Recombinant Methanocaldococcus jannaschii UPF0333 protein MJ1400 (MJ1400)

What is Methanocaldococcus jannaschii UPF0333 protein MJ1400?

Methanocaldococcus jannaschii UPF0333 protein MJ1400 is a membrane protein expressed by the hyperthermophilic methanarchaeon Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440). The protein belongs to the UPF0333 protein family and is encoded by the MJ1400 gene. Its amino acid sequence is MKFIMKFIKSNKGQISLEFSLLVMVVVLSAIIVSYYLIKTAIETRNANMDVINQSSNVAEKSLSNVT, with an expression region spanning positions 1-67 . The protein's specific biological function remains under investigation, but like many proteins from hyperthermophiles, it likely possesses adaptations for stability and function under extreme temperature conditions.

How should MJ1400 protein be stored for optimal stability?

MJ1400 protein requires specific storage conditions to maintain its structural integrity and biological activity. The recommended storage protocol is to keep the protein at -20°C for regular use, or at -80°C for extended storage periods. The protein is typically provided in a Tris-based buffer containing 50% glycerol, which helps protect the protein from freeze-thaw damage . It is critically important to avoid repeated freezing and thawing cycles, as these can lead to protein denaturation and loss of activity. For ongoing experiments, working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles, but should not be kept longer at this temperature to prevent degradation or contamination .

What are the key characteristics of Methanocaldococcus jannaschii as a model organism?

Methanocaldococcus jannaschii is an evolutionary deeply rooted hyperthermophilic methanarchaeon that offers unique advantages as a model organism for studying archaeal biology and extremophile adaptations . This organism belongs to the domain Archaea and is characterized by its ability to thrive in extreme environments, particularly high-temperature habitats. As a methanogen, it produces methane as a metabolic byproduct and utilizes specialized coenzymes like F420 for energy metabolism. The genome of M. jannaschii has been fully sequenced, revealing numerous genes encoding proteins with adaptations to extreme conditions, making it valuable for studying protein stability mechanisms, membrane biology in extreme environments, and the evolutionary history of life . M. jannaschii contains various specialized proteins, including oxidoreductases like FprA homologs (Mj_0732 and Mj_0748), which are involved in oxygen detoxification and represent interesting targets for functional studies .

How does the structure of MJ1400 compare to other membrane proteins from extremophiles?

Structural analysis of MJ1400 reveals distinctive features common to membrane proteins from hyperthermophilic organisms. The protein contains a significant proportion of hydrophobic amino acids, particularly in its transmembrane regions, which contribute to its stability in the membrane environment at extreme temperatures. When compared to homologous proteins from mesophilic organisms, MJ1400 likely exhibits increased internal hydrophobic interactions, ion pairing, and reduced loop regions—all adaptations that enhance thermostability .

Although the crystal structure of MJ1400 is not explicitly described in the available literature, structural homology modeling can be performed based on related proteins. For comparative analysis, researchers can reference the structural data available for other M. jannaschii membrane proteins or related proteins from thermophiles. For instance, the FprA protein from Methanothermobacter marburgensis (Mmar-FprA) has been crystallographically resolved and shares structural motifs with proteins from the same organism . The amino acid composition of MJ1400, with its hydrophobic stretches interspersed with charged residues at strategic positions, suggests a typical membrane protein topology with transmembrane helices anchored in the lipid bilayer.

What experimental approaches can be used to investigate the function of UPF0333 family proteins like MJ1400?

Investigating UPF0333 family proteins like MJ1400 requires a multi-faceted experimental approach combining genomic, proteomic, and biophysical methods. The research strategy should include:

• Comparative Genomic Analysis: Examining synteny and conserved genomic context across archaeal species to identify potential functional associations. This can reveal conserved gene neighborhoods that suggest functional relationships.

• Protein-Protein Interaction Studies: Implementing pull-down assays, two-hybrid systems adapted for archaeal proteins, or cross-linking experiments to identify interaction partners.

• Gene Knockout or Silencing: Utilizing the genetic systems available for M. jannaschii to create knockout mutants of MJ1400 and assess the resulting phenotype . This approach can reveal the physiological importance of the protein.

• Heterologous Expression Systems: Expressing MJ1400 in model organisms under controlled conditions to study its effects on cellular physiology.

• Structural Determination: Using X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy to resolve the three-dimensional structure, providing insights into potential functional motifs.

A methodological workflow for functional characterization would begin with bioinformatic analysis, followed by recombinant expression and purification, structural characterization, and finally in vitro and in vivo functional assays under conditions mimicking the native hyperthermophilic environment.

What are the challenges in expressing and purifying thermostable proteins like MJ1400 in heterologous systems?

Expression and purification of thermostable proteins from hyperthermophiles present unique challenges that require specialized approaches. The primary challenges include:

• Codon Usage Bias: M. jannaschii has distinct codon preferences that differ from common expression hosts like E. coli, potentially leading to translation inefficiency. This can be addressed through codon optimization of the synthetic gene construct.

• Protein Folding Environment: Thermophilic proteins may not fold correctly at mesophilic temperatures in standard expression hosts. Researchers should consider:

  • Expression at elevated temperatures (30-42°C)

  • Co-expression with archaeal chaperones

  • Use of specialized strains designed for membrane protein expression

• Membrane Integration: As a membrane protein, MJ1400 requires proper integration into the host membrane or inclusion bodies, which can be facilitated by fusion tags or specialized detergents during purification.

• Post-translational Modifications: Any archaeal-specific modifications may be absent in bacterial hosts, potentially affecting function and structure.

The purification strategy should employ a sequential approach:

• Cell lysis under conditions that preserve native-like environments

• Membrane fraction isolation

• Solubilization with mild detergents

• Affinity chromatography using tags incorporated during expression

• Size exclusion chromatography for final purification

A table comparing purification methods for thermostable membrane proteins:

What is the optimal protocol for conducting structural studies on MJ1400?

Conducting structural studies on MJ1400 requires a methodical approach that accounts for its membrane-associated nature and thermostable properties. The following protocol outlines the key steps:

• Sample Preparation:

  • Express MJ1400 with appropriate tags that don't interfere with structure

  • Employ gentle solubilization using mild detergents like DDM or LMNG

  • Concentrate to 5-10 mg/ml while monitoring aggregation

  • Assess sample homogeneity via dynamic light scattering

• Crystallization Trials:

  • Screen detergent-solubilized protein against sparse matrix conditions

  • Include thermostability assays to identify stabilizing conditions

  • Consider lipidic cubic phase crystallization for membrane proteins

  • Optimize promising conditions with fine gradient screens

• Alternative Structural Methods:

  • Cryo-EM: Particularly useful if MJ1400 forms complexes or resists crystallization

  • NMR Spectroscopy: For dynamic studies, focusing on specific domains

  • Small-angle X-ray scattering: To obtain low-resolution envelope structures

• Data Collection and Processing:

  • Collect diffraction data at synchrotron sources with microfocus beamlines

  • Process data accounting for potential twinning or anisotropy

  • Apply molecular replacement using related structures or employ experimental phasing

• Structure Validation:

  • Assess Ramachandran plots, bond angles, and clash scores

  • Verify membrane protein-specific parameters such as hydrophobic belt positioning

  • Compare with homologous structures where available

The key consideration throughout this process is maintaining the protein in a native-like environment while generating conditions conducive to structural determination. Temperature control is especially important when working with proteins from hyperthermophiles, as they may exhibit different conformational states at mesophilic versus thermophilic temperatures.

How can researchers design experiments to investigate protein-protein interactions involving MJ1400?

Investigating protein-protein interactions for MJ1400 requires specialized approaches that accommodate both its membrane-associated nature and its origin from a thermophilic organism. The experimental design should incorporate the following methodologies:

• Split-reporter Assays Modified for Thermophilic Conditions:

  • Adapt bacterial or yeast two-hybrid systems to function at elevated temperatures

  • Use thermostable reporter proteins to maintain activity under experimental conditions

  • Design constructs that properly display the MJ1400 interaction domains

• Co-immunoprecipitation with Thermostable Antibodies:

  • Generate antibodies against MJ1400 or use epitope tags that withstand high temperatures

  • Perform pull-downs under native-like conditions (pH, salt concentration, temperature)

  • Validate interactions via western blotting and mass spectrometry identification

• Proximity Labeling in Reconstituted Systems:

  • Engineer MJ1400 with biotin ligase tags for BioID or APEX2 approaches

  • Create membrane-mimetic environments using nanodiscs or liposomes

  • Conduct labeling reactions at temperatures mimicking native conditions

• Surface Plasmon Resonance (SPR) Analysis:

  • Immobilize purified MJ1400 on sensor chips with appropriate detergent conditions

  • Test interactions with candidate partners at various temperatures

  • Determine binding kinetics and thermodynamic parameters

• Cross-linking Mass Spectrometry:

  • Apply membrane-permeable crosslinkers to capture transient interactions

  • Perform crosslinking at elevated temperatures to mimic physiological conditions

  • Analyze crosslinked peptides using specialized mass spectrometry workflows

Data from these complementary approaches should be integrated to construct an interaction network. Particular attention should be paid to temperature-dependent interactions that may only occur under conditions mimicking the native hyperthermophilic environment. The SMART experimental design approach (Sequential Multiple Assignment Randomized Trial) can be valuable for optimizing experimental conditions efficiently .

What quality control measures should be implemented when working with recombinant MJ1400 protein?

Quality control for recombinant MJ1400 protein should follow a systematic protocol that ensures both identity and functional integrity. Researchers should implement the following measures:

• Protein Identity Verification:

  • SDS-PAGE analysis to confirm expected molecular weight (approximately 7-8 kDa based on the 67 amino acid sequence)

  • Western blotting with specific antibodies or tag detection

  • Mass spectrometry fingerprinting to confirm primary sequence

  • N-terminal sequencing for the first 5-10 amino acids

• Purity Assessment:

  • High-resolution gel electrophoresis with silver staining (aim for >95% purity)

  • Size exclusion chromatography profiles to detect aggregation or degradation

  • Dynamic light scattering to verify monodispersity and absence of aggregates

• Structural Integrity Evaluation:

  • Circular dichroism spectroscopy to assess secondary structure elements

  • Fluorescence spectroscopy to evaluate tertiary structure through intrinsic fluorescence

  • Thermal shift assays to determine stability and proper folding

  • Limited proteolysis to verify domain organization

• Functional Activity:

  • Membrane integration assays using liposomes or nanodiscs

  • Binding assays with predicted interaction partners

  • Activity assays based on predicted function (if known)

• Stability Monitoring:

  • Design accelerated stability studies at different temperatures

  • Monitor for degradation over time with SDS-PAGE and activity assays

  • Implement freeze-thaw stability testing to validate storage recommendations

Quality control data should be systematically recorded in a standardized format to ensure reproducibility across experiments and batches. For long-term projects, establishing an internal reference standard is recommended to allow batch-to-batch comparisons.

How should researchers interpret evolutionary conservation patterns in UPF0333 family proteins across archaeal species?

Interpreting evolutionary conservation patterns in UPF0333 family proteins requires a systematic approach combining bioinformatics tools with statistical analysis. Researchers should follow these methodological steps:

• Multiple Sequence Alignment (MSA) Construction:

  • Gather homologous sequences from diverse archaeal lineages using PSI-BLAST

  • Align sequences using algorithms optimized for membrane proteins (e.g., MAFFT with E-INS-i strategy)

  • Manually refine alignments focusing on transmembrane regions

  • Calculate conservation scores using methods like Jensen-Shannon divergence

• Phylogenetic Analysis:

  • Construct maximum likelihood trees using models specific for membrane proteins

  • Perform bootstrap analysis (>1000 replicates) to assess clade stability

  • Map taxonomic information to identify lineage-specific patterns

  • Calculate evolutionary rates using relative rate tests

• Structural Mapping of Conservation:

  • Project conservation scores onto structural models (homology models if experimental structures unavailable)

  • Identify conservation patterns in membrane-spanning versus loop regions

  • Analyze clustering of conserved residues as potential functional sites

  • Compare conservation between thermophilic and mesophilic homologs

• Functional Inference:

  • Apply mutual information analysis to identify co-evolving residue networks

  • Examine gene neighborhood conservation across species

  • Identify conserved motifs and compare to known functional domains

  • Use evolutionary trace methods to rank residue importance

When interpreting the results, researchers should consider that highly conserved residues likely play critical structural or functional roles. Conversely, variable regions may indicate adaptation to specific environmental niches or diversification of function. The pattern of conservation in membrane-spanning regions versus aqueous-exposed loops can provide insights into interaction interfaces or substrate-binding regions. Similar approaches have been successfully applied to other membrane proteins from M. jannaschii, such as FprA homologs, where conservation analysis revealed functional relationships with proteins like Mmar-FprA that share 67-82% sequence identity/similarity .

What statistical approaches are most appropriate for analyzing thermal stability data for MJ1400?

Analyzing thermal stability data for thermostable proteins like MJ1400 requires specialized statistical approaches that account for the unique properties of hyperthermophilic proteins. Researchers should implement the following methodological framework:

• Thermal Unfolding Curve Analysis:

  • Apply sigmoidal fitting models (Boltzmann or logistic) to thermal denaturation curves

  • Calculate melting temperature (Tm) with confidence intervals using non-linear regression

  • Use Bayesian statistics for complex unfolding patterns with multiple transitions

  • Implement maximum likelihood estimation for parameters when assumptions of normality aren't met

• Comparative Statistical Methods:

  • Apply paired t-tests or Wilcoxon signed-rank tests for comparing thermal stability under different conditions

  • Use ANOVA with post-hoc tests for multi-condition experiments

  • Implement mixed-effects models when analyzing repeated measures data

  • Calculate effect sizes (Cohen's d or similar) to quantify the magnitude of differences

• Multivariate Analysis for Complex Datasets:

  • Apply principal component analysis (PCA) to identify patterns in stability across multiple variables

  • Use hierarchical clustering to group similar stability profiles

  • Implement partial least squares regression for predictive modeling of stability determinants

  • Consider machine learning approaches for large datasets with multiple parameters

• Thermodynamic Parameter Calculation:

  • Derive ΔG, ΔH, and ΔS using van't Hoff or Gibbs-Helmholtz equations

  • Calculate activation energies using Arrhenius plots

  • Apply propagation of errors to determine confidence intervals for derived parameters

  • Use bootstrap resampling for robust parameter estimation

The choice of statistical approach should be guided by experimental design considerations similar to those used in adaptive intervention studies, where sequential analysis helps optimize parameters . When reporting results, researchers should include both descriptive statistics (mean, median, standard deviation) and inferential statistics (p-values, confidence intervals) while avoiding multiple testing problems through appropriate corrections.

How can researchers effectively compare experimental data on MJ1400 with computational predictions of its structure and function?

Effectively comparing experimental data with computational predictions for MJ1400 requires a systematic methodology that accounts for the strengths and limitations of both approaches. Researchers should implement the following framework:

When integrating computational and experimental approaches, researchers should consider the hierarchical nature of protein structure and function predictions. This hierarchical approach is similar to the mixed methods research design described in resource , which combines quantitative measurements with qualitative structural insights for comprehensive analysis.

How might MJ1400 protein be applied in biotechnology and what research would support these applications?

MJ1400, as a protein from a hyperthermophilic organism, presents several promising biotechnological applications that capitalize on its inherent stability and unique properties. Potential applications and supporting research directions include:

• Thermostable Biosensors Development:

  • Research Needs: Characterize ligand binding properties and signal transduction mechanisms

  • Methodology: Engineer MJ1400 with reporter domains that maintain function at high temperatures

  • Application Potential: Create biosensors that operate in extreme industrial conditions where conventional protein-based sensors would denature

• Membrane Protein Expression Systems:

  • Research Needs: Identify elements that contribute to efficient membrane integration

  • Methodology: Develop fusion constructs incorporating MJ1400 transmembrane domains

  • Application Potential: Enhance expression and stability of difficult-to-express membrane proteins for structural studies

• Protein Engineering Platforms:

  • Research Needs: Determine the structural basis for MJ1400's thermostability

  • Methodology: Apply directed evolution and rational design to transfer stability features to mesophilic proteins

  • Application Potential: Create engineered proteins with enhanced stability for industrial catalysis

• Nanobiotechnology Applications:

  • Research Needs: Characterize self-assembly properties and interaction with nanomaterials

  • Methodology: Develop protocols for incorporating MJ1400 into nanodiscs or artificial membranes

  • Application Potential: Create thermostable bioelectronic interfaces or drug delivery systems

• Evolutionary Biology Research Tools:

  • Research Needs: Establish phylogenetic relationships among UPF0333 proteins

  • Methodology: Develop assays to test functional conservation across evolutionary distance

  • Application Potential: Create tools for studying protein evolution under extreme conditions

Supporting research should follow rigorous experimental design principles, potentially adopting adaptive research methodologies as described in resource , which would allow for optimization of protocols based on intermediate results. This approach would be particularly valuable given the exploratory nature of many biotechnological applications.

What are the most promising approaches for elucidating the physiological role of MJ1400 in Methanocaldococcus jannaschii?

Elucidating the physiological role of MJ1400 in M. jannaschii requires a multi-faceted research approach that combines genetic, biochemical, and systems biology methods. The most promising methodological approaches include:

• Gene Knockout and Phenotypic Analysis:

  • Apply the recently developed genetic system for M. jannaschii to create MJ1400 knockout strains

  • Conduct comparative growth studies under various stress conditions (temperature, pressure, pH)

  • Perform metabolomic analysis to identify altered metabolic pathways in mutant strains

  • Measure fitness parameters like growth rate, survival, and adaptation capabilities

• Transcriptomics and Proteomics Integration:

  • Analyze expression patterns of MJ1400 under different environmental conditions

  • Identify co-regulated genes through RNA-Seq analysis

  • Perform differential proteomics comparing wild-type and knockout strains

  • Apply network analysis to position MJ1400 within cellular pathways

• Protein Localization and Dynamics:

  • Develop fluorescent protein fusions adapted for thermophilic conditions

  • Apply super-resolution microscopy to determine subcellular localization

  • Perform pulse-chase experiments to understand protein turnover rates

  • Investigate membrane microdomain associations

• Interactome Mapping:

  • Perform comprehensive protein-protein interaction screens

  • Validate key interactions using orthogonal methods

  • Reconstruct interaction networks and compare with known membrane protein complexes

  • Identify potential regulators or effectors of MJ1400

• Comparative Genomics Across Conditions:

  • Analyze genome-wide transposon mutagenesis data to identify synthetic lethal interactions

  • Compare gene conservation patterns across different methanogenic archaea

  • Correlate genomic features with specific environmental adaptations

  • Identify potential horizontal gene transfer events involving MJ1400

The research approach should be methodically structured using sequential multiple assignment randomized trials (SMART) design principles , which allow for adaptive investigation based on preliminary findings. This is particularly important when studying proteins of unknown function, as initial results may significantly redirect the research focus.

What emerging technologies might accelerate research on thermostable membrane proteins like MJ1400?

Research on thermostable membrane proteins like MJ1400 stands to benefit significantly from several emerging technologies that address current methodological limitations. The most promising technological advances include:

• Advanced Cryo-EM Methodologies:

  • Microcrystal electron diffraction (MicroED) for small membrane proteins

  • Time-resolved cryo-EM for capturing dynamic states

  • AI-enhanced particle picking and 3D reconstruction algorithms

  • Application Potential: Determine high-resolution structures of MJ1400 without the need for large crystals

• Integrated Structural Biology Platforms:

  • Hybrid methods combining NMR, X-ray, and cryo-EM data

  • Computational integration of sparse and diverse experimental constraints

  • In-cell structural biology approaches adapted for thermophiles

  • Application Potential: Generate comprehensive structural models incorporating dynamics and interactions

• Advanced Membrane Mimetics:

  • Designed nanodiscs with archaeal lipid compositions

  • Amphipathic polymers specifically developed for thermostable proteins

  • 3D-printed artificial membrane systems with controlled curvature

  • Application Potential: Maintain native-like environments for functional and structural studies

• Single-Molecule Technologies:

  • High-temperature-adapted single-molecule FRET

  • Nanopore-based single-molecule sensing platforms

  • Force spectroscopy methods for membrane protein unfolding

  • Application Potential: Characterize conformational dynamics and rare states of MJ1400

• Genome Editing and Synthetic Biology Tools:

  • CRISPR-Cas systems optimized for archaeal genomes

  • Thermostable fluorescent proteins and biosensors

  • Synthetic genomics approaches for minimal archaeal systems

  • Application Potential: Create tailored genetic variants for in vivo functional studies

Implementation of these technologies should follow methodological principles similar to those used in advanced research methods described in resource , which emphasizes the importance of selecting appropriate techniques based on research questions rather than technical convenience. The development of quantitative assays should draw inspiration from approaches used in clinical studies , adapting principles of biomarker assessment to the evaluation of protein function.