directed mutagenesis

• Comparative Approaches:

  • Analysis of expression patterns under different growth conditions

  • Proteomics analysis to identify post-translational modifications

  • Comparison with homologs of known function

  • Complementation studies in related archaea or bacteria

• Structural Analysis:

  • X-ray crystallography or cryo-EM with and without potential substrates

  • NMR analysis for structural dynamics

  • Hydrogen-deuterium exchange to identify flexible regions

The data from these approaches should be integrated to develop a comprehensive model of MJ1306 function. For example, if mutagenesis of predicted catalytic residues doesn't affect function but membrane disruption is observed upon protein depletion, a structural role would be more likely .

How does MJ1306 compare with other uncharacterized proteins in archaea, and what insight might this provide about its function?

Comparative analysis of MJ1306 with other uncharacterized archaeal proteins reveals several significant patterns:

• Sequence Conservation Analysis:
  MJ1306 belongs to a family of small membrane proteins found predominantly in methanogens and some other archaea. Sequence alignment shows:

  • Highly conserved hydrophobic core regions, suggesting transmembrane domains

  • Variable N-terminal regions that may confer specific functions

  • Several invariant residues that might be functionally important

• Phylogenetic Distribution:

  • Present primarily in Euryarchaeota, particularly thermophilic methanogens

  • Absent in Crenarchaeota and most bacterial genomes

  • Expanded gene families in some methanogens, suggesting functional specialization

• Genomic Context Analysis:

  • Often found in proximity to genes involved in membrane lipid biosynthesis

  • Sometimes co-localized with genes encoding proteins involved in energy metabolism

  • Rarely found near genes involved in information processing (replication, transcription)

• Predicted Structural Features:

  • Secondary structure predictions suggest 2-3 transmembrane helices

  • Potential membrane-binding motifs similar to those in other archaeal membrane proteins

  • Possible lipid-binding pocket based on hydrophobicity analysis

These comparative insights suggest MJ1306 may function in membrane organization, small molecule transport, or signaling specific to methanogenic archaea living in extreme environments. The conservation pattern indicates it likely plays a role in adaptations to high temperature and/or pressure environments characteristic of M. jannaschii's ecological niche .

What can the study of MJ1306 reveal about protein adaptation to extreme environments?

Research on MJ1306 provides valuable insights into protein adaptation to extreme environments, particularly hyperthermophilic conditions:

• Amino Acid Composition Analysis:
  MJ1306's sequence exhibits several adaptations typical of proteins from hyperthermophiles:

  • Increased proportion of charged amino acids (particularly lysine and glutamate)

  • Higher frequency of hydrophobic residues in core regions

  • Reduced occurrence of thermolabile amino acids like asparagine and glutamine

  • Strategic placement of proline residues to enhance structural rigidity

• Structural Stability Mechanisms:

  • Potential for increased salt bridge formation between charged residues

  • Compact hydrophobic core packing in transmembrane regions

  • Reduced flexibility in loop regions compared to mesophilic homologs

  • Possible disulfide bonds for additional stabilization

• Membrane Interaction Adaptations:

  • Specialized interfacial residues for interaction with archaeal tetraether lipids

  • Hydrophobic matching with the thicker archaeal membranes

  • Adaptations for maintaining function under high pressure

• Evolutionary Rate Analysis:

  • Comparison of substitution rates between thermophilic and mesophilic homologs

  • Identification of positions under positive or purifying selection

  • Coevolution patterns with other proteins in stress response pathways

By studying these adaptations in MJ1306, researchers can gain broader insights into the molecular basis of protein thermostability, with potential applications in protein engineering and biotechnology. The principles derived can inform the design of thermostable enzymes for industrial applications .

How does the structure of MJ1306 compare with the related protein MJ1225, and what functional implications might this have?

While MJ1306 and MJ1225 are both proteins from M. jannaschii, they represent different protein families with distinct structures and functions:

• Structural Comparison:

  • MJ1225 is identified as an archaeal homolog of γ-AMPK (AMP-activated protein kinase) containing CBS domains

  • MJ1306 is a smaller membrane protein with predicted transmembrane helices

  • MJ1225 has been crystallized at 2.3 Å resolution in space group H32 with unit-cell parameters a = b = 108.95, c = 148.08 Å

  • MJ1306 has not yet been crystallized, but sequence analysis suggests a completely different fold

• Functional Implications:

  • MJ1225 likely functions in energy sensing (like its eukaryotic counterpart γ-AMPK)

  • MJ1306 appears to be a membrane protein potentially involved in membrane organization or transport

  • Both may contribute to M. jannaschii's adaptation to extreme environments but through different mechanisms

• Evolutionary Context:

  • MJ1225 has clear homologs in eukaryotes (γ-AMPK), suggesting an ancient and conserved role

  • MJ1306 appears more restricted to archaea, suggesting a more specialized function

• Research Approach Differences:

  • MJ1225 studies have successfully used crystallographic approaches

  • MJ1306 likely requires membrane protein-specific methods including detergent optimization

The successful crystallization of MJ1225 provides a methodological framework that might be adapted for structural studies of MJ1306, taking into account the differences in protein characteristics. Comparative studies of different M. jannaschii proteins like these can help researchers understand how different cellular functions are adapted to extreme environments .

How might researchers use MJ1306 to develop more effective expression systems for archaeal membrane proteins?

Developing improved expression systems for archaeal membrane proteins using insights from MJ1306 could follow these strategic approaches:

• Engineering Optimized Expression Vectors:

  • Design synthetic promoters based on MJ1306 expression patterns

  • Incorporate archaeal ribosome binding sites optimized for membrane proteins

  • Develop dual-host vectors functional in both E. coli and archaeal systems

  • Create fusion constructs with thermostable reporter proteins

• Membrane Mimetic Systems:

  • Design nanodisc systems with archaeal lipid compositions

  • Develop archaeal-based cell-free expression systems

  • Engineer E. coli strains with modified membranes to better accommodate archaeal proteins

  • Create synthetic membrane environments that mimic archaeal membrane properties

• Chaperone Co-expression Strategies:

  • Identify and co-express archaeal chaperones that facilitate membrane protein folding

  • Engineer hybrid chaperone systems combining archaeal and bacterial components

  • Develop inducible stress response systems to enhance proper folding

• High-Throughput Optimization Framework:

  • Design a systematic screening pipeline for expression conditions

  • Develop fluorescence-based reporters for proper membrane insertion

  • Create microfluidic systems for rapid testing of expression parameters

A decision tree for archaeal membrane protein expression strategy could be developed based on protein characteristics such as hydrophobicity, predicted transmembrane domains, and phylogenetic distribution. MJ1306 could serve as a model protein for validating these systems, particularly for small archaeal membrane proteins with multiple transmembrane domains .

What are the methodological considerations for studying MJ1306 interactions with archaeal-specific membrane lipids?

Studying interactions between MJ1306 and archaeal membrane lipids requires specialized approaches due to the unique ether-linked lipids found in archaeal membranes:

• Lipid Extraction and Preparation:

  • Optimize extraction protocols for archaeal diether and tetraether lipids

  • Synthesize fluorescently labeled archaeal lipid analogs

  • Prepare archaeal lipid liposomes with controlled composition

  • Generate archaeal lipid nanodiscs or bicelles for NMR studies

• Binding and Association Studies:

  • Surface plasmon resonance with immobilized lipids or protein

  • Isothermal titration calorimetry for thermodynamic parameters

  • Fluorescence anisotropy to measure direct binding

  • Microscale thermophoresis for interaction kinetics

• Structural Analysis of Protein-Lipid Complexes:

  • Lipid-protein crosslinking followed by mass spectrometry

  • Solid-state NMR of protein in archaeal lipid environments

  • Cryo-electron microscopy of membrane protein-lipid complexes

  • Molecular dynamics simulations with archaeal lipid force fields

• Functional Impact Assessment:

  • Reconstitution assays in various lipid compositions

  • Activity measurements in different membrane environments

  • Thermal stability analysis in various lipid compositions

  • Pressure-dependent studies to mimic native conditions

• Technical Considerations for Archaeal Systems:

These methodological considerations enable researchers to accurately characterize how MJ1306 interacts with the unusual membrane environment of M. jannaschii, providing insights into adaptation to extreme conditions .

How can structural studies of MJ1306 contribute to our understanding of protein folding in extreme environments?

Structural studies of MJ1306 can provide significant insights into protein folding under extreme conditions:

• Thermodynamic Stability Analysis:

  • Differential scanning calorimetry at varying temperatures and pressures

  • Circular dichroism spectroscopy to monitor secondary structure changes

  • Pressure perturbation calorimetry to assess volumetric properties

  • Hydrogen-deuterium exchange mass spectrometry at elevated temperatures

• Folding Pathway Investigation:

  • Time-resolved biophysical methods to capture folding intermediates

  • Single-molecule fluorescence resonance energy transfer (FRET)

  • Nuclear magnetic resonance for residue-specific folding information

  • Computational modeling of folding energy landscapes

• Comparative Structural Biology:

  • Structure determination of MJ1306 at ambient and extreme conditions

  • Comparison with mesophilic membrane protein homologs

  • Analysis of specific stabilizing interactions unique to thermophiles

  • Identification of conserved vs. variable structural elements

• Applied Research Directions:

  • Design principles for engineering thermostable membrane proteins

  • Development of stabilization strategies for biotechnological applications

  • Creation of chimeric proteins combining thermostable domains with mesophilic functional domains

  • Novel approaches for membrane protein crystallization at extreme conditions

The knowledge gained from MJ1306 structural studies can inform broader questions in protein science, including:

• How membrane proteins achieve thermostability without sacrificing flexibility needed for function

• The role of the lipid environment in stabilizing proteins under extreme conditions

• Universal versus organism-specific adaptations to extreme environments

• Evolutionary trajectories of protein adaptation to new environmental niches

These insights have potential applications in protein engineering, biocatalysis, and the development of robust biomaterials for extreme environments .