Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1495 (MJ1495)

What is Methanocaldococcus jannaschii and why is it significant for research?

M. jannaschii has adapted to thrive in extreme conditions including temperatures ranging from 48-94°C, high pressure, and moderate salinity . Its genome consists of a large circular chromosome (1.66 mega base pairs with a G+C content of 31.4%), along with large and small circular extra-chromosomes . As a model extremophile, it has contributed significantly to our understanding of archaeal-specific metabolic pathways, particularly those involved in methanogenesis.

What biochemical characteristics define M. jannaschii's metabolism?

M. jannaschii is a strict hydrogenotrophic methanogen, capable of growth only on carbon dioxide and hydrogen as primary energy sources, unlike many other methanococci that can also utilize formate . Its genome encodes numerous hydrogenases, including:

• 5,10-methenyltetrahydromethanopterin hydrogenase

• Ferredoxin hydrogenase (eha)

• Coenzyme F420 hydrogenase

Research has demonstrated that M. jannaschii does not produce intermediates expected in the pentose phosphate pathway but instead utilizes the ribulose monophosphate (RuMP) pathway for ribose-5-phosphate biosynthesis . This represents a distinct metabolic adaptation compared to many other organisms.

What is currently known about the uncharacterized protein MJ1495?

MJ1495 is a protein from M. jannaschii that remains functionally uncharacterized. According to available data, the full-length protein consists of 292 amino acids (1-292aa) and has been successfully expressed as a recombinant protein in E. coli with an N-terminal His-tag . The protein has been assigned the UniProt accession number Q58890 .

Despite being uncharacterized, the protein's successful recombinant expression indicates it can be produced in sufficient quantities for biochemical and structural studies. The absence of detailed functional annotation suggests significant research opportunities remain for characterizing this protein's role in M. jannaschii biology.

What expression systems work best for recombinant production of M. jannaschii proteins?

Based on available research, E. coli remains the predominant expression system for recombinant production of M. jannaschii proteins, including MJ1495 . When designing expression strategies, researchers should consider:

While E. coli expression is practical for initial characterization, researchers should be aware that mesophilic expression of thermophilic proteins may result in structural differences compared to the native state, potentially requiring additional validation in systems that better mimic the native environment.

What experimental design considerations are crucial when studying thermostable proteins from M. jannaschii?

When designing experiments for thermostable proteins like MJ1495, researchers must account for several unique factors:

• Temperature-dependent activity assessment: Activity assays should be performed across a temperature range (50-95°C) that reflects M. jannaschii's natural environment. Standard protocols must be modified to account for buffer stability at high temperatures.

• Structural analysis under native conditions: Consider techniques that enable structural studies at high temperatures:

  • Circular dichroism with temperature ramping

  • Temperature-controlled NMR studies

  • Molecular dynamics simulations parameterized for thermophilic conditions

• Thermodynamic stability measurements: Implement differential scanning calorimetry (DSC) and thermal shift assays with extended temperature ranges to accurately capture the high melting temperatures characteristic of archaeal proteins.

• Functional reconstitution: Design in vitro systems that replicate the physiological conditions of M. jannaschii:

• Control experiments: Include well-characterized thermophilic proteins as positive controls and mesophilic homologs as comparative references.

How can researchers approach the functional characterization of an uncharacterized protein like MJ1495?

A comprehensive approach to characterizing MJ1495 should combine multiple complementary strategies:

• Bioinformatic analysis:

  • Sequence homology searches against characterized proteins

  • Structural prediction using AlphaFold2 or similar tools

  • Genomic context analysis (examining neighboring genes)

  • Phylogenetic profiling to identify co-evolving protein families

• Structural characterization:

  • X-ray crystallography or cryo-EM for high-resolution structure

  • Identification of potential catalytic sites or binding pockets

  • Comparison with structural databases to identify similar folds

• Biochemical activity screening:

  • Substrate screening panels based on metabolic pathways known in M. jannaschii

  • Activity-based protein profiling with chemical probes

  • Metabolomics analysis comparing wild-type and overexpression strains

  • Protein-protein interaction studies using thermostable pull-down assays

• In vivo functional studies:

  • Gene knockout or CRISPR interference in model methanogens

  • Heterologous complementation in related species

  • Transcriptomic analysis to identify co-regulated genes

The integration of these approaches can provide convergent evidence for functional assignments, even when individual methods yield ambiguous results.

How can researchers resolve contradictory data when characterizing archaeal proteins?

When facing contradictory results in archaeal protein characterization, researchers should implement a systematic troubleshooting approach:

• Evaluate experimental conditions:

  • Confirm that assay conditions reflect the native environment of M. jannaschii

  • Verify protein stability under the tested conditions using orthogonal methods

  • Consider the impact of heterologous expression on protein folding and function

• Implement multiple methodological approaches:

  • Apply both in vitro and in vivo techniques to cross-validate findings

  • Utilize different detection technologies to rule out method-specific artifacts

• Resolve structure-function relationships:

  • Compare the recombinant protein structure with computational predictions

  • Examine whether post-translational modifications might explain functional differences

  • Consider oligomeric state and protein-protein interactions

• Decision matrix for resolving contradictions:

• Data integration framework:

  • Assign confidence weights to different evidence types

  • Develop testable hypotheses that could explain contradictions

  • Design critical experiments specifically targeting the points of contradiction

What unique considerations apply when investigating potential metabolic roles of MJ1495?

M. jannaschii possesses unique metabolic pathways, particularly in the biosynthesis of ribose-5-phosphate via the ribulose monophosphate (RuMP) pathway rather than the pentose phosphate pathway . When investigating MJ1495's potential metabolic role:

• Metabolic context analysis:

  • Determine if MJ1495 is co-regulated with genes involved in known pathways

  • Map M. jannaschii's metabolic network and identify gaps where uncharacterized proteins might function

  • Pay particular attention to archaeal-specific pathways like methanogenesis

• Comparative metabolomics:

  • Develop targeted metabolomics methods optimized for thermophilic metabolites

  • Compare metabolite profiles between wild-type and MJ1495 mutant strains

  • Track isotope-labeled substrates to identify altered flux patterns

• Enzyme activity screening:

  • Design assays around M. jannaschii's unique metabolic intermediates, such as:

    • Ribulose monophosphate pathway intermediates

    • Methanogenic cofactors

    • Archaeal-specific lipid precursors

• Inteins consideration:

  • Verify whether MJ1495 contains inteins, as M. jannaschii proteins frequently harbor these elements

  • Ensure recombinant protein preparation accounts for potential protein splicing

How can researchers apply structural genomics approaches to study MJ1495?

Structural genomics provides powerful tools for understanding proteins like MJ1495:

• Integrative structural biology workflow:

  • Begin with computational structure prediction using AlphaFold2

  • Validate predictions with circular dichroism and limited proteolysis

  • Pursue high-resolution structures using X-ray crystallography or cryo-EM

  • Perform molecular dynamics simulations under thermophilic conditions

• Functional site identification:

  • Use computational tools to identify potential catalytic sites or binding pockets

  • Apply docking studies with metabolites from M. jannaschii

  • Design site-directed mutagenesis experiments to test functional hypotheses

• Structural comparison strategy:

  • Compare MJ1495 structure to proteins with known functions

  • Identify structural motifs shared with characterized proteins

  • Examine structural adaptations specific to thermophilic environments

• Structure-guided experimental design:

  • Develop truncation constructs based on domain predictions

  • Target conserved residues for mutagenesis

  • Design protein engineering approaches to test functional hypotheses