Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1223 (MJ1223)

What is Methanocaldococcus jannaschii and why is it significant in genomic research?

The organism is particularly significant as it represents extremophiles with remarkable adaptations to high temperatures, high pressure, and moderate salinity environments. Its genome consists of a large circular chromosome (1.66 megabase pairs, 31.4% G+C content), along with large and small extra-chromosomes. The sequencing of M. jannaschii has enabled the identification of numerous archaeal-specific metabolic pathways and served as a foundation for understanding unique archaeal biology .

What defines a protein as "uncharacterized" like MJ1223?

Uncharacterized proteins like MJ1223 belong to a class of gene products that either lack typical domains with known functions or contain domains whose functions have rarely been identified. Despite having a defined amino acid sequence and sometimes predicted structural features, these proteins have not been experimentally validated for their biological functions, cellular localization, interaction partners, or their roles in specific metabolic or signaling pathways .

The designation "uncharacterized" indicates that while the protein's existence is confirmed through genomic and/or proteomic analysis, its biochemical activities and physiological relevance remain to be determined. Many of these proteins are identified during whole-genome sequencing projects but lack homology to proteins with known functions, making their characterization a significant challenge and opportunity in functional genomics research .

What basic biochemical properties characterize the MJ1223 protein?

MJ1223 displays several notable biochemical properties that reflect its archaeal origin and potential membrane association:

As a protein from a thermophilic archaeon, MJ1223 likely possesses adaptations for functioning at high temperatures, which may include specific amino acid compositions, increased hydrophobic interactions, and structural features that enhance thermostability.

What expression systems are most suitable for recombinant production of MJ1223?

For archaeal proteins like MJ1223 from thermophilic organisms, specialized expression systems may be required to achieve proper folding and stability. Based on research approaches for similar proteins, the following expression systems are recommended:

• Escherichia coli-based systems with thermostability enhancements: Modified E. coli strains containing chaperones that assist in the folding of thermophilic proteins can improve expression yields. For archaeal membrane proteins like MJ1223, E. coli strains such as C41(DE3) or C43(DE3) specifically designed for membrane protein expression may be beneficial.

• Archaeal host expression systems: When feasible, expression in archaeal hosts like Thermococcus kodakarensis or Sulfolobus solfataricus can provide the native cellular environment more conducive to proper folding of archaeal proteins.

• Cell-free expression systems: For difficult-to-express proteins, cell-free systems using archaeal components can be effective, as they bypass issues related to toxicity or inclusion body formation.

The choice should be guided by the specific experimental goals and the physicochemical properties of MJ1223. For functional studies requiring native conformation, archaeal hosts or specialized E. coli strains designed for membrane proteins would be most appropriate, while high-yield production for structural studies might benefit from optimized E. coli systems with solubility-enhancing fusion partners .

What purification strategies are most effective for MJ1223 protein?

Given the membrane-associated characteristics of MJ1223, effective purification strategies should account for its hydrophobicity and potential thermostability:

For membrane proteins like MJ1223, the choice of detergent is particularly critical, as it must efficiently extract the protein from membranes while maintaining native conformation. The purification protocol should also include steps to verify that the protein retains its proper folding, particularly if functional assays are planned.

How can researchers properly store and handle recombinant MJ1223 protein?

Based on the product information provided, recombinant MJ1223 requires specific storage and handling conditions to maintain stability and functionality:

• Optimal storage conditions: Store at -20°C for regular use, or at -80°C for extended storage periods. The protein is supplied in a Tris-based buffer containing 50% glycerol .

• Handling precautions: Repeated freezing and thawing cycles should be strictly avoided as they can lead to protein denaturation and loss of function. Prepare appropriately sized working aliquots during initial thawing to minimize freeze-thaw cycles .

• Working aliquot management: Store working aliquots at 4°C for no longer than one week to maintain protein integrity .

• Temperature considerations: As MJ1223 originates from a thermophilic organism, the protein may exhibit unique stability characteristics at elevated temperatures, but standard cold-chain practices should still be followed for purified recombinant preparations.

• Buffer compatibility: If buffer exchange is required for specific experiments, maintain conditions that prevent protein aggregation, particularly considering the hydrophobic nature of this likely membrane-associated protein.

These storage and handling guidelines help ensure that experiments are conducted with structurally intact and functionally active protein, increasing reproducibility and reliability of research findings.

What analytical methods are recommended for characterizing MJ1223 structure?

For comprehensive structural characterization of an uncharacterized protein like MJ1223, a multi-technique approach is recommended:

For MJ1223 specifically, techniques that are effective for membrane proteins would be most informative, such as NMR with appropriate detergents, cryo-EM, or X-ray crystallography with lipidic cubic phase crystallization approaches. Additionally, computational structure prediction methods, including AlphaFold2, can provide valuable starting models for experimental validation.

What computational approaches can predict the function of uncharacterized proteins like MJ1223?

Several computational approaches can provide valuable insights into the potential functions of uncharacterized proteins like MJ1223:

• Sequence-based homology analyses: While MJ1223 may lack obvious homologs with known functions, sensitive sequence comparison tools like PSI-BLAST, HHpred, or HMMER can detect distant relationships that might suggest functional characteristics.

• Structural prediction and comparison: AlphaFold2 and RoseTTAFold can generate high-confidence structural models that, when compared against structural databases like DALI or CATH, may reveal structural similarities to functionally characterized proteins despite low sequence identity.

• Genomic context analysis: Examining the genomic neighborhood of MJ1223 in M. jannaschii and related species can provide clues about functional associations, as functionally related genes are often clustered or co-regulated.

• Protein-protein interaction prediction: Computational tools can predict potential interaction partners of MJ1223, suggesting possible functional pathways or complexes it might participate in.

• Transmembrane topology prediction: For membrane proteins like MJ1223, tools such as TMHMM, Phobius, or TOPCONS can predict the arrangement of transmembrane segments and topology, which can inform functional hypotheses .

These computational approaches should be considered as hypothesis-generating tools, with predictions requiring experimental validation through the methods discussed in other sections of this FAQ.

How can protein-protein interaction studies help elucidate MJ1223 function?

Protein-protein interaction studies are particularly valuable for elucidating the functions of uncharacterized proteins by contextualizing them within cellular pathways and complexes. For MJ1223, several approaches are recommended:

• Yeast two-hybrid (Y2H) screening: This method can identify potential interaction partners of MJ1223 from genomic libraries. Special consideration must be given to the membrane-associated nature of MJ1223, which may require modified Y2H systems designed for membrane proteins .

• Bimolecular Fluorescence Complementation (BiFC): This technique allows visualization of protein interactions in living cells by fusing complementary fragments of a fluorescent protein to potential interaction partners. When the proteins interact, the fluorescent protein fragments come together to produce a signal .

• Co-Immunoprecipitation (CoIP): This approach can confirm physical interactions between MJ1223 and candidate proteins identified through other methods. Epitope tagging of MJ1223 may be necessary for antibody recognition .

• GST-pulldown assays: These in vitro assays can validate direct protein-protein interactions using purified components, helping distinguish direct interactions from those requiring additional binding partners .

• Proximity-based labeling approaches: Methods like BioID or APEX can identify proteins in close proximity to MJ1223 in its native cellular environment, capturing even transient interactions.

For thermophilic archaeal proteins like MJ1223, these methods may require adaptation to account for high-temperature stability and proper folding. Identifying interaction partners can provide significant insights into potential functions and integrate MJ1223 into known cellular pathways or complexes.

How might the extreme environment of M. jannaschii inform hypotheses about MJ1223 function?

The extreme environmental conditions where Methanocaldococcus jannaschii thrives provide valuable context for generating hypotheses about MJ1223's function:

• Thermal adaptation: As M. jannaschii lives in environments with temperatures ranging from 48-94°C, MJ1223 likely plays a role in cellular processes specifically adapted to high temperatures. Its amino acid composition may reflect thermostability adaptations common in proteins from thermophiles .

• Pressure considerations: Given that M. jannaschii was isolated from deep-sea hydrothermal vents at 2600m depth, MJ1223 may be involved in cellular processes that function under high hydrostatic pressure conditions .

• Membrane functionality: The predicted transmembrane nature of MJ1223 suggests potential roles in:

  • Maintaining membrane integrity under extreme conditions

  • Specialized transport systems for thermophilic growth

  • Sensing environmental changes at hydrothermal vents

  • Energy conservation mechanisms unique to methanogenic archaea

• Methanogenesis connection: As M. jannaschii is a methanogenic archaeon that grows by producing methane from carbon dioxide and hydrogen, MJ1223 might participate in methanogenesis-related processes. Its genomic context relative to known methanogenesis genes could provide supporting evidence .

• Unique archaeal biology: The protein could be involved in archaeal-specific metabolic pathways or information processing systems that have been identified in M. jannaschii, such as novel amino acid synthesis pathways or archaeal-specific DNA replication mechanisms .

These environmentally-informed hypotheses can guide targeted experimental approaches to investigate MJ1223's function within the context of archaeal extremophile biology.

What metabolic pathways should be investigated in relation to MJ1223?

Based on the information available about Methanocaldococcus jannaschii and the characteristics of MJ1223, several metabolic pathways warrant investigation:

To investigate these pathways effectively, genetic manipulation systems for M. jannaschii or closely related archaea would be ideal, though challenging. Alternative approaches include heterologous expression systems with reconstituted pathway components or computational pathway modeling incorporating protein interaction data.

Are there homologs of MJ1223 in other archaeal species?

Comparative genomic analysis reveals important evolutionary patterns for MJ1223:

M. jannaschii was the first archaeon to have its genome sequenced, establishing strong evidence for the three domains of life . For MJ1223 specifically, homology analyses should focus on:

A comprehensive homology analysis would require specialized search algorithms capable of detecting distant relationships and should include assessment of both sequence and structural similarities across the archaeal domain.

How can phylogenetic analysis help understand MJ1223's role?

Phylogenetic analysis offers several insights into understanding the potential function of uncharacterized proteins like MJ1223:

• Evolutionary history reconstruction: By building phylogenetic trees of MJ1223 and its homologs, researchers can trace the protein's evolutionary history and identify patterns of conservation or divergence that correlate with specific archaeal adaptations or metabolic capabilities.

• Co-evolutionary patterns: Proteins that function together often show correlated evolutionary patterns. Identifying proteins whose phylogenetic profiles correlate strongly with MJ1223 can suggest functional relationships or involvement in shared pathways.

• Lateral gene transfer assessment: Phylogenetic analysis can reveal whether MJ1223 shows evidence of horizontal gene transfer between archaeal lineages or even between domains, which can provide clues about its function in different cellular contexts.

• Rate of sequence evolution: The rate at which MJ1223 has evolved compared to other proteins can indicate selective pressures and functional constraints. Slowly evolving regions often correspond to functionally critical domains.

• Ancestral sequence reconstruction: Computational reconstruction of ancestral MJ1223 sequences can highlight conserved residues that have been maintained throughout evolution, suggesting their functional importance.

When combined with structural predictions and experimental data, phylogenetic analysis creates an evolutionary context for understanding MJ1223's role and can guide targeted functional studies based on patterns observed across archaeal lineages.

What can domain conservation tell us about MJ1223's potential function?

Domain conservation analysis, even for uncharacterized proteins like MJ1223, can provide valuable functional insights:

• Motif identification: While MJ1223 may lack characterized domains, sensitive pattern recognition algorithms might identify short conserved motifs or sequence signatures shared with proteins of known function.

• Structural element conservation: Secondary structure predictions can reveal conserved structural elements that may correspond to functional regions, even when primary sequence conservation is limited.

• Transmembrane topology patterns: For membrane proteins like MJ1223, conservation of transmembrane segment arrangements across homologs can suggest functional constraints related to membrane integration or transport activities.

• Conserved residues in 3D space: Mapping conserved residues onto predicted 3D structures can identify spatial clusters that often correspond to functional sites, such as binding pockets or catalytic centers.

• Correlated mutation analysis: Residues that show correlated patterns of mutation across homologs often interact functionally or structurally, providing clues about intramolecular interactions important for MJ1223 function.

The absence of recognized domains doesn't preclude functional inference; instead, it necessitates more sophisticated computational approaches that can detect subtle patterns of conservation associated with specific biochemical functions.

What are the main challenges in expressing archaeal proteins in heterologous systems?

Expressing archaeal proteins like MJ1223 in heterologous systems presents several significant challenges:

• Codon usage bias: Archaeal codon preferences often differ substantially from those of common expression hosts like E. coli, potentially leading to translation inefficiency or errors.

• Post-translational modifications: Archaeal proteins may require specific post-translational modifications not available in bacterial or eukaryotic expression systems.

• Thermostability requirements: Proteins from thermophilic archaea like M. jannaschii (which thrives at 48-94°C) may fold incorrectly at the lower temperatures used for mesophilic expression hosts .

• Membrane protein integration: For membrane proteins like MJ1223, proper insertion into host membranes may be compromised by differences in membrane composition and protein insertion machinery.

• Protein toxicity: Expression of foreign membrane proteins often causes toxicity in host cells, limiting yield and viability.

Effective solutions include:

• Codon optimization for the expression host

• Co-expression with archaeal chaperones

• Use of specialized E. coli strains designed for membrane protein expression

• Tight regulation of expression levels to minimize toxicity

• Expression as fusion proteins with solubility-enhancing tags

• Low-temperature induction combined with extended expression periods

These strategies can be implemented individually or in combination to overcome the challenges associated with heterologous expression of archaeal proteins.

How can researchers address problems with protein solubility for thermophilic proteins?

Addressing solubility challenges for thermophilic proteins like MJ1223 requires specialized approaches:

For transmembrane proteins like MJ1223, amphipathic polymers (SMALPs) or nanodiscs can create native-like membrane environments that maintain proper folding and stability. Additionally, directed evolution approaches can be employed to engineer variants with improved expression and solubility while retaining the native function.

These strategies should be approached systematically, with empirical testing of multiple conditions to identify optimal solubilization and stabilization parameters for the specific protein.

How can researchers validate predicted functions experimentally?

Validating predicted functions for uncharacterized proteins like MJ1223 requires a multi-faceted experimental approach:

• Gene deletion/complementation studies: Where genetic systems exist, creating knockout strains and observing phenotypic changes can provide insights into the protein's role. Complementation with wild-type or mutant variants can confirm specific functions.

• Heterologous expression and functional assays: Expressing MJ1223 in a heterologous system and testing specific biochemical activities based on computational predictions can validate suspected functions. For membrane proteins, reconstitution in liposomes may be necessary for activity .

• Site-directed mutagenesis: Mutating predicted key residues and assessing the impact on function can validate their importance and provide mechanistic insights. Conserved residues identified through evolutionary analysis are prime targets.

• Protein interaction confirmation: Experimentally validating predicted protein interactions using methods like yeast two-hybrid, BiFC, CoIP, or GST-pulldown assays can place the protein in a functional context .

• Localization studies: Determining the subcellular localization can support functional hypotheses. For membrane proteins like MJ1223, confirming membrane integration and topology is particularly informative.

• Structural validation: Obtaining experimental structural data through X-ray crystallography, NMR, or cryo-EM and comparing it with computational predictions can validate structural hypotheses that inform function.

• Comparative functional analysis: Testing homologs from related species can provide additional evidence for conserved functions and evolutionary significance.

The validation strategy should be guided by the specific functional hypotheses generated through computational analysis and should incorporate multiple complementary approaches to build a comprehensive functional profile.

How can researchers address contradictory experimental results when studying uncharacterized proteins?

When facing contradictory results in the study of uncharacterized proteins like MJ1223, researchers should employ a structured approach to resolution:

• Systematic validation of experimental conditions: Discrepancies often arise from differences in experimental conditions. Carefully document and standardize:

  • Protein preparation methods and purity assessments

  • Buffer compositions and pH conditions

  • Temperature and pressure parameters

  • Presence of cofactors or binding partners

  • Time-dependent effects

• Cross-validation with multiple methodologies: Employ orthogonal techniques to investigate the same functional question, as different methods have distinct biases and limitations .

• Structural context consideration: Contradictory functional results may stem from conformational differences. Evaluate whether the protein adopts different structures under varied experimental conditions.

• Biological relevance assessment: For thermophilic archaeal proteins like MJ1223, ensure that experimental conditions reflect the native environment (high temperature, pressure, etc.) when possible .

• Controlled variable isolation: When contradictions arise, systematically isolate variables until the source of discrepancy is identified.

• Literature contradictions framework: Develop a structured approach for addressing contradictions in published results:

  • Evaluate methodological differences

  • Assess data quality and statistical validity

  • Consider biological context variations

  • Contact study authors for clarification when necessary

• Collaborative verification: Engage multiple laboratories to independently verify controversial results using standardized protocols.

By approaching contradictions as opportunities for deeper understanding rather than obstacles, researchers can often uncover nuanced aspects of protein function that might otherwise remain hidden.

What emerging technologies might accelerate functional characterization of proteins like MJ1223?

Several cutting-edge technologies show promise for advancing the functional characterization of uncharacterized proteins like MJ1223:

• AI-driven protein function prediction: Machine learning approaches trained on vast protein datasets can detect subtle patterns associated with specific functions, potentially offering more accurate predictions than traditional bioinformatics methods .

• High-throughput activity screening: Microfluidic platforms that enable thousands of enzymatic assays in parallel could systematically test MJ1223 against diverse substrates to identify potential activities.

• Single-molecule analysis techniques: Methods that observe individual protein molecules can detect rare conformational states or transient interactions that might be missed in bulk experiments.

• Cryo-electron tomography: This technique can visualize proteins in their native cellular context, potentially revealing associations and localizations that inform function.

• Long-read sequencing technologies: These can improve the optimization of genome annotation and quantification of transcriptome abundance, providing new opportunities for discovering functional relationships .

• In-cell structural biology: Techniques like in-cell NMR and fluorescence-based structural sensors enable structural studies in living cells, providing more biologically relevant data.

• Synthetic biology approaches: Reconstructing minimal systems with defined components can test hypothesized functions in controlled environments.

These technologies, particularly when combined in integrated approaches, have the potential to dramatically accelerate our understanding of uncharacterized proteins from extremophile organisms like M. jannaschii.

How might understanding MJ1223 contribute to broader archaea research?

Characterizing MJ1223 has the potential to make significant contributions to archaeal research in several dimensions:

• Expanding the functional annotation of archaeal genomes: As one of many uncharacterized proteins in archaeal genomes, functional insights into MJ1223 could establish patterns that facilitate annotation of other uncharacterized proteins, gradually filling gaps in our understanding of archaeal biology .

• Understanding archaeal membrane biology: As a likely membrane protein, MJ1223 characterization could reveal archaeal-specific aspects of membrane protein function and integration, which differ significantly from bacterial and eukaryotic systems.

• Extremophile adaptation mechanisms: Insights from MJ1223 could illuminate how proteins from thermophilic archaea are adapted to function under extreme conditions, contributing to our understanding of life in extreme environments .

• Evolution of protein functions: Tracking the evolutionary history of MJ1223 across archaeal lineages could reveal patterns of functional diversification or conservation that inform our understanding of protein evolution.

• Archaeal metabolic networks: Placing MJ1223 within the metabolic framework of M. jannaschii could help complete our picture of archaeal-specific metabolic pathways, particularly those related to methanogenesis or energy conservation in extreme environments .

• Biotechnological applications: Functional characterization might reveal properties valuable for biotechnological applications, such as thermostable enzymes or novel membrane transport systems.

By contributing to these broader areas, MJ1223 characterization extends beyond a single protein to inform our fundamental understanding of archaeal biology and evolution.

What interdisciplinary approaches might be most fruitful for studying MJ1223?

The most promising interdisciplinary approaches for studying uncharacterized proteins like MJ1223 combine techniques from multiple scientific domains:

• Structural biology + computational chemistry: Integrating experimental structural determination with molecular dynamics simulations and quantum calculations can provide insights into functional mechanisms at atomic resolution.

• Genomics + biochemistry: Combining comparative genomic analyses with targeted biochemical assays can efficiently test function predictions derived from evolutionary patterns.

• Synthetic biology + systems biology: Reconstructing minimal systems with MJ1223 and measuring system-wide responses can reveal emergent functions not apparent from isolated studies.

• Biophysics + evolutionary biology: Correlating biophysical properties with evolutionary conservation patterns can reveal how structural and functional constraints have shaped MJ1223 evolution.

• Environmental microbiology + molecular biology: Understanding MJ1223 in the context of hydrothermal vent ecosystems and extremophile adaptations can provide ecological relevance to molecular findings .

• Proteomics + metabolomics: Multi-omics approaches can capture the broader impact of MJ1223 on cellular physiology by measuring changes in the proteome and metabolome when the protein is modified.

These interdisciplinary approaches leverage the strengths of different scientific domains to overcome the significant challenges associated with characterizing proteins from extremophile archaea, potentially yielding insights that would be inaccessible through any single discipline.