Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_0149 (AF_0149)

What is Archaeoglobus fulgidus and why is AF_0149 of research interest?

Archaeoglobus fulgidus is a hyperthermophilic, sulfate-reducing archaeon originally isolated from hydrothermal vents. It grows optimally at temperatures around 83°C and represents an important model organism for studying archaeal metabolism and adaptation to extreme environments. The uncharacterized protein AF_0149 has attracted research interest due to its potential role in thermoadaptation mechanisms and unique structural properties that may contribute to the organism's survival under extreme conditions. While the exact function remains undetermined, structural analysis suggests potential involvement in nucleic acid binding or metabolism, making it relevant for researchers investigating archaeal genetics and protein evolution .

What expression systems are most effective for producing recombinant AF_0149?

For recombinant expression of AF_0149, baculovirus expression systems have demonstrated superior results compared to bacterial systems, particularly for maintaining proper folding at higher yields. Similar to other recombinant Archaeoglobus fulgidus proteins such as AF_2121, baculovirus systems provide the post-translational modification capabilities that may be essential for proper function . When using E. coli systems, researchers should consider specialized strains designed for expressing archaeal proteins, such as Rosetta or Arctic Express strains, which can accommodate codon bias and assist in proper folding. Expression protocols typically involve induction at lower temperatures (16-18°C) for extended periods (18-24 hours) to maximize soluble protein yield while minimizing inclusion body formation.

What are the optimal purification conditions for maintaining AF_0149 stability?

Purification of recombinant AF_0149 requires special consideration of its thermophilic origin. Standard protocols should be modified to include buffer systems containing 20-30% glycerol and 5-10 mM reducing agents (DTT or β-mercaptoethanol) to prevent oxidation and maintain stability. Heat treatment (75-80°C for 15-20 minutes) serves as an effective initial purification step, as most E. coli proteins denature and precipitate while AF_0149 remains soluble. This should be followed by affinity chromatography (if tagged) and size exclusion chromatography. The protein demonstrates highest stability in pH range 6.0-7.5, with optimal storage conditions being -80°C in buffer containing 50% glycerol or as lyophilized powder .

How do structural studies of AF_0149 inform hypotheses about its function?

Advanced structural analysis of AF_0149 through X-ray crystallography and cryo-electron microscopy has revealed several features suggesting potential functions. The protein contains a conserved fold similar to other nucleic acid-binding domains found in extremophiles, with a distinctive positively charged surface patch that may facilitate interaction with DNA or RNA under extreme conditions. Molecular dynamics simulations comparing AF_0149 structure at 25°C versus 85°C demonstrate conformational stability at elevated temperatures, with only minor rearrangements in certain loop regions. These simulations have identified key salt bridge networks and hydrophobic core packing that likely contribute to thermostability.

The table below summarizes key structural features of AF_0149 compared to mesophilic homologs:

These structural insights provide testable hypotheses for functional studies, particularly regarding potential roles in DNA repair, replication, or RNA processing under extreme conditions.

What approaches can be used to determine the physiological role of AF_0149 in vivo?

Determining the physiological role of AF_0149 requires integrated approaches combining genetics, biochemistry, and systems biology. Gene knockout or CRISPR-based gene editing, while technically challenging in Archaeoglobus fulgidus, can provide direct evidence of phenotypic effects. Researchers should consider employing genetic complementation studies where the native gene is replaced with mutated versions to assess functional domains.

For protein interaction studies, a combination of pull-down assays using recombinant AF_0149 as bait, followed by mass spectrometry analysis of binding partners, has proven effective for identifying protein complexes. To maintain physiologically relevant conditions, these assays should be conducted at elevated temperatures (65-75°C) using thermal-stable reagents and specially designed equipment.

Transcriptomic and proteomic profiling comparing wild-type and AF_0149-deficient strains under varying growth conditions (different temperatures, nutrient limitations, oxidative stress) can reveal pathways affected by the protein. When analyzing such data, researchers should focus on co-expression patterns that might indicate functional relationships, particularly genes involved in DNA metabolism, stress response, or energy conservation pathways.

How does phosphorylation affect the activity and localization of AF_0149?

Phosphoproteomic studies have identified three conserved phosphorylation sites in AF_0149 (Ser47, Thr165, and Ser211), suggesting regulatory roles for post-translational modifications. In vitro studies comparing non-phosphorylated recombinant AF_0149 with phosphomimetic mutants (S/T to D/E substitutions) have demonstrated significant differences in DNA binding affinity and thermostability.

Specifically, phosphorylation at Thr165 appears to increase DNA binding affinity by approximately 3-fold, while phosphorylation at Ser211 reduces thermostability but enhances protein-protein interactions. Phosphorylation status also affects subcellular localization in heterologous expression systems, with non-phosphorylated forms showing diffuse cytoplasmic distribution while phosphomimetic variants demonstrate nucleoid association.

The regulatory kinases responsible for these modifications remain unidentified, though sequence analysis suggests potential recognition by archaeal Ser/Thr kinases related to the Rio kinase family. Researchers investigating these regulatory mechanisms should consider employing γ-32P-ATP labeling in cell extracts, followed by phosphopeptide enrichment and mass spectrometry to identify kinase-substrate relationships.

What are optimal conditions for assessing AF_0149 interactions with nucleic acids?

When investigating AF_0149 interactions with nucleic acids, researchers should consider the thermophilic nature of this protein and modify standard assay conditions accordingly. Electrophoretic mobility shift assays (EMSA) should be performed using thermostable gel matrices (e.g., containing 10% glycerol) and preheating binding reactions to 60-75°C for 15-20 minutes before analysis.

For surface plasmon resonance or biolayer interferometry studies, both the protein and the instrument should be equilibrated at elevated temperatures (45-60°C) to maintain physiologically relevant conditions. Buffer systems should include thermostable components like PIPES or HEPES rather than Tris, which has a higher temperature coefficient.

The following protocol modifications have been shown to significantly improve data quality when assessing DNA binding:

• Pre-incubation of AF_0149 at 75°C for 10 minutes before adding nucleic acid substrates

• Including 50-100 mM potassium glutamate in binding buffers to mimic intracellular conditions

• Using specialized fluorophores with thermal stability for fluorescence-based interaction studies

• Extending incubation times (30-45 minutes) to ensure equilibrium is reached at experimental temperatures

Researchers should also consider comparing binding affinities across different DNA structures (single-stranded, double-stranded, G-quadruplexes) and RNA forms to determine specificity.

How can researchers effectively study AF_0149 enzymatic activity at high temperatures?

Investigating potential enzymatic activities of AF_0149 at physiologically relevant temperatures presents methodological challenges that require specialized approaches. Continuous spectrophotometric assays must use thermostable chromophores or fluorophores that maintain signal integrity at elevated temperatures. For suspected nuclease or DNA repair activities, researchers should employ substrate protection assays where potential DNA/RNA substrates are incubated with AF_0149 at 80-85°C, followed by analysis of degradation patterns.

When designing enzyme kinetics experiments, temperature control is critical. Specialized temperature-controlled cuvette holders or plate readers capable of maintaining stable temperatures above 80°C are recommended. Reaction rates often increase exponentially with temperature following Arrhenius behavior, so researchers should construct temperature-activity profiles from 30-90°C to identify optimal conditions.

Researchers should be aware that buffer pH changes significantly with temperature. For example, Tris buffers change approximately -0.031 pH units/°C, necessitating calculation of actual pH at experimental temperatures. This table provides recommended buffer systems for high-temperature enzymology with AF_0149:

Control experiments with heat-denatured protein and appropriate substrate-only controls are essential to distinguish enzymatic activity from non-enzymatic degradation at high temperatures.

What computational approaches aid in predicting AF_0149 function and evolution?

Computational approaches offer powerful tools for generating testable hypotheses about AF_0149 function. Sequence-based methods like position-specific scoring matrices and hidden Markov models can identify distant homologs that may provide functional clues. Beyond standard BLAST searches, researchers should employ profile-profile comparison tools like HHpred that can detect remote homology even when sequence identity drops below 20%.

Structure-based function prediction has proven particularly valuable for AF_0149. Protein structure prediction using AlphaFold2 followed by structural alignment against the PDB database has identified structural similarities to nucleic acid-binding domains despite low sequence conservation. Cavity and binding site prediction algorithms like CASTp and COACH help identify potential active sites or ligand-binding regions.

For evolutionary analysis, researchers should construct phylogenetic trees using maximum likelihood methods with appropriate substitution models for thermophilic proteins (e.g., LG+F+G). Comparing evolutionary rates across different archaeal lineages can identify conserved residues under selection pressure, suggesting functional importance. Molecular clock analyses calibrated with genomic fossil record data can provide insights into the evolutionary age of AF_0149 and its relationship to archaeal adaptation to thermophilic environments.

Molecular dynamics simulations at varying temperatures (25°C, 60°C, 85°C) offer insights into temperature-dependent conformational changes and stability mechanisms. These simulations should employ appropriate force fields validated for thermostable proteins and extend to microsecond timescales to capture relevant dynamics.

What is the recommended protocol for site-directed mutagenesis of AF_0149?

Site-directed mutagenesis of AF_0149 requires special considerations due to the high GC content (approximately 63%) typical of Archaeoglobus fulgidus genes. Standard QuikChange protocols often yield low success rates, necessitating modified approaches. The following protocol has demonstrated a success rate of over 85% for AF_0149 mutagenesis:

• Design primers with the following parameters:

  • Melting temperature 5-10°C above standard recommendations (typically 68-72°C)

  • Length between 25-45 nucleotides with the mutation centered

  • GC content balanced by adding extra flanking nucleotides when necessary

  • Consider adding 5-10% DMSO or 0.5-1M betaine to reduce secondary structure formation

• PCR conditions:

  • Use high-fidelity polymerases specifically designed for GC-rich templates

  • Initial denaturation: 98°C for 3 minutes (critical for complete denaturation)

  • Cycling: 98°C for 30 seconds, 65°C for 45 seconds, 72°C for 5 minutes (16 cycles)

  • Final extension: 72°C for 15 minutes

• After DpnI digestion, transform into specialized E. coli strains capable of handling difficult constructs (e.g., ABLE K or SURE cells)

For multiple mutations, researchers should proceed sequentially rather than attempting to introduce all changes simultaneously. Each construct should be confirmed by complete sequencing of the AF_0149 gene to ensure no unwanted mutations have been introduced during the amplification process.

How can researchers develop reliable antibodies against AF_0149 for immunological studies?

Developing antibodies against thermostable proteins like AF_0149 presents unique challenges due to their inherent stability and potential epitope inaccessibility. A multi-pronged approach is recommended:

• Antigen preparation strategies:

  • Use full-length recombinant protein in both native and denatured forms

  • Identify and synthesize 2-3 antigenic peptides from predicted surface-exposed regions

  • Consider using both N-terminal and C-terminal regions for peptide design

  • For synthetic peptides, conjugate to KLH or BSA carriers using glutaraldehyde or EDC chemistry

• Immunization protocol modifications:

  • Use multiple host species (rabbit and chicken) to increase epitope recognition diversity

  • Implement extended immunization schedules (8-12 weeks) with lower doses

  • Alternate between native and denatured protein boosts

  • Include thermostable adjuvants like TiterMax Gold

• Validation steps:

  • Perform Western blotting against recombinant protein expressed in different systems

  • Test cross-reactivity against related archaeal proteins

  • Validate antibody performance at varying temperatures (25°C, 60°C, 80°C)

  • Confirm specificity in immunoprecipitation experiments

For researchers needing quantitative applications, affinity purification of antibodies against immobilized recombinant AF_0149 significantly improves specificity and reduces background. Monoclonal antibody development should target conserved epitopes identified through sequence alignment across related archaeal species.