Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_0864 (AF_0864)

What is Archaeoglobus fulgidus and why is protein AF_0864 significant for research?

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon that has attracted significant research interest due to its unique metabolic capabilities and evolutionary position . The uncharacterized protein AF_0864 (UniProt ID: O29397) represents an important research target within this organism as it may hold clues to novel biological functions adapted to extreme environments.

The protein consists of 126 amino acids with the sequence: MLERPTGVTVLAILYVLAAVFFFLAAAVSGYLAQVASTTQLGEIPYAELFFAFSGIFFSITGTVWLITAYGLWKGRGWGWWLAVIFTAFGLISSLLSLPKGVVGIVVLGAILYYLTRRHVREFFGV . Analysis of this sequence suggests AF_0864 may be a membrane protein with multiple transmembrane domains, which makes it particularly interesting for researchers studying archaeal membrane biology and protein function in extreme environments.

What structural and biochemical characteristics of AF_0864 should researchers be aware of?

The AF_0864 protein exhibits several key structural features that researchers should consider:

• Membrane association: The amino acid sequence contains predominantly hydrophobic regions interspersed with charged residues, suggesting multiple transmembrane domains .

• Protein size: The full-length protein spans 126 amino acids with an estimated molecular weight of approximately 14-15 kDa, though the His-tagged recombinant version may appear slightly larger in gel electrophoresis analyses .

• Expression system: The recombinant protein is expressed in E. coli, which may affect post-translational modifications compared to the native archaeal environment .

• Purification approach: The protein is supplied with an N-terminal His-tag to facilitate purification and detection, which should be considered when designing binding or functional assays .

For experimental work, researchers should note that the protein is supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . The presence of hydrophobic regions may necessitate specialized handling protocols to maintain protein solubility and native conformation.

How should recombinant AF_0864 protein be stored and reconstituted for experimental use?

Proper storage and reconstitution are critical for maintaining AF_0864 protein functionality in experimental settings:

Storage protocol:

• Store lyophilized powder at -20°C to -80°C upon receipt.

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles.

• Working aliquots can be stored at 4°C for up to one week .

• Long-term storage requires aliquoting with 5-50% glycerol (final concentration) and storage at -20°C to -80°C .

Reconstitution methodology:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom.

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• For optimal stability, add glycerol to a final concentration of 50% .

• If protein aggregation occurs, consider using mild detergents or alternative buffer systems suitable for membrane proteins.

Researchers should validate protein stability after reconstitution by running SDS-PAGE or functional assays before proceeding with complex experiments. When designing long-term studies, prepare sufficient aliquots during initial reconstitution to ensure consistent protein quality throughout the research project.

How can researchers design effective experiments to characterize the function of AF_0864?

Designing experiments to characterize an uncharacterized protein requires a systematic approach:

Step-by-step experimental design framework:

• Define clear research questions and hypotheses:

  • Based on sequence analysis, hypothesize potential functions (e.g., transporter, signaling protein)

  • Formulate null hypothesis (H0): "AF_0864 does not function as a membrane transporter"

  • Formulate alternate hypothesis (H1): "AF_0864 functions as a membrane transporter"

• Identify and control variables:

  • Independent variables: protein concentration, buffer composition, temperature

  • Dependent variables: binding affinity, transport activity, protein-protein interactions

  • Control for extraneous variables: protein purity, buffer effects, temperature fluctuations

• Select appropriate experimental treatments:

  • Design systematic manipulations of conditions (pH range: 5.0-9.0, temperature range: 25-85°C)

  • Include appropriate positive and negative controls

  • Plan for biological and technical replicates

• Implementation strategies:

  • Begin with sequence-based predictions and structural modeling

  • Progress to in vitro binding/activity assays

  • Advance to reconstitution in liposomes or expression systems if membrane function is hypothesized

This methodical approach ensures that experiments produce reliable, interpretable data about AF_0864 function while controlling for the unique challenges of working with archaeal proteins.

What protein labeling and visualization techniques are most suitable for AF_0864 studies?

For effective tracking and visualization of AF_0864 in experimental systems, researchers should consider:

Labeling techniques suitable for AF_0864:

• His-tag detection: The recombinant protein already contains an N-terminal His-tag, enabling:

  • Immunodetection using anti-His antibodies

  • Purification via nickel affinity chromatography

  • Visualization using fluorescently labeled anti-His antibodies

• Light-catalyzed protein modification: Recent advances in protein editing chemistry can be applied:

  • Targeting tryptophan residues in AF_0864 sequence with difluoromethyl radicals

  • Creating carbon-carbon bonds to generate aldehyde groups

  • Using the aldehydes as attachment points for fluorescent labels

• Location tracking methodologies:

  • The aldehyde groups generated through light-catalyzed modification create a fluorescence shift

  • This enables tracking of protein localization without additional bulky tags

  • The technique is suitable for applications in live cell imaging if AF_0864 is expressed in a cellular system

Application considerations:

• For hyperthermophilic proteins like AF_0864, ensure the stability of both the protein and label at higher temperatures

• Verify that labeling does not interfere with potential membrane association

• Consider dual-labeling approaches for co-localization studies with potential interaction partners

These techniques provide researchers with multiple options for visualizing and tracking AF_0864 in various experimental contexts while minimizing disruption to protein function.

What expression systems are optimal for studying AF_0864 for functional characterization?

Selecting the appropriate expression system is crucial for functional studies of archaeal proteins:

Expression system comparison for AF_0864 studies:

Methodological recommendations:

• Begin with E. coli expression for initial characterization and structural studies

• Progress to archaeal expression systems for functional studies requiring authentic folding environments

• Consider temperature-adapted experimental conditions that match the native environment of Archaeoglobus fulgidus

• For membrane proteins like AF_0864, evaluate the need for detergents or membrane mimetics during purification and functional assays

The commercial recombinant AF_0864 is expressed in E. coli , which provides a good starting point for preliminary studies, but researchers should consider alternative expression platforms as their functional characterization progresses.

How might AF_0864 relate to other characterized proteins in Archaeoglobus fulgidus?

Understanding the relationship between AF_0864 and other characterized proteins in A. fulgidus requires comparative analysis:

Comparative analysis with characterized A. fulgidus proteins:

The genome of A. fulgidus contains several well-characterized proteins, including a novel ferric reductase that has been purified to homogeneity . This ferric reductase:

• Is a homodimer with 18 kDa subunits (40 kDa native form)

• Uses NADH and NADPH as electron donors

• Requires FMN or FAD as catalytic intermediates

• Represents approximately 0.75% of soluble protein

While AF_0864 is currently uncharacterized, sequence analysis suggests it differs significantly from the ferric reductase:

• AF_0864 appears to be a membrane protein rather than a soluble protein

• Its size (126 amino acids) is smaller than the ferric reductase subunit

• Lacks obvious cofactor binding motifs for FMN or FAD

Research approaches to establish relationships:

• Conduct co-expression studies to identify potential interactions between AF_0864 and known proteins

• Perform comparative genomic analyses across archaeal species to identify conserved genetic contexts

• Investigate transcriptomic data to identify co-regulated genes that may function in the same pathway

• Apply protein-protein interaction techniques such as pull-down assays using the His-tagged AF_0864 as bait

Understanding the relationship between AF_0864 and characterized proteins may provide crucial insights into its biological role within the unique sulfate-reducing metabolism of A. fulgidus.

What approaches can be used to resolve contradictory experimental data when characterizing AF_0864?

When faced with contradictory data during AF_0864 characterization, researchers should follow a systematic resolution approach:

Methodological framework for resolving contradictory data:

• Validation of experimental parameters:

  • Verify protein quality through multiple analytical methods (e.g., SDS-PAGE, mass spectrometry)

  • Confirm experimental conditions match the thermophilic nature of the source organism

  • Ensure buffer compositions are compatible with predicted protein properties

• Systematic variation of experimental conditions:

  • Design a factorial experiment varying multiple parameters (temperature, pH, salt concentration)

  • Apply statistical design of experiments (DoE) methodology to identify significant factors

  • Test for interactions between variables that may explain contradictory results

• Alternative hypothesis generation:

  • Consider multiple potential functions rather than a single hypothesis

  • Develop orthogonal assays that test protein function through different mechanisms

  • Evaluate whether the protein requires specific co-factors or interaction partners

• Independent verification approaches:

  • Use complementary techniques to address the same research question

  • Collaborate with laboratories using different methodological approaches

  • Compare in vitro results with in silico predictions and evolutionary analyses

This structured approach enables researchers to systematically explore contradictions in experimental data and develop a coherent understanding of AF_0864 function despite initial ambiguities.

How can protein editing techniques be applied to study the function of AF_0864?

Advanced protein editing techniques offer powerful approaches to study AF_0864 function:

Application of cutting-edge protein editing to AF_0864:

Recent advances in light-catalyzed protein editing can be particularly valuable for studying uncharacterized proteins like AF_0864:

• Tryptophan-targeted modification:

  • AF_0864 contains tryptophan residues that can be selectively modified using light-catalyzed reactions

  • Difluoromethyl radicals can be used to create carbon-carbon bonds to tryptophan

  • This reaction can introduce various functional groups while maintaining protein integrity

• Aldehyde group introduction:

  • The two-step process creates aldehyde (formyl) groups on the protein

  • These aldehyde groups serve as versatile attachment points for:

    • Fluorescent probes for localization studies

    • Affinity tags for interaction studies

    • Cross-linking agents for structural analysis

• Methodological advantages:

  • The small size of modifications minimizes disruption to protein function

  • The technique works under mild conditions compatible with protein stability

  • The approach can be applied to live cells, enabling in situ studies

• Experimental design considerations:

  • Control reactions to confirm modification specificity

  • Verify that modifications do not disrupt predicted transmembrane domains

  • Compare function of modified and unmodified protein to assess impact

These protein editing approaches provide researchers with tools to introduce specific modifications to AF_0864, enabling studies of structure-function relationships and interaction networks that would otherwise be challenging for an uncharacterized protein.

What bioinformatic approaches are recommended for predicting the function of AF_0864?

Comprehensive bioinformatic analysis represents a critical first step in characterizing uncharacterized proteins like AF_0864:

Recommended bioinformatic analysis pipeline:

• Sequence analysis tools:

  • Position-Specific Iterative BLAST (PSI-BLAST) to identify distant homologs

  • Multiple sequence alignment to identify conserved residues

  • Hydropathy analysis to confirm transmembrane regions (particularly relevant for AF_0864)

  • Motif identification to detect functional domains

• Structural prediction methodologies:

  • Secondary structure prediction (e.g., PSIPRED)

  • Tertiary structure modeling using AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations to assess stability at high temperatures

  • Ligand binding site prediction to identify potential functional regions

• Comparative genomic approaches:

  • Analyze gene neighborhood across archaeal species

  • Identify conserved gene clusters that may indicate functional relationships

  • Examine horizontal gene transfer patterns

• Integration of existing experimental data:

  • Mine proteomics datasets for expression patterns

  • Analyze transcriptomic data for co-expression networks

  • Review metabolomic data for potential substrates or products

This comprehensive bioinformatic analysis provides a foundation for generating testable hypotheses about AF_0864 function, guiding experimental design, and interpreting results in a broader biological context.

How should researchers interpret experimental results for an uncharacterized protein like AF_0864?

Interpreting experimental results for uncharacterized proteins requires a structured analytical approach:

Framework for result interpretation:

• Contextual analysis:

  • Compare results to predicted function from bioinformatic analyses

  • Consider the hyperthermophilic, sulfate-reducing lifestyle of A. fulgidus

  • Evaluate consistency with known membrane protein functions

• Validation through multiple methodologies:

  • Confirm key findings using orthogonal experimental approaches

  • Assess reproducibility across different experimental conditions

  • Consider the impact of the recombinant expression system on results

• Stepwise interpretation process:

  • Begin with direct observations (e.g., binding affinity, localization)

  • Progress to functional implications (e.g., potential transport activity)

  • Extend to biological significance (e.g., role in cellular processes)

  • Consider evolutionary context (e.g., conservation across archaeal species)

• Addressing experimental limitations:

  • Acknowledge constraints of working with recombinant proteins

  • Consider potential artifacts from His-tagging or expression system

  • Evaluate temperature effects on experimental outcomes

  • Assess whether membrane environment was adequately replicated

This structured approach ensures that interpretations remain grounded in experimental evidence while acknowledging the inherent challenges of working with uncharacterized proteins from extremophilic organisms.

What statistical methods are most appropriate for analyzing AF_0864 protein interaction data?

Statistical approaches for AF_0864 interaction studies:

• Preliminary data analysis:

  • Normality testing to determine appropriate parametric/non-parametric tests

  • Outlier detection and management protocols

  • Transformation methods for non-normal distributions

• Comparative statistical methods:

  • Student's t-test or ANOVA for comparing binding under different conditions

  • Non-parametric alternatives (Mann-Whitney U or Kruskal-Wallis) when assumptions aren't met

  • Paired analyses for before/after experimental designs

• Binding data analysis:

  • Non-linear regression for binding curve fitting

  • Statistical comparison of binding parameters (Kd, Bmax)

  • Bootstrap resampling for parameter confidence intervals

• Advanced analytical approaches:

  • Principal Component Analysis (PCA) for multivariate data

  • Hierarchical clustering of interaction profiles

  • Network analysis for complex interaction datasets

• Experimental design considerations:

  • Power analysis to determine sample size requirements

  • Multiple testing correction (e.g., Bonferroni, FDR) for large datasets

  • Randomization and blocking strategies to control extraneous variables

What emerging technologies show promise for further characterizing AF_0864?

Several cutting-edge technologies offer new avenues for AF_0864 characterization:

• Cryo-electron microscopy (Cryo-EM): Particularly valuable for membrane proteins like AF_0864, enabling structural determination without crystallization.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Provides insights into protein dynamics and conformational changes under various conditions, including high temperatures relevant to A. fulgidus.

• Nanodiscs and membrane mimetics: Advanced systems for studying membrane proteins in near-native environments, potentially resolving functional characteristics of AF_0864.

• Single-molecule techniques: FRET and other single-molecule approaches that can detect conformational changes and interactions with potential substrates or partners.

• Native mass spectrometry: For detecting non-covalent interactions and potential ligands that might be missed in traditional approaches.

Researchers should consider these emerging technologies when designing comprehensive characterization strategies for AF_0864, particularly when traditional approaches yield limited insights.

How might knowledge of AF_0864 contribute to our understanding of archaeal biology?

The characterization of AF_0864 has potential to advance several areas of archaeal biology:

• Membrane biology in extremophiles: As a predicted membrane protein, AF_0864 may reveal adaptations that enable membrane function at high temperatures and pressures.

• Evolution of uncharacterized protein families: Detailed characterization provides insights into the function of homologous proteins across archaeal lineages.

• Metabolic adaptations in sulfate-reducing archaea: May reveal unique aspects of energy metabolism in these specialized organisms.

• Archaeal protein structure-function relationships: Contributes to our understanding of how archaeal proteins maintain stability and function under extreme conditions.

• Comparative genomics across domains of life: Characterization enables more accurate annotation of homologous proteins in diverse genomes.