Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJECL16 (MJECL16)

What is MJECL16 and what are its basic characteristics?

MJECL16 is an uncharacterized protein from the thermophilic methanogenic archaeon Methanocaldococcus jannaschii. It is a small protein consisting of 70 amino acids with the sequence: MSILISNKQFNHGLKDEFATKKDLELLEERILRYVDNKFNQLDKKIDRTFYLLVFFIILWVSREAFFYLI . As part of the M. jannaschii proteome, it originates from an organism first isolated from submarine hydrothermal vents at depths of approximately 2600 meters near the western coast of Mexico . The protein's function remains largely uncharacterized, though its presence in an extremophile suggests possible roles in adaptation to extreme environments including high temperature, high pressure, and moderate salinity .

How is recombinant MJECL16 typically produced for research?

Recombinant MJECL16 is typically produced in E. coli expression systems with an N-terminal histidine tag to facilitate purification . The full-length protein (amino acids 1-70) is expressed and then purified using affinity chromatography techniques that exploit the His-tag. Following expression and purification, the protein is generally supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For expression, researchers typically use vectors containing the MJECL16 gene sequence optimized for E. coli codon usage, as the native archaeal codons may not be efficiently translated in bacterial systems.

What storage and handling conditions are recommended for recombinant MJECL16?

For optimal stability and activity, recombinant MJECL16 should be stored as a lyophilized powder at -20°C to -80°C upon receipt . After reconstitution, it is recommended to aliquot the protein to avoid repeated freeze-thaw cycles which can lead to protein degradation and loss of functional properties. Working aliquots can be stored at 4°C for up to one week .

The recommended reconstitution procedure involves:

• Brief centrifugation of the vial prior to opening

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (typically 50% is recommended) for long-term storage

• Storage of aliquoted samples at -20°C/-80°C

What approaches can be used to elucidate the function of MJECL16?

As an uncharacterized protein, determining MJECL16's function requires multiple complementary approaches:

• Bioinformatic Analysis: Employ sequence-based predictions including hidden Markov models, protein threading, and homology modeling to predict structural features and possible functions. Since M. jannaschii was the first archaeon to have its genome sequenced, comparative genomics with other archaeal species may reveal conserved functional domains .

• Structural Studies: X-ray crystallography or NMR spectroscopy can provide atomic-level structural information. For a small 70-amino acid protein like MJECL16, NMR may be particularly suitable.

• Gene Neighborhood Analysis: Examining genes adjacent to MJECL16 in the M. jannaschii genome might provide clues about its function through guilt-by-association principles.

• Expression Pattern Analysis: Determining when and under what conditions MJECL16 is expressed in M. jannaschii may provide functional hints.

• Protein-Protein Interaction Studies: Techniques such as pull-down assays, yeast two-hybrid screening, or cross-linking coupled with mass spectrometry can identify interaction partners.

• Gene Knockout/Knockdown Studies: Though challenging in archaea, CRISPR-Cas systems have been adapted for some archaeal species to study gene function.

How might the extreme environment of M. jannaschii influence MJECL16's structure and function?

M. jannaschii lives in extreme conditions including temperatures of 48-94°C, high hydrostatic pressure (260 atmospheres), and moderate salinity . These conditions likely influence MJECL16's properties in several ways:

• Thermal Stability: The protein likely possesses adaptations for thermostability, which may include increased hydrophobic core packing, additional salt bridges, disulfide bonds, or increased secondary structure elements.

• Pressure Adaptation: High-pressure environments can favor protein conformations with smaller volumes. MJECL16 may exhibit unusual compressibility properties or functional pressure dependence.

• Membrane Association: The amino acid sequence of MJECL16 suggests it may have hydrophobic regions (particularly toward the C-terminus with the sequence FFIILWVSREAFFYLI), potentially indicating membrane association . This could be related to maintaining membrane integrity under extreme conditions.

• Protein Folding: Chaperones and other folding machinery in extremophiles often have unique properties. When expressing MJECL16 in mesophilic hosts like E. coli, the protein may not attain its native conformation without specific co-factors or conditions.

What is known about potential inteins or post-translational modifications in MJECL16?

Methanocaldococcus jannaschii contains a large number of inteins (protein splicing elements), with 19 discovered in one study . While specific information about inteins or post-translational modifications in MJECL16 is not explicitly mentioned in the available literature, researchers should consider:

• Intein Analysis: Examine the MJECL16 sequence for potential intein insertion sites based on known archaeal intein consensus sequences.

• Post-translational Modification Search: Mass spectrometry analysis of native MJECL16 compared to recombinant versions may reveal archaeal-specific modifications absent in E. coli-expressed protein.

• Functional Impact: If modifications exist, they may be critical for protein function, potentially explaining why recombinant versions might show different activities than native protein.

• Methodological Approaches: To identify potential modifications, researchers could use targeted mass spectrometry approaches, including electron-transfer dissociation (ETD) or electron-capture dissociation (ECD) for fragmentation, which better preserve labile modifications.

Expression Optimization Table:

Purification Strategy:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with buffers containing Tris/PBS at pH 8.0 .

• Secondary Purification: Size exclusion chromatography or ion exchange chromatography to remove contaminating proteins and aggregates.

• Quality Control: Assess purity via SDS-PAGE (target >90% purity) , and verify protein identity by mass spectrometry or western blotting.

• Functional Assessment: Develop activity assays based on predicted functions or binding partners to ensure the recombinant protein is properly folded.

• Special Considerations: For a thermophilic protein like MJECL16, consider performing purification steps at elevated temperatures or including thermostability screening to ensure native-like conformation.

How can researchers address the challenge of working with an uncharacterized protein in functional assays?

Working with uncharacterized proteins presents unique challenges that require systematic approaches:

• Functional Screening Battery:

  • Nucleic acid binding assays (EMSA, filter binding)

  • Enzymatic activity screens (hydrolase, transferase, isomerase activities)

  • Metal binding assays using ITC or differential scanning fluorimetry

  • Membrane association tests using liposome flotation assays

• Environmental Response Profiling:

  • Test protein stability and activity across temperature ranges (25-95°C)

  • Examine pressure effects using specialized high-pressure equipment

  • Assess activity in various salt concentrations and pH conditions

• Comparative Analysis:

  • Express and characterize homologs from related species

  • Use phylogenetic profiling to identify co-occurring genes/proteins

  • Compare expression patterns with known stress-response proteins

• Structural Analysis-Guided Experiments:

  • Generate structural models to identify potential active sites

  • Design targeted mutations to test functional hypotheses

  • Use structure-based virtual screening to identify potential ligands

What controls should be included when studying the potential role of MJECL16 in thermoadaptation?

When investigating MJECL16's potential role in thermoadaptation, the following controls are essential:

• Negative Controls:

  • Homologous proteins from mesophilic (non-thermophilic) archaea

  • Denatured MJECL16 protein

  • Empty vector controls in expression systems

• Positive Controls:

  • Known thermostable proteins from M. jannaschii

  • Characterized thermostability factors (e.g., specific chaperones)

• Experimental Controls:

  • Thermal shift assays comparing MJECL16 stability to control proteins

  • Circular dichroism measurements at various temperatures to monitor structural changes

  • Activity assays at different temperatures with standardized components

• Genetic Context Controls:

  • Expression of MJECL16 in mesophilic hosts with/without thermoadaptation genetic elements

  • Co-expression experiments with known thermostability factors

How should researchers interpret sequence homology findings for MJECL16?

Sequence homology analysis for uncharacterized proteins like MJECL16 requires careful interpretation:

What are the considerations when analyzing potential membrane association of MJECL16?

The C-terminal region of MJECL16 (FFIILWVSREAFFYLI) contains hydrophobic residues that might indicate membrane association . When analyzing this possibility:

• Computational Analysis:

  • Apply multiple transmembrane prediction algorithms (TMHMM, Phobius, MEMSAT)

  • Use hydropathy plots and amphipathicity analysis

  • Predict membrane interaction motifs (e.g., amphipathic helices)

• Experimental Approaches:

  • Membrane fractionation of native or heterologous expression systems

  • Fluorescence microscopy with tagged MJECL16 to visualize localization

  • Liposome binding assays with varying lipid compositions

• Interpretation Challenges:

  • Archaeal membranes contain unique lipids (e.g., isoprenoid-based rather than fatty acid-based)

  • High-temperature adaptations may alter typical membrane interaction patterns

  • Recombinant expression may not correctly localize the protein without archaeal-specific factors

• Integrative Assessment: Combine computational predictions, experimental localization, and functional assays to build a coherent model of membrane association.

How can researchers distinguish between direct and indirect effects when studying MJECL16 in heterologous systems?

When expressing MJECL16 in non-native systems (e.g., E. coli), distinguishing direct and indirect effects presents challenges:

• Concentration Controls:

  • Express MJECL16 at varying levels to establish dose-dependent relationships

  • Use inducible promoters to control timing and level of expression

• Domain Mapping:

  • Create truncation variants to pinpoint functional regions

  • Design point mutations in predicted functional sites

• Interaction Validation:

  • Confirm direct interactions using multiple methods (pull-down, crosslinking, FRET)

  • Perform competition experiments with purified components

• In vitro Reconstitution:

  • Recreate observed effects with purified components to demonstrate direct action

  • Systematically add components to identify minimum requirements for activity

• Control Proteins:

  • Use structurally similar but functionally distinct proteins as negative controls

  • Create function-disrupting mutants as comparative controls

What approaches are recommended for studying potential protein-protein interactions of MJECL16?

For investigating MJECL16's interaction network, multiple complementary approaches should be considered:

• In vitro Methods:

  • Pull-down assays using recombinant His-tagged MJECL16

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Native mass spectrometry to identify complex composition

• Cellular Methods:

  • Bacterial two-hybrid systems (potentially more suitable than yeast systems for archaeal proteins)

  • Co-immunoprecipitation from heterologous expression systems

  • Proximity labeling approaches (BioID, APEX) to identify neighboring proteins

• Computational Prediction:

  • Protein-protein docking simulations

  • Co-evolution analysis to identify potential interaction partners

  • Integration of gene neighborhood and expression correlation data

• Challenging Aspects:

  • Temperature considerations (interactions may only occur at elevated temperatures)

  • Archaeal-specific interactions may not be detected in mesophilic systems

  • Transient interactions might require crosslinking approaches

How can researchers explore the relationship between MJECL16 and the unique genomic features of M. jannaschii?

M. jannaschii contains interesting genomic features including repetitive elements and a three-component genome structure (chromosome plus two extrachromosomal elements) . To explore MJECL16's relationship with these features:

• Genomic Context Analysis:

  • Determine MJECL16's location relative to repetitive elements (LR/SR segments)

  • Investigate whether MJECL16 is located on the main chromosome or on one of the extrachromosomal elements

  • Examine synteny with related archaea to understand evolutionary context

• Expression Coordination:

  • Analyze whether MJECL16 expression correlates with genes near repetitive elements

  • Investigate potential co-regulation with other genes in stress response pathways

• Functional Connections:

  • Test for interactions between MJECL16 and proteins encoded by genes near repetitive elements

  • Explore potential roles in genome maintenance or extrachromosomal element functions

• Experimental Approaches:

  • ChIP-seq to identify potential DNA binding sites if MJECL16 has DNA interaction capability

  • RNA-seq under varying conditions to identify co-expression patterns

  • Genetic manipulation (if possible in M. jannaschii or model archaeal systems) to test functional hypotheses

What techniques are most appropriate for studying MJECL16's potential role in extremophile adaptation?

To investigate MJECL16's contribution to M. jannaschii's extremophile lifestyle:

Methodological Approach Table:

• Comparative Studies:

  • Express MJECL16 homologs from mesophilic, thermophilic, and hyperthermophilic species

  • Compare stability and activity profiles across environmental conditions

  • Identify specific residues or motifs associated with extremophile adaptation

• Stress Response Analysis:

  • Examine MJECL16 expression changes during various stress conditions

  • Test protective effects of MJECL16 on other cellular components under extreme conditions

  • Investigate potential chaperone-like activities or stabilizing interactions

• Structural Stabilization Studies:

  • Identify intramolecular interactions contributing to stability (hydrogen bonds, salt bridges)

  • Test the impact of mutations at key stabilizing positions

  • Examine potential stabilizing post-translational modifications in native protein

What emerging technologies could advance understanding of MJECL16's function?

Several cutting-edge technologies show promise for elucidating the function of uncharacterized proteins like MJECL16:

• AI-Driven Structure Prediction:

  • AlphaFold2 and RoseTTAFold can provide high-confidence structural models

  • Structure-based functional inference may identify active sites or binding regions

  • Molecular dynamics simulations under extreme conditions can reveal stability mechanisms

• Single-Molecule Techniques:

  • Single-molecule FRET to detect conformational changes under various conditions

  • Optical tweezers to measure protein mechanical stability

  • High-speed AFM to visualize dynamic interactions with partners or substrates

• Advanced Mass Spectrometry:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

  • Native MS to identify complex formation and stoichiometry

  • Crosslinking MS to map interaction networks

• In situ Approaches:

  • Cryo-electron tomography to visualize native cellular localization

  • Proximity labeling in heterologous systems

  • Live-cell imaging with archaeal-adapted fluorescent tags

How might studying MJECL16 contribute to broader understanding of archaeal biology?

MJECL16 research could advance several areas of archaeal biology:

• Evolutionary Insights:

  • Uncharacterized proteins represent a significant portion of archaeal genomes

  • Functional characterization may reveal archaeal-specific pathways or mechanisms

  • Understanding unique proteins contributes to models of archaeal evolution and adaptation

• Extremophile Adaptation Mechanisms:

  • Small proteins like MJECL16 may represent specialized adaptation factors

  • Could reveal novel stabilization mechanisms applicable to protein engineering

  • May uncover unique stress response pathways in extremophiles

• Archaeal Cell Biology:

  • If membrane-associated, could provide insights into archaeal membrane organization

  • May reveal archaeal-specific protein localization mechanisms

  • Could identify unique regulatory networks in archaea

• Biotechnological Applications:

  • Novel thermostable proteins have significant biotechnological potential

  • Understanding archaeal proteins may enable engineering of extremophile properties

  • Could lead to development of new research tools for high-temperature applications