Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1361 (MJ1361)

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

Methanocaldococcus jannaschii is an autotrophic archaeon originally isolated from a sediment sample collected at the base of a 2600m deep "white smoker" chimney located at 21°N on the East Pacific Rise . This extremophile grows under remarkable conditions: pressures up to more than 500 atmospheres and temperatures ranging from 48-94°C, with an optimal growth temperature near 85°C . As a strict anaerobe, it produces methane as part of its metabolic processes, making it a methanogen .

The significance of M. jannaschii extends to multiple research areas. Its complete genome (1.66-megabase pair) was sequenced, along with its 58- and 16-kilobase pair extrachromosomal elements, revealing 1738 predicted protein-coding genes . As one of the phylogenetically deeply rooted methanogens living in deep-sea hydrothermal vents, it represents an organism that derives energy exclusively from hydrogenotrophic methanogenesis, considered one of the oldest respiratory metabolisms on Earth . This makes M. jannaschii invaluable for evolutionary studies, extremophile biology, and understanding archaeal protein structure and function at extreme conditions.

What is currently known about the uncharacterized protein MJ1361?

MJ1361 is an uncharacterized protein from M. jannaschii with a complete amino acid sequence of 292 amino acids . The protein's exact function remains undetermined, as suggested by its "uncharacterized" designation. The amino acid sequence is available and has been used to produce recombinant versions of the protein for research purposes .

From the available data, we know that MJ1361 is encoded on the main chromosome of M. jannaschii. The protein sequence contains regions that may provide clues to its structure and function, though specific domain annotations and structural characterizations are still developing areas of research. Current research efforts are focused on expression, purification, and characterization of this protein to elucidate its biological role within the extremophilic context of M. jannaschii.

How does the extremophilic nature of M. jannaschii influence research approaches for MJ1361?

The extremophilic nature of M. jannaschii has profound implications for research on MJ1361. The native protein evolved to function optimally at high temperatures (near 85°C) and high pressures (up to 500 atmospheres), conditions that would denature most mesophilic proteins . This presents both challenges and opportunities for researchers.

When working with recombinant MJ1361, researchers must consider:

• Protein stability and folding: The recombinant protein may require high temperatures to achieve its native conformation and optimal activity. Standard room-temperature assays might not reveal the protein's true functional capabilities.

• Expression systems: Traditional E. coli expression systems may not produce properly folded protein, necessitating specialized conditions or alternative expression hosts.

• Purification strategies: Heat treatment can often be used as a purification step, as the thermostable MJ1361 will remain soluble while most E. coli proteins denature and precipitate.

• Activity assays: Enzyme activity, binding studies, or other functional assays should be conducted across a range of temperatures, including those approaching the optimal growth temperature of M. jannaschii (85°C).

• Structural studies: The protein structure may reveal adaptations specific to thermostability, such as increased ionic interactions, hydrophobic packing, or disulfide bonding.

These considerations make MJ1361 not only an object of study for its unknown function but also a model for understanding protein adaptation to extreme environments.

What expression systems have been successfully used for recombinant production of MJ1361?

While the search results don't specifically mention expression systems used for MJ1361, successful expression of archaeal proteins from M. jannaschii typically employs the following systems:

• E. coli expression systems: Most commonly used due to their simplicity and high yield. For archaeal proteins like MJ1361, specialized E. coli strains designed for expressing proteins with rare codons (such as Rosetta or CodonPlus strains) may be necessary due to codon bias differences between archaea and bacteria.

• Vector selection: Expression vectors with strong inducible promoters (T7, tac) are typically used. For thermostable proteins like those from M. jannaschii, vectors allowing cold-shock induction can sometimes improve folding by slowing down the expression process.

• Fusion tags: Affinity tags such as His6, GST, or MBP can facilitate purification. The choice of tag can significantly affect solubility and yield of the recombinant protein.

For optimal expression of MJ1361, researchers should consider:

Given the thermophilic nature of M. jannaschii, heat treatment (60-75°C) of the crude cell lysate can be an effective initial purification step, as most E. coli proteins will denature while MJ1361 should remain soluble.

What are the basic physicochemical properties of MJ1361?

Based on the amino acid sequence provided in the search results , the basic physicochemical properties of MJ1361 can be calculated or predicted:

These properties are important considerations for experimental design, particularly for purification strategies. The basic properties suggest that MJ1361:

• Is a medium-sized protein amenable to standard expression and purification techniques

• Has a relatively basic pI, which affects its behavior in ion exchange chromatography

• Likely possesses high thermal stability, reflecting its origin from a hyperthermophilic organism

• May require glycerol for stability in storage, as indicated in the recommended storage buffer

Researchers should validate these predicted properties experimentally, as post-translational modifications or structural factors may influence the actual behavior of the protein.

What structural motifs or domains have been identified in MJ1361?

While the search results don't explicitly identify specific structural motifs or domains in MJ1361, researchers can employ various bioinformatic approaches to predict functional regions:

Sequence analysis of MJ1361 using tools like InterPro, Pfam, SMART, or CDD might reveal conserved domains or motifs. Based on the amino acid sequence provided , several features may be present:

• Potential nucleic acid binding regions: The sequence contains multiple lysine and arginine residues (particularly in the regions "YPIKTRYIKRGEN" and "MPKEETLKHKQ"), which are often associated with nucleic acid binding domains.

• Hydrophobic core regions: Segments like "GVLAYLCYYWSK" and "GIISGIGVFGFILGR" contain clusters of hydrophobic residues that may form structural cores in the folded protein.

• Potential enzymatic motifs: The sequence "VVVMVADTDATY" contains aspartic acid residues that are common in various enzyme active sites.

To fully characterize the structural elements of MJ1361, researchers should consider:

• Secondary structure prediction using tools like PSIPRED or JPred

• 3D structure modeling using homology modeling or ab initio approaches

• Conducting experimental structure determination via X-ray crystallography, NMR, or cryo-EM

• Limited proteolysis experiments to identify stable domains within the protein

These approaches would provide insights into how MJ1361's structure relates to its yet-undetermined function in M. jannaschii.

How does temperature affect the folding and stability of recombinant MJ1361?

Temperature effects on folding:

• Low-temperature expression: May improve solubility but might lead to misfolded states or incomplete folding if the protein requires higher temperatures to achieve proper conformation.

• Heat activation: Many proteins from hyperthermophiles require a heat treatment step to attain their fully folded, active conformation after expression in mesophilic hosts.

Temperature effects on stability:
Proteins from hyperthermophiles like M. jannaschii typically show a stability curve with:

• Increased rigidity at mesophilic temperatures (20-40°C)

• Optimal flexibility and function at high temperatures (60-90°C)

• Denaturation at extremely high temperatures (generally >90-100°C)

Researchers studying MJ1361 should consider:

Understanding these temperature effects is crucial for proper handling, storage, and functional characterization of MJ1361.

Are there homologous proteins to MJ1361 in other archaeal or bacterial species?

Identifying homologs of MJ1361 across different species is essential for functional prediction and evolutionary analysis. While the search results don't specifically mention homologs, researchers can employ several approaches to identify them:

• BLAST searches: Using MJ1361's sequence to query protein databases can identify similar proteins across the tree of life.

• Profile-based searches: Tools like PSI-BLAST, HMMer, or profile HMMs can detect more distant homologs based on position-specific scoring matrices or hidden Markov models.

• Structural homology: Even with low sequence similarity, structural homology can be detected using tools like HHPred or Phyre2.

Potential types of homologs might include:

• Orthologs: Direct functional equivalents in other species, particularly in closely related methanogenic archaea

• Paralogs: Related proteins within M. jannaschii that arose through gene duplication

• Functionally similar proteins: Proteins with similar functions but divergent sequences in distantly related organisms

To systematically analyze homology relationships, researchers should compile data in a format similar to:

Genomic context analysis can provide additional functional insights, as genes with related functions often cluster together in prokaryotic genomes. Examining the genetic neighborhood of MJ1361 in M. jannaschii and its homologs in other species may reveal functional associations.

What are the recommended protocols for expression and purification of recombinant MJ1361?

Based on general practices for recombinant archaeal proteins and information from the search results, here is a recommended protocol for expression and purification of MJ1361:

Expression Protocol:

• Cloning:

  • Clone the MJ1361 gene into a suitable expression vector (e.g., pET series)

  • Include a purification tag (His6 recommended based on common practice)

  • Verify the construct by sequencing

• Transformation and Expression:

  • Transform into an E. coli expression strain (BL21(DE3) or Rosetta(DE3))

  • Grow cultures at 37°C to mid-log phase (OD600 ≈ 0.6-0.8)

  • Induce with 0.5 mM IPTG

  • Shift to lower temperature (18-25°C) for overnight expression

Purification Protocol:

• Cell Lysis:

  • Harvest cells by centrifugation

  • Resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Lyse cells by sonication or high-pressure homogenization

• Heat Treatment (exploit thermostability):

  • Heat clarified lysate to 65-75°C for 15-20 minutes

  • Remove precipitated E. coli proteins by centrifugation

• Affinity Chromatography:

  • Apply supernatant to Ni-NTA column

  • Wash with buffer containing 20-40 mM imidazole

  • Elute with 250-300 mM imidazole

• Further Purification:

  • Size exclusion chromatography using a Superdex 75/200 column

  • Ion exchange chromatography if additional purity is required

• Storage:

  • Exchange into Tris-based buffer with 50% glycerol

  • Store at -20°C for short-term or -80°C for long-term preservation

  • Avoid repeated freeze-thaw cycles

Protein Quality Assessment:

• SDS-PAGE to verify size and purity

• Western blot to confirm identity

• Mass spectrometry for precise molecular weight determination

• Circular dichroism to assess secondary structure content

• Dynamic light scattering to evaluate homogeneity

This protocol leverages the thermostable nature of MJ1361 as a purification advantage while following standard recombinant protein practices.

How can researchers assess the structural integrity of purified MJ1361?

Assessing the structural integrity of purified MJ1361 is crucial to ensure that experiments are conducted with properly folded, functionally relevant protein. Several complementary techniques can be employed:

Spectroscopic Methods:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm): Provides information about secondary structure content (α-helices, β-sheets)

  • Near-UV CD (250-350 nm): Reports on tertiary structure and environment of aromatic residues

  • Thermal melt CD: Monitors unfolding transitions as temperature increases

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence (MJ1361 contains tryptophans in its sequence)

  • Changes in fluorescence emission maximum indicate alterations in protein folding

  • Can be used to monitor thermal or chemical denaturation

• Fourier Transform Infrared Spectroscopy (FTIR):

  • Provides complementary information about secondary structure

  • Particularly useful for proteins with high β-sheet content

Hydrodynamic Methods:

• Size Exclusion Chromatography (SEC):

  • Confirms proper oligomeric state and absence of aggregation

  • Multi-angle light scattering (SEC-MALS) provides absolute molecular weight

• Dynamic Light Scattering (DLS):

  • Measures particle size distribution

  • Monitors homogeneity and potential aggregation

  • Particularly useful before crystallization attempts

Thermal Stability Analysis:

• Differential Scanning Calorimetry (DSC):

  • Measures heat changes during protein unfolding

  • Determines melting temperature (Tm) and enthalpy of unfolding

  • For thermostable proteins like MJ1361, may require high-temperature capability

• Thermal Shift Assays:

  • Uses fluorescent dyes (e.g., SYPRO Orange) that bind to hydrophobic regions exposed during unfolding

  • Can be performed in a qPCR instrument for high-throughput analysis

Functional Assessments:

• Activity assays (once function is determined)

• Ligand binding studies using techniques like isothermal titration calorimetry (ITC) or microscale thermophoresis (MST)

For a thermostable protein like MJ1361, researchers should perform these analyses at both standard and elevated temperatures to properly characterize the protein's stability profile across the relevant temperature range for M. jannaschii (48-94°C) .

What analytical techniques are most suitable for characterizing MJ1361's potential enzymatic activities?

Since MJ1361 is an uncharacterized protein, a systematic approach to identify potential enzymatic activities is necessary. Based on genomic context and sequence homology insights, the following analytical techniques can be employed:

Activity Screening Approaches:

• Broad-spectrum enzymatic assays:

  • Test for common enzymatic activities (hydrolase, transferase, oxidoreductase)

  • Use substrate libraries with colorimetric or fluorometric readouts

  • Screen across different pH values and temperatures (including the M. jannaschii optimal temperature of ~85°C)

• Nucleic acid interaction assays:

  • DNA/RNA binding assays (gel shift, fluorescence anisotropy)

  • Nuclease activity assays (given that M. jannaschii has genes for DNA degradation, like MJ1434)

  • Helicase or chaperone activity tests

• Metabolic pathway-related assays:

  • Focus on methanogenesis-related reactions (M. jannaschii's primary energy-generating pathway)

  • Test activities related to archaeal-specific metabolic processes

Advanced Analytical Techniques:

• Mass Spectrometry-Based Approaches:

  • Enzyme activity detection by mass spectrometry (EADMS)

  • Activity-based protein profiling (ABPP)

  • Substrate identification through untargeted metabolomics

• Structural Biology Methods:

  • X-ray crystallography with potential substrates or substrate analogs

  • NMR for detecting substrate binding and structural changes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify binding regions

• Computational Predictions:

  • Active site prediction through structural modeling

  • Substrate docking simulations

  • Quantum mechanics/molecular mechanics (QM/MM) simulations of potential reactions

Systematic Activity Testing Protocol:

For each condition, researchers should test:

• Hydrolytic activities against various substrates

• Redox reactions with potential electron donors/acceptors

• Transferase activities with relevant metabolic intermediates

• Binding of common cofactors and metabolites

Given M. jannaschii's extremophilic nature, enzyme assays should be conducted under anaerobic conditions at high temperatures, ideally replicating the organism's native environment as closely as possible.

What computational approaches can help predict the function of MJ1361?

Given the uncharacterized nature of MJ1361, computational approaches offer valuable insights into potential functions. Researchers can employ a multi-layered strategy combining various predictive methods:

Sequence-Based Approaches:

• Motif and Domain Identification:

  • Search against domain databases (Pfam, InterPro, CDD)

  • Identify functional motifs using PROSITE or PRINTS

  • Detect distant homologies using profile-based methods (PSI-BLAST, HMMer)

• Evolutionary Analysis:

  • Phylogenetic profiling to identify co-evolving genes

  • Conservation analysis to identify functionally important residues

  • Analysis of synonymous/non-synonymous substitution rates

• Genomic Context:

  • Examine neighboring genes in the M. jannaschii genome

  • Identify operons or gene clusters that might suggest functional relationships

  • Compare genomic context across related archaeal species

Structure-Based Approaches:

• Homology Modeling:

  • Generate 3D structural models using templates from related proteins

  • Use threading approaches like I-TASSER or Phyre2 when sequence homology is low

  • Validate models using quality assessment tools (PROCHECK, VERIFY3D)

• Structural Comparison:

  • Compare predicted structure to known protein structures using DALI or TM-align

  • Identify structural similarities that might not be apparent from sequence alone

  • Analyze structural motifs associated with specific functions

• Binding Site Prediction:

  • Identify potential active sites using tools like CASTp or POCASA

  • Predict ligand binding pockets and their properties

  • Use FTMap or SiteMap to predict binding hot spots

Integrated Approaches:

• Protein-Protein Interaction Prediction:

  • Use methods like STRING or PIPS to predict interaction partners

  • Infer function through guilt-by-association with known proteins

• Protein Function Prediction Servers:

  • Employ meta-servers like COFACTOR or ProFunc that integrate multiple approaches

  • Use machine learning approaches trained on diverse features

• Pathway Mapping:

  • Map potential functions to known biochemical pathways in archaea

  • Focus on methanogenesis and other pathways critical to M. jannaschii

By applying these computational approaches systematically and integrating the results, researchers can generate testable hypotheses about MJ1361's function that can guide experimental design.

If attempting to crystallize MJ1361 for structural studies, what special considerations apply to proteins from hyperthermophiles?

Crystallizing proteins from hyperthermophiles like M. jannaschii presents unique challenges and opportunities. The following considerations should guide crystallization attempts for MJ1361:

Advantages of Crystallizing Thermostable Proteins:

• Intrinsic Stability:

  • Higher conformational rigidity often leads to better crystal formation

  • Reduced flexibility may result in higher-resolution diffraction

  • Greater resistance to oxidation during crystallization process

• Enhanced Solubility:

  • Many thermostable proteins remain soluble at high concentrations

  • Often less prone to non-specific aggregation

  • May allow for a wider range of crystallization conditions

Special Considerations and Strategies:

• Temperature Optimization:

  • Test crystallization at elevated temperatures (30-60°C)

  • Consider temperature as an additional crystallization parameter

  • Use temperature-controlled crystallization systems

• Buffer Selection:

  • Choose buffers with stability at higher temperatures

  • Consider buffers that mimic the intracellular environment of M. jannaschii

  • Test a wider pH range, as extremophile proteins often function in unusual pH environments

• Additive Screening:

  • Include ions found in deep-sea hydrothermal vents (Mg²⁺, Fe²⁺/³⁺)

  • Test specific cofactors that might stabilize the native conformation

  • Consider archaeal-specific lipids or membrane components

• Surface Engineering:

  • If initial crystallization attempts fail, consider surface entropy reduction

  • Modify surface-exposed lysine clusters to alanines to promote crystal contacts

  • Design constructs with flexible termini removed

Practical Crystallization Protocol:

Alternative Structural Approaches:
If crystallization proves challenging, consider:

• Cryo-electron microscopy, especially if MJ1361 forms oligomeric assemblies

• NMR spectroscopy for structure determination in solution

• Small-angle X-ray scattering (SAXS) for low-resolution envelope determination

The thermostability of MJ1361 can be advantageous during data collection, potentially allowing for room-temperature data collection which can reveal functionally relevant conformational states that might be obscured in cryo-cooled crystals.

How do researchers address the challenge of determining function for uncharacterized proteins like MJ1361?

Determining the function of uncharacterized proteins like MJ1361 requires an integrated, multidisciplinary approach that combines computational predictions with targeted experimental validation:

Systematic Functional Discovery Workflow:

• Initial Computational Prediction:

  • Generate hypotheses about function using bioinformatic approaches

  • Identify potential binding partners, substrates, or activities

  • Classify the protein into broad functional categories

• Strategic Experimental Design:

  • Develop experiments to test multiple functional hypotheses in parallel

  • Prioritize experiments based on computational confidence scores

  • Design controls that can distinguish between alternative functions

• Iterative Refinement:

  • Use initial experimental results to refine computational models

  • Narrow the functional search space based on positive or negative results

  • Develop increasingly specific experiments as knowledge accumulates

Experimental Approaches for Functional Determination:

• Gene Context Analysis:

  • Examine the genomic neighborhood of MJ1361 in M. jannaschii

  • Identify operons or gene clusters that might suggest function

  • Compare with syntenic regions in related organisms

• Gene Deletion or Silencing:

  • If a genetic system exists for M. jannaschii , create deletion mutants

  • Assess phenotypic changes under various growth conditions

  • Perform transcriptomic or metabolomic profiling of mutants

• Protein Interaction Studies:

  • Identify binding partners through pull-down assays or yeast two-hybrid

  • Characterize protein complexes using mass spectrometry

  • Map interaction networks to infer function from known partners

• Substrate Identification:

  • Use activity-based protein profiling to identify potential substrates

  • Screen metabolite libraries for binding using thermal shift assays

  • Apply metabolomic approaches to identify accumulating substrates in deletion mutants

Case Study Approach:

For uncharacterized archaeal proteins, researchers can examine successful functional determination case studies. For example, with MJ1361:

• Start with sequence analysis and structural modeling

• Generate recombinant protein and confirm proper folding

• Screen for enzymatic activities based on predicted functional class

• Validate in vivo relevance through genetic approaches where possible

• Confirm biological context through physiological studies

The key to success is maintaining a flexible, iterative approach where computational and experimental methods inform each other, gradually narrowing the functional possibilities until the biological role of MJ1361 is determined.

What can comparative genomics reveal about the potential function of MJ1361?

Comparative genomics provides powerful insights into the potential function of uncharacterized proteins by examining patterns of conservation, gene neighborhood, and co-evolution across species. For MJ1361, researchers can employ several comparative genomic approaches:

Conservation Analysis:

• Phylogenetic Distribution:

  • Determine which lineages possess MJ1361 homologs

  • Map presence/absence patterns onto the archaeal phylogenetic tree

  • Correlate with ecological niches and metabolic capabilities

• Sequence Conservation Patterns:

  • Identify highly conserved residues across homologs

  • Map conservation onto structural models to locate potential functional sites

  • Analyze patterns of conservative versus non-conservative substitutions

Genomic Context Analysis:

• Gene Neighborhood Conservation:

  • Examine genes consistently found adjacent to MJ1361 homologs

  • Identify conserved operons or gene clusters across species

  • Look for functional relationships among neighboring genes

• Fusion Events:

  • Search for proteins where MJ1361-like domains are fused with domains of known function

  • These fusion events can suggest functional relationships (Rosetta stone principle)

Co-Evolution Analysis:

• Gene Co-occurrence Patterns:

  • Identify genes that show similar phylogenetic profiles to MJ1361

  • These may participate in the same biological process

• Correlated Mutations:

  • Detect co-evolving residues within MJ1361 and between MJ1361 and other proteins

  • These can indicate functional or physical interactions

Comparative Experimental Data:

By integrating these comparative genomic approaches, researchers can develop more informed hypotheses about MJ1361's function. For example, if MJ1361 homologs are consistently found near genes involved in methanogenesis across multiple methanogenic archaea, this would suggest a potential role in this pathway, which is central to M. jannaschii's metabolism .

How does the adaptation to extreme environments influence protein structure and function in M. jannaschii proteins like MJ1361?

The adaptation of M. jannaschii to extreme environments (high temperature, high pressure, strict anaerobiosis) has profound effects on protein structure and function. For MJ1361, these adaptations likely include:

Thermostability Adaptations:

• Amino Acid Composition:

  • Increased proportion of charged amino acids (Glu, Arg, Lys) for ionic interactions

  • Higher content of hydrophobic residues in the protein core

  • Reduced occurrence of thermolabile residues (Asn, Gln, Cys, Met)

• Structural Features:

  • More extensive ion pair networks, especially salt bridges

  • Tighter hydrophobic packing in the protein core

  • Reduced surface-to-volume ratio

  • Shortened loop regions with increased rigidity

• Secondary Structure Preferences:

  • Higher proportion of α-helices compared to β-sheets

  • More extensive hydrogen bonding networks

  • Optimized helix dipole stabilization

Pressure Adaptation Mechanisms:

• Structural Modifications:

  • Reduced internal cavities and voids

  • Multimeric assembly stabilization

  • Modified subunit interactions in oligomeric proteins

• Functional Adaptations:

  • Potential altered reaction volumes for enzymatic activities

  • Modified conformational changes during catalytic cycles

  • Pressure-resistant active site architecture

Metabolic Context Adaptations:

• Cofactor Binding:

  • Specialized binding pockets for archaeal-specific cofactors

  • Adaptations for methanogenesis-related molecules

  • Modified metal coordination environments

• Protein-Protein Interactions:

  • Specialized interfaces for interactions at high temperatures

  • Stabilized complexes that maintain function under extreme conditions

Analyzing MJ1361 for Extremophilic Adaptations:

Examining MJ1361's sequence reveals features consistent with thermophilic adaptation:

• High content of charged residues (numerous Lys, Arg, Glu residues)

• Presence of extensive hydrophobic regions (e.g., "GVLAYLCYYWSK" and "GIISGIGVFGFILGR")

• Limited occurrence of thermolabile residues

These adaptations not only ensure stability under extreme conditions but may also provide clues about MJ1361's function. Proteins involved in core metabolic processes in extremophiles often show more pronounced adaptive features than those involved in secondary functions.

What insights can be gained from studying MJ1361 in the context of archaeal evolution?

Studying MJ1361 in the context of archaeal evolution can provide valuable insights into both protein function and the broader evolutionary history of archaea:

Archaeal Protein Evolution:

• Archaeal-Specific Adaptations:

  • Identifying features unique to archaeal homologs versus bacterial counterparts

  • Understanding protein adaptations specific to the archaeal cellular environment

  • Characterizing archaeal-specific functional innovations

• Domain Architecture Evolution:

  • Tracing the acquisition or loss of domains throughout archaeal evolution

  • Identifying fusion events that link MJ1361-like domains with other functional modules

  • Mapping the diversification of related protein families

Evolutionary Origin and History:

• Phylogenetic Placement:

  • Determining if MJ1361 represents an ancient protein present in the last archaeal common ancestor

  • Assessing whether it was horizontally transferred between lineages

  • Evaluating its conservation across different archaeal phyla

• Selection Pressure Analysis:

  • Calculating Ka/Ks ratios to identify regions under purifying or positive selection

  • Correlating selection patterns with functional importance

  • Determining if MJ1361 has undergone adaptive evolution in certain lineages

Implications for Early Life:

• Deep Branching Position:

  • M. jannaschii occupies a phylogenetically deep position among methanogens

  • MJ1361 might represent a protein adapted to conditions resembling early Earth

  • Study may provide insights into proteins functioning in primitive biological systems

• Methanogenesis Evolution:

  • If MJ1361 is involved in methanogenesis, it contributes to understanding one of Earth's oldest metabolic pathways

  • May reveal adaptations specific to archaeal energy conservation mechanisms

  • Could illuminate the evolution of anaerobic metabolism

Comparative Analysis Table:

By placing MJ1361 in this evolutionary context, researchers can not only better understand its specific function but also contribute to broader questions about protein evolution in extremophiles, the development of archaeal-specific metabolic pathways, and potentially even the conditions of early life on Earth.

What challenges are typically encountered when working with recombinant proteins from hyperthermophiles, and how can they be addressed?

Working with recombinant proteins from hyperthermophiles like M. jannaschii presents several unique challenges. Here are the major obstacles researchers face with proteins like MJ1361 and strategies to overcome them:

Challenge 1: Expression in Mesophilic Hosts

Problem: Codon bias differences, toxicity to host, improper folding at lower temperatures.

Solutions:

• Use codon-optimized synthetic genes for E. coli expression

• Employ specialized E. coli strains (Rosetta, CodonPlus) designed for rare codons

• Test multiple expression vectors with different promoter strengths

• Use cold-shock inducible systems and lower expression temperatures

• Consider archaeal expression systems for particularly challenging proteins

Challenge 2: Proper Folding and Activity

Problem: Proteins evolved to fold at high temperatures may misfold at lower temperatures.

Solutions:

• Implement post-expression heat treatment (60-80°C) to promote proper folding

• Co-express with chaperones from thermophilic organisms

• Use fusion partners that enhance solubility (SUMO, MBP, thioredoxin)

• Refold from inclusion bodies under controlled temperature ramping

• Include appropriate metal ions or cofactors during refolding

Challenge 3: Activity Assessment

Problem: Standard assay conditions may not reveal the true activity of thermophilic enzymes.

Solutions:

• Perform assays at elevated temperatures (60-85°C) that mimic native conditions

• Use thermostable assay components and buffers

• Implement specialized equipment for high-temperature enzyme assays

• Design control experiments with known thermostable enzymes

• Consider pressure effects for deep-sea organisms like M. jannaschii

Challenge 4: Stability During Storage and Handling

Problem: While generally more stable, improper handling can still compromise protein quality.

Solutions:

• Store in optimized buffers with glycerol as recommended for MJ1361

• Avoid repeated freeze-thaw cycles

• Consider lyophilization for long-term storage

• Validate protein integrity after storage using activity assays and structural analysis

Challenge 5: Structural Studies

Problem: Crystallization conditions optimal for mesophilic proteins may not work for thermophilic proteins.

Solutions:

• Screen crystallization conditions at elevated temperatures

• Include ligands or substrates that may stabilize specific conformations

• Consider surface entropy reduction for improved crystal packing

• Employ cryo-EM as an alternative approach

Practical Implementation Table:

By implementing these specialized approaches, researchers can overcome the unique challenges presented by hyperthermophilic proteins like MJ1361 and successfully characterize their structural and functional properties.

How should researchers approach the challenge of determining substrate specificity for an uncharacterized protein like MJ1361?

Determining substrate specificity for an uncharacterized protein like MJ1361 requires a systematic approach combining computational predictions and experimental screening. Here's a comprehensive strategy:

Computational Substrate Prediction:

• Structural Analysis:

  • Identify potential binding pockets and active sites

  • Analyze electrostatic and hydrophobic properties of these sites

  • Compare with known structures of functionally characterized proteins

• Homology-Based Prediction:

  • Identify distant homologs with known substrate preferences

  • Analyze conservation of substrate-binding residues

  • Use homology to generate initial substrate hypotheses

• Docking Simulations:

  • Perform virtual screening of metabolite libraries

  • Dock potential substrates into predicted binding sites

  • Rank compounds based on binding energy predictions

Experimental Substrate Identification:

• Broad-Spectrum Substrate Screening:

  • Design a hierarchical screening approach, starting with substrate classes

  • Test substrate categories (e.g., sugars, amino acids, nucleotides, lipids)

  • Narrow down to specific compounds based on initial hits

• Activity-Based Methods:

  • Activity-based protein profiling with probe libraries

  • Substrate activity screening using compound libraries

  • Monitor product formation using mass spectrometry or spectroscopic methods

• Binding Assays:

  • Thermal shift assays to identify stabilizing ligands

  • Isothermal titration calorimetry for direct binding measurements

  • Surface plasmon resonance for binding kinetics

  • Fluorescence-based binding assays for high-throughput screening

Substrate Validation Approaches:

• Enzyme Kinetics:

  • Determine kinetic parameters (Km, kcat, catalytic efficiency)

  • Compare efficiency across potential substrates

  • Test at multiple temperatures (including M. jannaschii's optimal growth temperature)

• Structural Confirmation:

  • Co-crystallization with substrates or substrate analogs

  • NMR titration experiments to map binding interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

• Mutagenesis Studies:

  • Mutate predicted substrate-binding residues

  • Measure effects on catalysis and binding

  • Establish structure-function relationships

Substrate Screening Strategy Table:

For MJ1361, researchers should begin with computational predictions based on sequence analysis and structural modeling, followed by experimental validation using thermal stability assays and activity screens with potential substrate classes. Given M. jannaschii's extremophilic lifestyle, special attention should be paid to unique metabolites found in hyperthermophilic methanogens.

What interdisciplinary approaches are most valuable for characterizing proteins like MJ1361 from extremophilic organisms?

Characterizing proteins from extremophiles like M. jannaschii requires interdisciplinary approaches that span multiple scientific disciplines. For MJ1361, integrating these diverse perspectives can provide comprehensive insights:

Integrative Approaches for MJ1361 Characterization:

• Structural Biology and Biophysics:

  • High-resolution structure determination (X-ray crystallography, cryo-EM, NMR)

  • Biophysical characterization under extreme conditions

  • Analysis of conformational dynamics at high temperatures

  • Specialized techniques for studying thermostable proteins

• Computational Biology:

  • Molecular dynamics simulations at elevated temperatures

  • Quantum mechanics/molecular mechanics for reaction mechanism prediction

  • Machine learning approaches for function prediction

  • Systems biology modeling of metabolic pathways

• Environmental Microbiology:

  • Understanding the ecological context of M. jannaschii

  • Simulating deep-sea hydrothermal vent conditions in laboratory settings

  • Correlating protein function with environmental adaptations

  • Studying protein expression under different environmental stresses

• Evolutionary Biology:

  • Comparative genomics across archaeal lineages

  • Reconstruction of ancestral protein sequences

  • Molecular clock analyses to date protein origins

  • Correlation of protein features with evolutionary history

• Biotechnology and Synthetic Biology:

  • Engineering MJ1361 for enhanced stability or altered specificity

  • Development of applications leveraging thermostability

  • Creation of chimeric proteins with mesophilic homologs

  • Expression optimization in heterologous hosts

Collaborative Research Framework:

Implementation Strategy:

• Cross-disciplinary team formation:

  • Core expertise in protein biochemistry and structural biology

  • Computational specialists for in silico analysis

  • Microbiology experts for physiological context

  • Evolutionary biologists for phylogenetic perspective

• Integrated experimental design:

  • Parallel computational and experimental approaches

  • Iterative refinement of hypotheses

  • Multi-scale analysis from atomic to systems level

• Technology integration:

  • High-temperature bioreactors for native-like conditions

  • Advanced imaging for protein localization in archaeal cells

  • High-performance computing for simulation under extreme conditions

  • Mass spectrometry for comprehensive interaction mapping

By leveraging these interdisciplinary approaches, researchers can develop a comprehensive understanding of MJ1361's structure, function, and evolutionary significance. This integrated strategy is particularly valuable for proteins from extremophiles, where traditional approaches developed for mesophilic proteins may be insufficient to capture the unique adaptations and functional characteristics of these fascinating biomolecules.