Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_2291 (AF_2291)

What is Archaeoglobus fulgidus and why is AF_2291 significant for research?

Archaeoglobus fulgidus is a hyperthermophilic, sulfate-reducing archaeon that thrives in extreme environments with temperatures between 60-95°C. It belongs to the domain Archaea and has become a model organism for studying adaptations to extreme conditions. AF_2291 is one of several uncharacterized proteins in this organism that may provide insights into unique biochemical pathways or structural adaptations enabling survival in extreme environments. Similar hypothetical proteins from A. fulgidus, such as AF2241, have been shown to adopt interesting structural folds with potential novel functions, suggesting AF_2291 may likewise have distinctive properties worthy of investigation . The research significance of AF_2291 lies in its potential to reveal new insights into archaeal biology, protein evolution, and possibly novel enzymatic activities adapted to extreme conditions.

What is known about the physical properties of recombinant AF_2291?

Recombinant AF_2291 is available as a His-tagged protein with purity greater than 90% as determined by SDS-PAGE analysis . While detailed physical characterization specific to AF_2291 is limited in the current literature, we can draw parallels from studies of other A. fulgidus proteins. For instance, many proteins from hyperthermophilic archaea exhibit remarkable thermostability, often retaining structure and function at temperatures exceeding 80°C. Based on comparable proteins from this organism, AF_2291 likely exhibits resistance to denaturation under high temperatures, potentially possesses disulfide bonds or salt bridges that contribute to structural stability, and may have a compact folding pattern optimized for extreme environments.

How does AF_2291 relate to other characterized proteins in Archaeoglobus fulgidus?

While direct comparative studies between AF_2291 and other A. fulgidus proteins are not extensively documented in the provided search results, we can draw some connections based on research on other proteins from this organism. For example, AF2241, another hypothetical protein from A. fulgidus, has been characterized as adopting a cyclophilin-like fold with a beta-barrel core composed of eight beta-strands, one alpha-helix, and one 3(10) helix . Despite structural similarity to human cyclophilin A, AF2241 likely has distinct biological functions based on differences in the putative active site . Similarly, AF_1257 has been characterized as a tRNA methyltransferase that interacts with AF_RS01245 (AfTrm112) . These examples illustrate the diversity of protein structures and functions within this organism, suggesting that AF_2291 might likewise have unique structural or functional properties yet to be fully characterized.

What are the optimal conditions for expressing recombinant AF_2291 in E. coli?

Based on successful expression strategies for other A. fulgidus proteins, the optimal conditions for expressing recombinant AF_2291 in E. coli typically involve using specialized expression strains such as BL21(DE3) or Rosetta(DE3) that are designed to accommodate the codon usage bias found in archaeal genes. Drawing from methodologies used for similar proteins, expression should be conducted at lower temperatures (16-25°C) following induction to promote proper folding, despite A. fulgidus being a thermophile. The expression vector should contain a strong promoter (such as T7) and appropriate fusion tags to facilitate purification.

For example, with the related A. fulgidus protein AF_1257, researchers successfully used a pET21-a vector with the gene cloned between NdeI and XhoI sites, incorporating a C-terminal His6-tag . Induction protocols typically employ 0.5-1.0 mM IPTG when culture density reaches OD600 of 0.6-0.8, followed by expression at reduced temperatures for 16-20 hours to maximize protein yield while maintaining solubility. For thermostable proteins like those from A. fulgidus, inclusion of heat shock steps (50-60°C for 20-30 minutes) during purification can help eliminate less stable E. coli proteins.

What purification strategies are most effective for obtaining high-purity AF_2291?

The most effective purification strategy for AF_2291 likely involves a multi-step approach. Based on successful purification of other His-tagged archaeal proteins, initial capture should utilize immobilized metal affinity chromatography (IMAC) with Ni-NTA or TALON resins. The commercially available AF_2291 is reported to achieve >90% purity through appropriate purification methods . The specific protocol would typically include:

• Cell lysis under native conditions using either sonication or high-pressure homogenization in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors.

• IMAC purification with a gradient elution using increasing imidazole concentrations (typically 20-250 mM).

• A heat treatment step (65-80°C for 15-20 minutes) can be particularly effective for A. fulgidus proteins, as it denatures most E. coli proteins while leaving the thermostable archaeal protein intact.

• Size exclusion chromatography as a polishing step, using buffers optimized for downstream applications.

• Optional ion exchange chromatography if higher purity is required for crystallographic or other structural studies.

For maximal stability during purification, buffers typically include glycerol (5-10%) and potentially reducing agents like DTT or β-mercaptoethanol to maintain any critical thiol groups in the reduced state.

What are the challenges in expressing membrane-associated archaeal proteins and how can they be overcome?

If AF_2291 has membrane-associated properties (which is not explicitly stated in the available data), researchers would face several challenges in its expression and purification. Membrane proteins from archaea present particular difficulties due to their hydrophobicity, the differences in membrane composition between archaea and expression hosts like E. coli, and potential toxicity to the host cells.

To overcome these challenges, several approaches can be employed:

• Use of specialized expression vectors with tightly controlled promoters to prevent toxic accumulation during the growth phase.

• Incorporation of fusion partners that enhance solubility, such as MBP (maltose-binding protein), SUMO, or Mistic.

• Expression in specialized E. coli strains designed for membrane proteins, such as C41(DE3) or C43(DE3).

• Co-expression with archaeal chaperones to facilitate proper folding.

• Use of mild detergents (DDM, LDAO, or Fos-choline derivatives) during lysis and purification to solubilize the protein while maintaining native structure.

• Alternative expression systems such as cell-free protein synthesis that can directly incorporate the protein into nanodiscs or liposomes.

• If traditional approaches fail, expression in archaeal hosts that provide the native membrane environment might be necessary, though these systems are less developed than E. coli-based expression.

What structural determination methods are most suitable for AF_2291?

For structural determination of AF_2291, researchers should consider multiple complementary approaches based on successful studies of similar archaeal proteins. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy represent the gold standard methods, with cryo-electron microscopy (cryo-EM) emerging as a powerful alternative for certain protein classes.

For NMR studies, the approach used for AF2241 provides a valuable template, where structural determination revealed a cyclophilin-like fold with a beta-barrel core . This approach is particularly effective for smaller proteins (typically <30 kDa). The methodology typically involves:

• Expression in minimal media supplemented with 15N-ammonium chloride and 13C-glucose to produce isotopically labeled protein.

• Collection of a suite of 2D and 3D NMR experiments (HSQC, NOESY, TOCSY, etc.) to assign resonances and derive distance constraints.

• Computational structure calculation using software packages like CYANA or XPLOR-NIH.

For X-ray crystallography, researchers would need to:

• Screen numerous crystallization conditions using commercial kits to identify initial hits.

• Optimize promising conditions by varying precipitant concentration, pH, temperature, and additives.

• Incorporate selenomethionine for phase determination if molecular replacement is not feasible.

• Collect diffraction data at synchrotron facilities and process using standard crystallographic software.

The choice between these methods should be guided by protein size, stability, and behavior during initial characterization.

How can computational approaches assist in predicting the structure and function of AF_2291?

Computational approaches offer valuable insights for uncharacterized proteins like AF_2291, particularly when experimental structural data is limited. A comprehensive computational strategy would include:

• Homology modeling based on related proteins of known structure, similar to how AF2241 was found to have structural similarity to human cyclophilin A despite low sequence identity . Modern tools like AlphaFold2 or RoseTTAFold can produce remarkably accurate models even for proteins with distant homologs.

• Structural motif recognition to identify conserved folds that might suggest function, as demonstrated by the identification of the cyclophilin-like fold in AF2241 through structural similarity searches .

• Active site prediction through analysis of conserved residues and electrostatic potential mapping, which was effective in identifying a putative active site in AF2241 composed of 9 conserved residues in a negatively charged pocket .

• Molecular dynamics simulations to assess structural stability under different temperature conditions, particularly relevant for thermostable proteins from A. fulgidus.

• Protein-protein interaction prediction using tools like PRISM or InterPreTS to identify potential binding partners.

• Functional annotation using integrated approaches that combine structure prediction with genomic context analysis, phylogenetic profiling, and co-expression data.

These computational approaches can generate testable hypotheses about AF_2291's structure and function that guide subsequent experimental design.

What insights can be gained from comparing AF_2291 to structurally characterized proteins like AF2241?

Comparative analysis between AF_2291 and structurally characterized proteins like AF2241 can provide valuable insights into potential structural features and functions. AF2241 was found to adopt a cyclophilin-like fold despite low sequence similarity to human cyclophilin A . This finding illustrates the phenomenon of divergent evolution, where proteins maintain structural similarity while sequence identity diminishes over evolutionary time.

Key insights that might be gained from such comparisons include:

• Identification of conserved structural motifs that might indicate shared ancestry or convergent evolution to perform similar functions in extreme environments.

• Analysis of the electrostatic surface properties, as AF2241 contains a negatively charged pocket with 9 conserved residues that likely constitutes its active site, distinct from the PPIase catalytic site of human cyclophilin A .

• Recognition of structural adaptations that contribute to thermostability, such as increased hydrophobic core packing, additional salt bridges, or disulfide bonds.

• Prediction of oligomerization states and potential interaction interfaces by comparing quaternary structures.

• Identification of substrate binding pockets or cavities that might suggest potential ligands or reaction specificity.

For meaningful comparison, structural alignment tools (such as DALI or TM-align) should be employed to quantify structural similarity, followed by detailed analysis of conserved residues in potential functional regions.

What enzymatic assays would be appropriate to test potential functions of AF_2291?

Without specific knowledge of AF_2291's function, a systematic approach to enzymatic characterization is necessary. Based on studies of other uncharacterized archaeal proteins, the following assays would be appropriate to explore potential functions:

• General Enzymatic Activity Screens:

  • Hydrolase activity assays using fluorogenic or chromogenic substrates

  • Oxidoreductase activity with various electron acceptors/donors

  • Transferase activity assays with isotopically labeled substrates

  • Isomerase activity tests with appropriate model substrates

• Targeted Assays Based on Structural Similarity:
  If structural analysis reveals similarity to proteins like AF2241 (which has a cyclophilin-like fold), specific assays for peptidyl-prolyl isomerase activity would be warranted, despite the lack of conservation in catalytic residues . Similarly, if homology to methyltransferases like AF_1257 is detected, assays for methyl transfer to various substrates (RNA, DNA, proteins) should be performed .

• Thermophile-Specific Considerations:
  All assays should be conducted at elevated temperatures (60-85°C) reflective of A. fulgidus' natural environment, with appropriate controls for substrate stability. Buffer systems with high temperature stability (phosphate, HEPES, MOPS) should be used, and potential cofactors stable at high temperatures should be included.

• Activity Modulation Tests:

  • Metal dependency using EDTA chelation followed by metal ion supplementation

  • Redox sensitivity using reducing and oxidizing agents

  • pH optima determination across a range relevant to hyperthermophiles (typically pH 5-8)

How can protein-protein interaction studies help elucidate the function of uncharacterized proteins like AF_2291?

Protein-protein interaction (PPI) studies can provide critical insights into the functional role of uncharacterized proteins by placing them within cellular pathways and complexes. For AF_2291, several methodologies are particularly relevant:

• Pull-down Assays and Co-immunoprecipitation:
  Using the His-tagged recombinant AF_2291 as bait to identify interaction partners from A. fulgidus lysate, followed by mass spectrometry identification of bound proteins.

• Yeast Two-Hybrid Screening:
  Though challenging for thermophilic proteins, modified Y2H systems with thermostable components can screen for interactors using A. fulgidus genomic libraries.

• Proximity Labeling:
  Fusion of AF_2291 with enzymes like BioID or APEX2 can identify proximal proteins in reconstituted systems or heterologous hosts.

• Surface Plasmon Resonance or Bio-Layer Interferometry:
  These techniques can quantify binding kinetics with candidate interactors identified through other methods.

• Crosslinking Mass Spectrometry:
  This approach can capture transient interactions and provide structural information about the binding interface.

An informative example of the value of interaction studies comes from research on AF_1257, which interacts with AF_RS01245 (AfTrm112) . This interaction enhances the enzymatic activity of AF_1257, demonstrating how binding partners can modulate protein function . Similar studies with AF_2291 could reveal whether it functions independently or requires interaction partners for full activity.

What approaches are effective for studying the potential role of AF_2291 in extremophile adaptation?

Investigating AF_2291's potential role in extremophile adaptation requires an integrated approach examining both the protein's intrinsic properties and its genomic/cellular context:

• Comparative Genomics:
  Analysis of AF_2291 conservation across archaea from different thermal environments can indicate if it's specifically associated with hyperthermophily. Presence in other extremophiles (halophiles, acidophiles) would suggest broader roles in stress adaptation.

• Transcriptomic and Proteomic Profiling:
  Examining expression levels of AF_2291 under various stress conditions (temperature shifts, oxidative stress, nutrient limitation) can indicate regulatory patterns consistent with stress response roles.

• Gene Deletion or Silencing:
  If genetic systems are available for A. fulgidus or related archaea, creating knockout strains can reveal phenotypic effects under various growth conditions.

• Thermostability Analysis:
  Detailed characterization of AF_2291's thermal stability using differential scanning calorimetry, circular dichroism thermal melts, or thermal shift assays can identify unusual stability features that might contribute to extremophile adaptation.

• Structural Features Analysis:
  Identification of structural elements that contribute to thermostability, such as compacted hydrophobic cores, surface salt bridge networks, or reduced loop regions, as has been observed in other thermophilic proteins.

• Heterologous Expression Studies:
  Testing whether expression of AF_2291 in mesophilic hosts confers any increased tolerance to heat or other stresses can indicate direct contributions to stress resistance.

How might AF_2291 be utilized in biotechnological applications?

Proteins from hyperthermophilic archaea like A. fulgidus have significant biotechnological potential due to their extreme stability. While the specific function of AF_2291 remains uncharacterized, its potential applications can be extrapolated from properties common to archaeal proteins and specific insights from related proteins:

• Biocatalysis at Extreme Conditions:
  If enzymatic activity is confirmed, AF_2291 could serve as a catalyst for industrial processes requiring high temperatures, extending reaction lifetimes and reducing contamination risks.

• Protein Engineering Platform:
  The thermostable scaffold of AF_2291 could serve as a starting point for directed evolution of novel activities that benefit from enhanced stability.

• Structural Biology Research Tool:
  Like other archaeal proteins that adopt unique folds (such as AF2241's cyclophilin-like structure ), AF_2291 could serve as a model system for studying protein folding principles and stability mechanisms.

• Molecular Biology Reagents:
  If AF_2291 exhibits nucleic acid binding or modifying activities (similar to the tRNA methyltransferase activity of AF_1257 ), it could potentially be developed into tools for molecular biology applications requiring thermostable enzymes.

• Biomaterial Development:
  The structural stability of hyperthermophilic proteins makes them potentially valuable components in engineered biomaterials requiring resistance to denaturation.

The development of these applications would require thorough functional and structural characterization, followed by optimization for specific use cases.

What are the challenges in resolving conflicting structural predictions for proteins like AF_2291?

Resolving conflicting structural predictions for uncharacterized proteins like AF_2291 presents several challenges that require methodological rigor to address:

How can integrated -omics approaches advance our understanding of AF_2291's biological context?

Integrated -omics approaches provide powerful tools to contextualize uncharacterized proteins like AF_2291 within the broader biological system of A. fulgidus:

• Multi-omics Data Integration:
  Combining genomic, transcriptomic, proteomic, and metabolomic data to place AF_2291 within functional networks. This approach can reveal co-expression patterns, metabolic pathway associations, and potential regulatory relationships.

• Comparative Genomics Analysis:
  Examining the genomic neighborhood of AF_2291 across multiple archaeal species can identify conserved gene clusters that suggest functional relationships, similar to how genomic context analysis helped characterize tRNA modification enzymes in archaea .

• Proteogenomic Mapping:
  Integration of proteomic data with genome annotations can verify the translation of AF_2291, identify post-translational modifications, and potentially correct gene models if necessary.

• Interactome Analysis:
  High-throughput protein-protein interaction studies, when combined with transcriptomic data, can identify condition-specific interaction networks involving AF_2291.

• Phylogenetic Profiling:
  Correlating the presence/absence patterns of AF_2291 with other genes across diverse archaeal genomes can identify functionally related proteins that co-evolve with AF_2291.

• Structural Proteomics Integration:
  Combining structural predictions or experimental structures with interaction data to model macromolecular complexes, as demonstrated by studies on protein complexes like the Trm11-Trm112 interaction in archaea .

• Systems Biology Modeling:
  Integrating multiple data types to develop predictive models of cellular processes that include AF_2291, potentially revealing emergent properties not obvious from single-omics approaches.

The use of such integrated approaches has proven valuable for other archaeal proteins, such as the characterization of Trm11 and Trm112 proteins involved in tRNA modification across archaeal species .

What buffer systems and storage conditions maintain optimal stability of recombinant AF_2291?

For thermostable proteins from A. fulgidus, buffer systems and storage conditions require special considerations to maintain stability and activity:

• Buffer Composition:

  • Recommended primary buffers: HEPES, phosphate, or MOPS buffers (50-100 mM) with pH 7.0-8.0, which maintain pH stability at elevated temperatures

  • Salt concentration: 150-300 mM NaCl to mimic physiological ionic strength and reduce aggregation

  • Stabilizing additives: 5-10% glycerol, 1-2 mM DTT or TCEP as reducing agents if the protein contains cysteine residues

  • Consider including divalent cations (Mg2+, Mn2+) at 1-5 mM if metal-dependent activity is suspected

• Storage Conditions:

  • Short-term storage (1-2 weeks): 4°C in the above buffer with addition of 0.02% sodium azide to prevent microbial growth

  • Long-term storage: Flash-freeze aliquots in liquid nitrogen and store at -80°C with 15-20% glycerol as cryoprotectant

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Thermal Considerations:

  • Heat stability testing should be performed to determine if AF_2291 benefits from periodic heat treatment (e.g., 60-80°C for 10-15 minutes), which can reestablish proper folding

  • For functional assays, pre-incubation at the optimal growth temperature of A. fulgidus (approximately 83°C) may be necessary for full activity

• Concentration Effects:

  • Monitor for concentration-dependent aggregation using dynamic light scattering

  • If aggregation occurs, consider additives such as arginine (50-100 mM) or non-detergent sulfobetaines

These recommendations are based on general practices for hyperthermophilic proteins and should be optimized specifically for AF_2291 through stability screening experiments.

What considerations are important when designing experiments to compare AF_2291 with homologous proteins from mesophilic organisms?

When comparing AF_2291 with potential homologs from mesophilic organisms, several experimental design considerations are crucial to ensure valid comparisons:

• Temperature Optimization:

  • Design experiments with parallel reactions at both optimal temperatures (e.g., 80°C for AF_2291, 37°C for mesophilic homologs)

  • Include activity vs. temperature profiles (20-90°C) to characterize the full thermal response curve of each protein

• Substrate Stability Control:

  • Ensure substrates remain stable at elevated temperatures or account for thermal degradation in reaction kinetics

  • Consider using thermostable substrate analogs when necessary

• Normalization Strategies:

  • Compare activities at physiologically relevant temperatures for each organism rather than at a single standard temperature

  • Use relative activities (percentage of maximum) when comparing thermal profiles

  • Calculate temperature coefficients (Q10) and activation energies for quantitative comparison

• Structural Comparison Controls:

  • Include structural characterization (CD spectroscopy, thermal denaturation curves) alongside functional assays

  • Monitor protein stability throughout the assay duration at each temperature

• Evolutionary Context:

  • Include proteins from thermophilic, mesophilic, and psychrophilic organisms to establish evolutionary trends

  • When possible, include proteins from organisms representing different evolutionary distances from A. fulgidus

• Buffer Consistency:

  • Use buffer systems with minimal temperature-dependent pH changes

  • Account for temperature effects on buffer pKa values

  • Match ionic strength rather than exact salt concentration across temperatures

• Analysis Framework:

  • Apply Arrhenius plots to compare temperature dependence of reaction rates

  • Consider using temperature-adapted enzyme models that account for both activation and inactivation processes

These considerations will help ensure that observed differences reflect genuine adaptations rather than experimental artifacts.

What mass spectrometry approaches are most appropriate for characterizing post-translational modifications in AF_2291?

Characterizing post-translational modifications (PTMs) in archaeal proteins like AF_2291 requires specialized mass spectrometry approaches optimized for the unique modification patterns found in these organisms:

• Sample Preparation Strategies:

  • Multiple protease digestion approach: Use complementary proteases beyond trypsin (e.g., chymotrypsin, GluC, AspN) to generate overlapping peptide coverage

  • Enrichment techniques: For phosphorylation, use titanium dioxide or immobilized metal affinity chromatography; for glycosylation, use lectin affinity or hydrazide chemistry

  • Native purification conditions: Minimize sample processing steps to retain labile modifications

• MS Instrumentation and Methods:

  • High-resolution instruments: Orbitrap or Q-TOF mass analyzers for accurate mass determination

  • Fragmentation techniques: Combine collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) for complementary fragmentation patterns

  • Data-independent acquisition (DIA) for comprehensive PTM mapping

• Archaeal-Specific Considerations:

  • Search for archaeal-specific modifications: N-terminal acetylation, methylation, and unusual archaeal-specific modifications like hypusine and archaeosine

  • Temperature-induced modifications: Non-enzymatic modifications that may occur under high-temperature growth conditions

  • Modified search parameters: Adjust search engines to account for archaeal codon usage and modification patterns

• Quantitative Approaches:

  • Stable isotope labeling to quantify modification stoichiometry

  • Multiple reaction monitoring (MRM) for targeted quantification of key modifications

  • Label-free quantification using extracted ion chromatograms

• Validation Strategies:

  • Site-directed mutagenesis of putative modification sites

  • Antibodies against specific modifications (where available)

  • Synthetic peptide standards with and without modifications

• Integrated Analysis:

  • Correlate identified PTMs with structural models to assess functional implications

  • Compare modification patterns across growth conditions to identify regulatory PTMs

These approaches would provide comprehensive characterization of PTMs in AF_2291, potentially revealing regulatory mechanisms specific to hyperthermophilic archaea.