Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_1225 (AF_1225)

What is Archaeoglobus fulgidus AF_1225 protein and what is known about its structure?

AF_1225 is an uncharacterized protein from the hyperthermophilic archaeon Archaeoglobus fulgidus. The mature protein spans amino acids 19-212 of the complete sequence and has been successfully expressed as a recombinant protein with an N-terminal His tag in E. coli expression systems . The protein has a UniProt ID of O29043. The full amino acid sequence of the mature protein is:

DIVSVNVTDTATLTVGDSCMYFVDNLQPSINATPGEYEVKIGINCTPGLKEVYADGSVLA QIDVNETTIDYQAYAASLEKENLALQKEVESLKEKLKISQEQIETLKSQLEDLQNKAKML GIQNELQKQQIEELQKKLERAKTELQKKKSDLDELEEKIRELNRQSSIYRLATFFMVSLF VGSFVALVFVARKE

While the tertiary structure has not been fully elucidated via crystallography or NMR, preliminary analysis of the amino acid sequence suggests potential membrane-associated domains in the C-terminal region, indicated by the hydrophobic amino acid cluster "FFMVSLF VGSFVALVFVARKE". This suggests AF_1225 may function as a membrane-associated protein, though further structural studies are needed to confirm this hypothesis.

What is the cellular localization of AF_1225 protein in Archaeoglobus fulgidus?

To experimentally determine the cellular localization, researchers should consider cellular fractionation experiments comparing cytosolic, membrane, and periplasmic fractions of A. fulgidus, followed by western blotting with anti-AF_1225 antibodies. Alternatively, expressing AF_1225 with fluorescent protein tags (ensuring thermostability for use in the hyperthermophilic A. fulgidus) might allow for in vivo visualization of localization patterns.

How is the recombinant AF_1225 protein typically prepared for research applications?

The recombinant AF_1225 protein is typically prepared as a His-tagged construct expressed in E. coli expression systems. The mature protein (amino acids 19-212) is fused to an N-terminal His tag to facilitate purification . After expression, the protein is typically purified using affinity chromatography with Ni-NTA or similar matrices that bind the His tag.

The purified protein is generally provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For storage stability, the protein is typically lyophilized in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . This formulation helps maintain protein integrity during freeze-thaw cycles.

For experimental use, it is recommended to reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and add glycerol to a final concentration of 5-50% for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain protein activity and structural integrity.

What approaches should be used to investigate potential binding partners of AF_1225?

Several complementary approaches are recommended for investigating potential binding partners of AF_1225:

• Pull-down assays: Using the His-tagged recombinant AF_1225 protein as bait, perform pull-down assays with cell lysates from A. fulgidus. After washing to remove non-specific binding, elute the protein complexes and analyze by mass spectrometry to identify potential interacting partners.

• Yeast two-hybrid screening: While this approach has limitations for archaeal proteins, a modified system using thermostable components could be employed to screen for potential protein-protein interactions.

• Co-immunoprecipitation: Using antibodies specific to AF_1225, immunoprecipitate the protein from A. fulgidus extracts and identify co-precipitating proteins by mass spectrometry.

• Crosslinking studies: Utilize chemical crosslinkers in intact cells or cell extracts to stabilize transient protein-protein interactions before immunoprecipitation and mass spectrometry analysis.

• Surface plasmon resonance (SPR): Use purified AF_1225 immobilized on a sensor chip to detect binding to candidate partners and determine binding kinetics.

To validate potential interactions, researchers should confirm results using at least two independent methods and include appropriate controls (e.g., unrelated proteins of similar size and charge characteristics) to ensure specificity of the interactions.

How should researchers design experiments to characterize the potential enzymatic activity of AF_1225?

As AF_1225 remains uncharacterized functionally, designing experiments to identify potential enzymatic activities requires a multi-faceted approach:

• Sequence-based prediction: Use bioinformatics tools like BLAST, Pfam, and PROSITE to identify conserved domains that might suggest enzymatic function. Compare with characterized proteins from other extremophiles.

• Activity screening panel: Prepare a panel of assays testing common enzymatic activities (hydrolase, transferase, oxidoreductase, etc.) using purified recombinant AF_1225 under conditions mimicking the natural hyperthermophilic environment (80-85°C, slightly acidic pH, high salinity).

• Substrate screening: Test the protein against a library of potential substrates, including various sugars, lipids, nucleic acids, and small peptides, monitoring for catalytic conversion.

• Metal dependency: Test activity in the presence of different metal ions (Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺, etc.) as many archaeal enzymes require metal cofactors for catalytic activity.

• Point mutations: Once potential active sites are identified, create site-directed mutants to confirm their role in catalysis.

For each assay, include appropriate positive and negative controls, and perform experiments in triplicate to ensure reproducibility. Temperature dependence studies (activity measured at different temperatures) can provide insights into optimal conditions and confirm the expected thermostable nature of this archaeal protein.

What are the challenges in expressing functional AF_1225 in heterologous systems, and how can they be addressed?

Expressing archaeal proteins like AF_1225 in heterologous systems presents several challenges:

• Codon usage bias: A. fulgidus and E. coli have different codon preferences. This can be addressed by optimizing the AF_1225 coding sequence for E. coli expression or using strains with rare tRNAs.

• Protein folding at mesophilic temperatures: As A. fulgidus is hyperthermophilic, its proteins may not fold correctly at lower temperatures used for E. coli growth. Potential solutions include:

  • Expression in psychrophilic bacteria at higher temperatures (30-37°C)

  • Co-expression with archaeal chaperones

  • In vitro refolding of inclusion bodies at elevated temperatures

• Post-translational modifications: If AF_1225 requires archaeal-specific modifications, expression in E. coli may yield non-functional protein. Consider:

  • Using archaeal expression systems like Sulfolobus solfataricus

  • Cell-free expression systems supplemented with archaeal extracts

• Protein solubility: The hydrophobic C-terminal region of AF_1225 suggests membrane association , which may cause solubility issues. Strategies include:

  • Expression as fusion proteins (MBP, SUMO, etc.)

  • Use of specialized detergents during purification

  • Truncation constructs removing hydrophobic regions for soluble domain characterization

• Protein stability: For storage and handling, including 6% trehalose in the buffer formulation has been shown to enhance stability . Additionally, storing the protein at -80°C with 50% glycerol helps maintain structural integrity.

How should researchers design experiments to investigate the potential role of AF_1225 in thermoadaptation?

To investigate AF_1225's potential role in thermoadaptation, consider the following experimental design:

• Comparative analysis across temperature gradients:

  • Create temperature-sensitive A. fulgidus strains with varied AF_1225 expression levels

  • Measure growth rates and survival at different temperatures (70°C, 80°C, 85°C, 90°C)

  • Compare protein abundance using quantitative proteomics across these conditions

• Structural stability assessment:

  • Perform differential scanning calorimetry (DSC) on purified AF_1225 to determine melting temperature (Tm)

  • Compare with homologous proteins from mesophilic organisms

  • Investigate stabilizing interactions through molecular dynamics simulations

• Gene knockout/knockdown studies:

  • Create AF_1225 deletion mutants or use CRISPR interference to reduce expression

  • Assess phenotypic changes in growth and survival at elevated temperatures

  • Perform transcriptomic analysis to identify compensatory mechanisms

• Interactome analysis:

  • Identify protein interaction partners at different temperatures

  • Determine if AF_1225 participates in temperature-dependent protein complexes

  • Investigate if it functions as a chaperone or co-chaperone

• Heterologous expression impact:

  • Express AF_1225 in mesophilic bacteria or archaea

  • Evaluate if it confers enhanced thermotolerance

  • Assess changes in membrane fluidity and cellular stress responses

Include appropriate controls in all experiments, such as wild-type strains and strains expressing unrelated archaeal proteins. Statistical analysis should include at least three biological replicates per condition and appropriate tests (ANOVA, t-tests) to determine significance of observed differences.

What controls should be included when studying AF_1225 protein-protein interactions?

When studying protein-protein interactions involving AF_1225, the following controls are essential:

• Negative controls:

  • Empty vector/bait protein alone in pull-down assays

  • Unrelated His-tagged protein of similar size

  • Denatured AF_1225 protein to control for non-specific interactions

  • Pre-immune serum or isotype control antibodies for immunoprecipitation experiments

• Positive controls:

  • Known interacting protein pairs from A. fulgidus

  • Artificial constructs with documented interaction domains

• Specificity controls:

  • Competition assays with unlabeled AF_1225

  • Dose-response experiments with varying concentrations of interacting partners

  • Analysis of interaction under different salt concentrations to distinguish ionic from hydrophobic interactions

• Technical validation:

  • Reverse pull-down experiments (using the identified partner as bait)

  • Confirmation by at least two independent methods (e.g., pull-down and SPR)

  • Demonstration of interaction in near-native conditions

• Biological relevance controls:

  • Co-localization studies in A. fulgidus

  • Phenotypic analysis of interaction-deficient mutants

  • Correlation of interaction with functional outcomes

All experiments should be performed in triplicate with appropriate statistical analysis to ensure reproducibility. Temperature-dependent interactions should be assessed at physiologically relevant temperatures (80-85°C) when possible, or extrapolated from studies at lower temperatures.

What methodological approaches can help determine if AF_1225 functions as part of a larger protein complex?

Several complementary methodological approaches can help determine if AF_1225 functions as part of a larger protein complex:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

  • Extract protein complexes under non-denaturing conditions

  • Separate complexes based on size while maintaining native interactions

  • Identify AF_1225-containing complexes via western blotting or mass spectrometry

• Size Exclusion Chromatography (SEC):

  • Fractionate cellular extracts based on molecular weight

  • Analyze fractions by western blotting to identify AF_1225

  • Compare elution profile with known size standards to estimate complex size

• Sucrose Gradient Ultracentrifugation:

  • Separate complexes based on sedimentation coefficient

  • Analyze fractions for AF_1225 presence

  • Compare with sedimentation of known complexes

• Crosslinking Mass Spectrometry (XL-MS):

  • Treat live cells or extracts with crosslinking agents

  • Digest and analyze by MS to identify crosslinked peptides

  • Reconstruct protein complex architecture from crosslinking constraints

• Co-immunoprecipitation with Quantitative Proteomics:

  • Immunoprecipitate AF_1225 under native conditions

  • Identify co-precipitating proteins by mass spectrometry

  • Quantify stoichiometry of complex components

• Cryo-electron Microscopy:

  • Purify native complexes containing AF_1225

  • Visualize structure by cryo-EM

  • Determine subunit composition and arrangement

To ensure reliable results, validate findings from at least two independent methods and include appropriate controls. Consider how complex formation might be affected by environmental conditions relevant to A. fulgidus (temperature, pH, salt concentration).

How can researchers resolve contradictory data regarding AF_1225 function across different experimental platforms?

When faced with contradictory data regarding AF_1225 function across different experimental platforms, consider the following systematic approach:

• Evaluate methodological differences:

  • Compare protein preparation methods (buffer conditions, tags, purification procedures)

  • Assess experimental conditions (temperature, pH, salt concentration)

  • Review reagent quality and assay sensitivity across studies

• Consider biological context:

  • Native vs. recombinant protein differences

  • In vivo vs. in vitro discrepancies

  • Presence/absence of necessary cofactors or binding partners

• Statistical reanalysis:

  • Perform meta-analysis when multiple datasets are available

  • Evaluate statistical power of each study

  • Consider batch effects and experimental noise

• Reconciliation experiments:

  • Design experiments specifically addressing contradictions

  • Systematically vary conditions to identify factors causing discrepancies

  • Use orthogonal methods to validate findings

• Biological validation:

  • Test predictions from each competing model in A. fulgidus

  • Create site-directed mutants affecting function

  • Correlate molecular findings with physiological outcomes

When reporting results, transparently acknowledge contradictions in the literature and provide a detailed methodological framework that allows others to replicate your findings. Consider how environmental factors specific to hyperthermophilic archaea might affect protein function differently than in standard laboratory conditions.

What bioinformatic approaches should be used to predict potential functions of AF_1225?

A comprehensive bioinformatic strategy to predict potential functions of AF_1225 should include:

• Sequence-based analyses:

  • PSI-BLAST searches against diverse databases to identify distant homologs

  • Multiple sequence alignment with homologs to identify conserved residues

  • Domain prediction using Pfam, SMART, InterPro, and CDD

  • Motif identification using PROSITE and MEME

• Structural prediction:

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold

  • Structural alignment with known proteins using DALI or VAST

  • Active site prediction based on structural templates

  • Molecular dynamics simulations to assess flexibility and potential binding sites

• Genomic context analysis:

  • Examination of operonic structure in A. fulgidus

  • Comparative genomics across archaeal species

  • Gene neighborhood conservation and co-evolution patterns

  • Phylogenetic profiling to identify co-occurring genes

• Network approaches:

  • Protein-protein interaction prediction

  • Gene co-expression analysis across available archaeal transcriptomes

  • Metabolic pathway mapping and gap analysis

• Integrated functional prediction:

  • Machine learning approaches combining multiple features

  • Bayesian integration of diverse evidence types

  • Text mining of scientific literature for related proteins

For each prediction, calculate confidence scores and validate key predictions experimentally. Be particularly attentive to archaeal-specific functions that might not be well-represented in standard databases dominated by bacterial and eukaryotic proteins.

How should expression data for AF_1225 be normalized across different experimental conditions?

Normalizing expression data for AF_1225 across different experimental conditions requires careful consideration of several factors:

• RT-qPCR normalization:

  • Select multiple reference genes stable under your experimental conditions

  • Use the geometric mean of at least three reference genes

  • Validate reference gene stability using algorithms like geNorm or NormFinder

  • Apply the 2^(-ΔΔCt) method with efficiency correction

• Proteomics normalization:

  • Use total protein normalization for Western blotting

  • For mass spectrometry, consider methods like:

    • Total ion current (TIC) normalization

    • Global normalization strategies (e.g., median normalization)

    • Spiked-in standards of known concentration

  • Always include loading controls appropriate for your subcellular fraction

• RNA-Seq normalization:

  • Apply appropriate normalization methods like:

    • TPM (Transcripts Per Million)

    • RPKM/FPKM (Reads/Fragments Per Kilobase Million)

    • DESeq2 normalization

  • Account for gene length and GC content biases

• Cross-platform normalization:

  • When comparing across technologies (e.g., microarray vs. RNA-seq)

  • Use quantile normalization or similar methods

  • Consider batch effect correction algorithms like ComBat

• Environmental condition considerations:

  • For extremophiles like A. fulgidus, account for how extreme conditions might affect reference genes

  • Validate normalization under all experimental conditions

  • Consider using external RNA/protein spike-ins as absolute references

For all normalization approaches, clearly document your methodology and justify your choice of normalization strategy in publications. Include raw data and normalized data to allow others to apply alternative normalization methods if needed.

What are the most promising research directions for elucidating the function of AF_1225?

Based on current knowledge about AF_1225, several promising research directions emerge:

• Structural biology approaches:

  • Determination of high-resolution crystal or cryo-EM structure

  • Structural comparisons with characterized proteins

  • Identification of potential active sites or binding pockets

• Genetic manipulation in native context:

  • Development of more efficient genetic tools for A. fulgidus

  • Creation of conditional knockouts or knockdowns

  • CRISPR-Cas9 based genome editing to introduce mutations

• Environmental response profiling:

  • Transcriptomic and proteomic analysis of AF_1225 expression under various stresses

  • Correlation with specific environmental adaptations

  • Comparative analysis across archaeal species from different niches

• Protein interaction network mapping:

  • Comprehensive interactome analysis under different conditions

  • Identification of protein complexes and their dynamics

  • Investigation of potential moonlighting functions

• Functional reconstitution:

  • In vitro reconstitution of potential biochemical activities

  • Liposome incorporation to test membrane-associated functions

  • Cell-free expression systems for functional testing

These directions should be pursued in parallel to build a comprehensive understanding of AF_1225 function. Collaborative approaches combining expertise in structural biology, genetics, biochemistry, and computational biology will likely yield the most significant insights into this uncharacterized protein.

How can knowledge about AF_1225 contribute to our understanding of archaeal evolution and adaptation?

Understanding AF_1225 can provide significant insights into archaeal evolution and adaptation through several avenues:

• Evolutionary conservation patterns:

  • Phylogenetic distribution across archaea and potential horizontal gene transfer events

  • Rates of sequence evolution compared to other proteins

  • Identification of positive selection signatures in specific lineages

• Thermoadaptation mechanisms:

  • Structural features contributing to thermostability

  • Comparison with homologs from mesophilic archaea

  • Potential role in stabilizing other cellular components

• Archaeal-specific biochemistry:

  • Unique functional mechanisms not found in bacteria or eukaryotes

  • Contribution to archaeal-specific metabolic pathways

  • Insights into archaeal membrane biology if membrane-associated

• Environmental adaptation:

  • Correlation of AF_1225 variants with specific ecological niches

  • Expression patterns across environmental gradients

  • Contribution to stress responses beyond temperature

• Ancient protein functions:

  • Potential insights into protein functions in the last archaeal common ancestor

  • Relevance to early life evolution on a hot, primitive Earth

  • Comparison with minimal functional requirements in modern cells

By systematically investigating these aspects, researchers can place AF_1225 in the broader context of archaeal biology and evolution, potentially revealing fundamental principles of adaptation to extreme environments that may have applications in biotechnology and our understanding of the limits of life.

What are the practical applications of research on AF_1225 in biotechnology and medicine?

Research on AF_1225 offers several potential practical applications in biotechnology and medicine:

• Enzyme technology:

  • If enzymatic activity is identified, potential use as a thermostable biocatalyst

  • Applications in industrial processes requiring high-temperature reactions

  • Protein engineering to enhance catalytic efficiency or substrate specificity

• Structural biology tools:

  • Development of thermostable protein scaffolds for biotechnology

  • Design of heat-resistant fusion proteins for purification

  • Creation of stable protein domains for crystallography

• Extremozyme applications:

  • PCR and DNA amplification technologies if nucleic acid binding properties exist

  • Bioremediation processes in high-temperature environments

  • Industrial processes requiring thermostable proteins

• Membrane protein insights:

  • The C-terminal hydrophobic region suggests membrane association

  • Potential applications in designing stable membrane proteins

  • Insights for thermostable biosensors or membrane-based technologies

• Archaeal systems biology:

  • Understanding archaeal protein networks

  • Development of synthetic biology tools for extremophiles

  • Design of minimal cells based on archaeal systems