Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_0891 (AF_0891)

What is the basic structural information available for AF_0891?

AF_0891 is an uncharacterized protein from Archaeoglobus fulgidus with 91 amino acids in its full-length form. The protein is identified in UniProt with the accession number O29371. The amino acid sequence is: MDTYAIISFLLGIAFGFLRRGKEDRAKIIEVIFVSLLLGLVSGIALSHAVLDGAGWGEFVKAFGLIVAALIYAIFFAAGTYLGDLLEKLRK . Based on this sequence information, the protein appears to have hydrophobic regions, suggesting it may be a membrane-associated protein. Computational structure predictions would be necessary to determine potential secondary and tertiary structural elements.

What expression systems are most effective for recombinant production of AF_0891?

For recombinant expression of AF_0891, Escherichia coli has been successfully used as a host system . When designing expression strategies, researchers should consider the following methodological approaches:

• Vector selection: pET-based vectors with T7 promoter systems have proven effective for archaeal protein expression

• Tag incorporation: His-tagging facilitates purification via metal affinity chromatography

• Growth conditions: Given that AF_0891 comes from a hyperthermophile (growth optimum 83°C), expression at higher temperatures (30-37°C) in E. coli may improve folding

• Codon optimization: Consider optimizing codons for E. coli expression, particularly if expression yields are low

For challenging membrane-associated proteins, alternative expression systems such as cell-free systems or eukaryotic hosts may be explored if E. coli expression yields inadequate results.

How should researchers approach the purification of recombinant AF_0891?

Purification of recombinant AF_0891 should follow a systematic approach considering its potential membrane association:

• Cell lysis: For potential membrane proteins, consider detergent-based lysis methods

• Affinity chromatography: His-tagged AF_0891 can be purified using nickel or cobalt resin columns

• Buffer optimization: Test buffers containing different salt concentrations (150-500 mM NaCl) and pH ranges (7.0-8.5)

• Storage conditions: The recombinant protein is typically stored in Tris-based buffer with 50% glycerol for stability

For quality control, researchers should perform SDS-PAGE analysis to confirm purity and Western blotting to verify identity. Mass spectrometry can provide additional confirmation of the recombinant protein sequence.

What are the optimal experimental designs for functional characterization of an uncharacterized protein like AF_0891?

When approaching the functional characterization of an uncharacterized protein like AF_0891, researchers should consider implementing a multi-faceted experimental design strategy:

• Independent measures design: When testing different conditions or treatments that might affect AF_0891 function, assign different test subjects to each condition . For example, when investigating potential binding partners, use separate reaction vessels for each candidate interactor rather than sequential testing.

• Repeated measures design: For experiments requiring multiple measurements over time (such as thermal stability or activity assays), use the same protein preparation across all timepoints to minimize variability .

• Matched pairs design: When comparing AF_0891 with homologous proteins from other organisms, design experiments that test both proteins under identical conditions to enable direct comparison .

A comprehensive functional characterization workflow should include:

• Bioinformatic analysis (sequence homology, conserved domains)

• Structural characterization (crystallography or cryo-EM if possible)

• Biochemical assays (binding partners, enzymatic activity)

• Cellular localization studies (if antibodies are available)

How should researchers approach experimental controls when working with AF_0891?

Establishing appropriate controls is critical for experiments involving uncharacterized proteins like AF_0891:

• Positive controls: Include well-characterized proteins from the same organism (such as AF_RFC or AF_PCNA ) in parallel experiments to validate methodologies.

• Negative controls: Use buffer-only or irrelevant protein controls to establish baseline measurements.

• Technical controls:

  • For binding assays: Test binding to empty matrix/beads

  • For activity assays: Heat-inactivated AF_0891

  • For structural studies: Reference proteins with known structures

• Biological relevance controls:

  • Test native vs. recombinant protein behaviors if native protein is available

  • Compare with homologous proteins from related organisms

Each experiment should be performed with at least three biological replicates and appropriate statistical analysis to ensure reproducibility and reliability of results.

What computational approaches can predict potential functions of AF_0891?

For uncharacterized proteins like AF_0891, computational approaches offer valuable insights into potential functions:

• Sequence analysis tools:

  • BLAST and multiple sequence alignments to identify distant homologs

  • Motif/domain prediction using PROSITE, Pfam, InterPro

  • Transmembrane topology prediction using TMHMM, Phobius

• Structure prediction and analysis:

  • AlphaFold or RoseTTAFold for tertiary structure prediction

  • Structure comparison with characterized proteins using DALI or PDBeFold

  • Active site prediction using CASTp or POOL

• Genomic context analysis:

  • Examining the genomic neighborhood of AF_0891 in Archaeoglobus fulgidus

  • Comparative genomics across archaeal species

• Protein-protein interaction prediction:

  • Using STRING database or PIPE prediction tools

  • Co-evolution analysis to identify potential interaction partners

The confidence in computational predictions can be assessed using metrics similar to the pLDDT score used in AlphaFold predictions, where scores above 90 indicate high confidence, 70-90 moderate confidence, and below 70 low confidence in structural predictions .

How can researchers investigate protein-protein interactions involving AF_0891?

Investigating potential protein-protein interactions for AF_0891 requires a systematic approach:

• Pull-down assays:

  • Use His-tagged AF_0891 as bait protein

  • Incubate with Archaeoglobus fulgidus cell lysate

  • Identify binding partners by mass spectrometry

• Yeast two-hybrid screening:

  • Clone AF_0891 into bait vectors

  • Screen against archaeal genomic library

  • Validate interactions using secondary assays

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI):

  • Immobilize purified AF_0891 on sensor chips

  • Test interaction with candidate partners

  • Determine binding kinetics and affinity constants

• Cross-linking mass spectrometry:

  • Use chemical cross-linkers to capture transient interactions

  • Identify interaction sites through MS/MS analysis

For each approach, researchers should consider the extreme growth conditions of Archaeoglobus fulgidus (hyperthermophilic, 83°C optimal growth ) and how this might affect protein-protein interactions when performed at standard laboratory temperatures.

What approaches are most effective for studying membrane-associated proteins from hyperthermophilic archaea?

Based on the amino acid sequence, AF_0891 appears to have hydrophobic regions suggesting possible membrane association . Studying membrane proteins from hyperthermophiles presents unique challenges:

• Detergent selection and optimization:

  • Test various detergents (DDM, LDAO, digitonin) for extraction efficiency

  • Optimize detergent concentration for stability vs. solubilization

  • Consider lipid nanodisc reconstitution for native-like environment

• Temperature considerations:

  • Perform binding and functional assays at elevated temperatures (60-80°C)

  • Use thermostable reagents and buffers

  • Design temperature-gradient experiments to determine optimal conditions

• Structural studies:

  • Cryo-EM may be preferable to crystallography for membrane proteins

  • Consider lipidic cubic phase crystallization if attempting X-ray studies

  • Use molecular dynamics simulations to model membrane interactions

• Functional reconstitution:

  • Liposome reconstitution with archaeal lipids if available

  • Giant unilamellar vesicle (GUV) formation for microscopy studies

  • Planar lipid bilayer experiments for channel/transporter function assessment

How does the extreme thermophilic nature of Archaeoglobus fulgidus affect experimental approaches for studying AF_0891?

The hyperthermophilic origin of AF_0891 (Archaeoglobus fulgidus grows optimally at 83°C ) presents both challenges and opportunities:

• Buffer and reagent selection:

  • Use thermostable buffers (HEPES, phosphate) rather than Tris (temperature-sensitive pKa)

  • Select thermostable enzymes for enzymatic assays (consider enzymes from Thermotoga maritima as used in studies of other Archaeoglobus proteins )

  • Use heat-stable reagents for all experiments intended to characterize native function

• Activity assays:

  • Design temperature-controlled experiments to compare activity at mesophilic (20-40°C) vs. thermophilic (60-85°C) temperatures

  • Incorporate appropriate controls at each temperature

• Structural stability:

  • Perform differential scanning calorimetry (DSC) to determine melting temperature

  • Use circular dichroism (CD) to monitor structural changes across temperature ranges

  • Compare stability in various buffer conditions

• Evolutionary considerations:

  • Compare AF_0891 with homologs from mesophilic archaea and bacteria

  • Analyze sequence features that may contribute to thermostability (increased ionic interactions, compact hydrophobic core, reduced surface loops)

What techniques should be employed to investigate the subcellular localization of AF_0891?

Determining the subcellular localization of AF_0891 is crucial for understanding its function:

• Immunolocalization approaches:

  • Generate specific antibodies against purified recombinant AF_0891

  • Perform immunogold electron microscopy on Archaeoglobus fulgidus cells

  • Use fluorescent antibodies for confocal microscopy (with appropriate fixation for archaeal cells)

• Biochemical fractionation:

  • Separate membrane, cytosolic, and other cellular fractions

  • Detect AF_0891 by Western blotting in different fractions

  • Perform protease protection assays to determine topology

• Fluorescent protein fusions:

  • Create GFP or mCherry fusions if genetic tools are available for Archaeoglobus

  • Consider temperature-stable fluorescent protein variants

  • Observe localization in live cells

• Heterologous expression systems:

  • Express AF_0891 in model organisms with established localization tools

  • Compare localization patterns with native system predictions

Due to the hydrophobic regions in its sequence , AF_0891 may be associated with membranes, possibly playing a role in membrane integrity at high temperatures.

How can researchers effectively compare AF_0891 with homologous proteins from other archaeal species?

For comparative analysis of AF_0891 with homologs:

• Sequence-based approaches:

  • Perform BLAST searches against archaeal genomes

  • Generate multiple sequence alignments using MUSCLE or CLUSTAL

  • Identify conserved residues and motifs across phylogenetic distances

• Structure-based comparisons:

  • Compare predicted structural models of homologs

  • Identify structurally conserved regions that may indicate functional sites

  • Use structure alignment tools like DALI or TM-align

• Genomic context analysis:

  • Compare gene neighborhoods across multiple archaeal genomes

  • Identify conserved gene clusters that may indicate functional relationships

  • Look for co-evolution patterns

• Experimental validation:

  • Select representative homologs for recombinant expression

  • Compare biochemical properties under standardized conditions

  • Test functional complementation where genetic systems exist

The evolutionary conservation pattern may provide valuable insights into the functional importance of specific residues or regions within AF_0891.

What methodological approaches should researchers use when investigating the potential role of AF_0891 in extremophile adaptation?

To investigate AF_0891's potential role in extremophile adaptation:

• Comparative genomics across temperature gradients:

  • Compare AF_0891 homologs from hyperthermophilic, thermophilic, and mesophilic archaea

  • Identify amino acid substitution patterns correlated with optimal growth temperatures

  • Analyze codon usage and GC content for temperature adaptation signatures

• Site-directed mutagenesis studies:

  • Target residues unique to hyperthermophilic homologs

  • Measure effects on protein stability and function

  • Create chimeric proteins with domains from mesophilic homologs

• Expression analysis:

  • Study expression levels of AF_0891 under different temperature conditions

  • Compare with other known temperature-responsive genes

  • Investigate potential regulatory mechanisms

• Phenotypic analysis:

  • Where genetic tools exist, attempt gene deletion or silencing

  • Assess phenotypic consequences under various temperature conditions

  • Test complementation with homologs from different thermal environments

How can structural studies of AF_0891 contribute to understanding archaeal membrane biology?

Structural studies of AF_0891 could provide significant insights into archaeal membrane biology:

• High-resolution structural determination approaches:

  • X-ray crystallography (challenging for membrane proteins)

  • Cryo-electron microscopy (single-particle or tomography)

  • NMR spectroscopy for specific domains or fragments

• Computational modeling approaches:

  • Molecular dynamics simulations in archaeal-like membranes

  • Coarse-grained simulations for longer timescale events

  • Protein-lipid interaction predictions

• Structure-function correlation:

  • Map conserved residues onto the structure

  • Identify potential functional sites or interaction interfaces

  • Design structure-guided mutagenesis experiments

• Comparative structural biology:

  • Compare AF_0891 structure with bacterial or eukaryotic membrane proteins

  • Identify archaeal-specific structural features

  • Relate structural elements to extremophile adaptation

Understanding the structure of AF_0891 could reveal adaptation mechanisms for protein stability and function in archaeal membranes at high temperatures, potentially informing biotechnological applications in extreme conditions.

What approaches should researchers use to resolve contradictory experimental data about AF_0891 function?

When faced with contradictory experimental results regarding AF_0891 function:

• Systematic error identification:

  • Review experimental conditions (temperature, pH, salt, detergents)

  • Examine protein preparation methods (tags, purification approach)

  • Consider organism-specific factors (growth conditions, strain variations)

• Independent verification approaches:

  • Use multiple complementary techniques to test the same hypothesis

  • Collaborate with independent laboratories for validation

  • Apply different experimental designs to test robustness

• Computational reconciliation:

  • Use modeling and simulation to explain apparently contradictory results

  • Develop mechanistic models that could account for different observations

  • Identify potential confounding variables through statistical analysis

• Controlled variable isolation:

  • Design experiments that systematically isolate each variable

  • Create a decision tree of experiments to narrow down sources of variation

  • Consider using microfluidic or high-throughput approaches for parameter optimization

Researchers should report conflicting results transparently in publications, proposing testable hypotheses to explain discrepancies rather than selectively reporting only consistent findings.