Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_1455 (AF_1455)

What is Archaeoglobus fulgidus and why is protein AF_1455 significant for research?

Archaeoglobus fulgidus is a hyperthermophilic, sulfate-reducing archaeon originally isolated from marine hydrothermal vents with an optimal growth temperature of 83°C. This organism has gained significant research attention due to its extremophilic properties and unique molecular adaptations. The uncharacterized protein AF_1455 represents one of many proteins in the A. fulgidus proteome with unknown function, making it an important target for structural and functional characterization. Similar to other proteins in Archaeoglobus species, such as the well-studied Argonaute protein (AfAgo), AF_1455 may have unique structural properties that contribute to thermostability and potentially novel biochemical functions . Research on AF_1455 contributes to our broader understanding of extremophile biology, protein evolution, and potentially identifies new enzymes with biotechnological applications.

What expression systems are most effective for recombinant AF_1455 production?

The optimal expression system for recombinant AF_1455 depends on research objectives and downstream applications. For structural studies requiring high purity and native folding, expression in E. coli BL21(DE3) strains with a pET-based vector system incorporating a His-tag for purification has shown reasonable success . When expressing thermophilic proteins like AF_1455, researchers should consider the following methodological approach:

• Test multiple expression strains (BL21, Rosetta, Arctic Express)

• Evaluate different induction temperatures (16°C, 25°C, 37°C)

• Vary IPTG concentrations (0.1-1.0 mM)

• Assess solubility enhancement with co-expression of chaperones

The table below summarizes typical expression conditions that have been effective for similar archaeal proteins:

What purification strategies yield the highest purity for recombinant AF_1455?

Purification of recombinant AF_1455 typically follows a multi-step chromatographic approach, beginning with immobilized metal affinity chromatography (IMAC) for His-tagged protein . For highest purity, researchers should implement the following methodological sequence:

• Initial capture using Ni-NTA affinity chromatography under native conditions

• Intermediate purification with ion exchange chromatography (typically Q-Sepharose)

• Polishing step using size exclusion chromatography (Superdex 75/200)

• Heat treatment (75-80°C for 20 minutes) to exploit thermostability and remove E. coli contaminants

Researchers should monitor protein purity by SDS-PAGE after each step and adjust buffer conditions based on AF_1455's predicted isoelectric point. The heat treatment step is particularly effective for archaeal proteins like AF_1455, as their thermostable nature allows selective precipitation of E. coli host proteins while retaining target protein activity. For specialized applications requiring tag removal, incorporate a protease cleavage site (TEV or PreScission) between the tag and target protein, followed by a second IMAC step to separate the cleaved protein from the tag.

What structural characterization techniques are most informative for AF_1455 analysis?

Structural characterization of AF_1455 should employ a complementary suite of techniques to elucidate its three-dimensional structure and functional domains. Based on approaches used for similar proteins from Archaeoglobus fulgidus, such as AfAgo, the following methodological workflow is recommended:

• Secondary structure analysis using circular dichroism (CD) spectroscopy to determine α-helix and β-sheet content

• Thermal stability assessment using differential scanning calorimetry (DSC) to determine melting temperature

• Crystallization trials for X-ray crystallography, focusing on conditions successful for other A. fulgidus proteins

• Cryo-electron microscopy for visualization of potential protein complexes

• Nuclear magnetic resonance (NMR) spectroscopy for solution structure determination, if protein size permits

When analyzing structural data, researchers should compare results with known structures of other A. fulgidus proteins, such as the Argonaute protein that contains MID and PIWI domains . Similar to AfAgo, AF_1455 may form heterodimeric complexes with other proteins encoded within the same operon, necessitating co-expression studies to capture physiologically relevant conformations.

How should recombinant AF_1455 be stored to maintain stability and activity?

Storage conditions significantly impact the stability and activity of recombinant proteins, particularly those from thermophilic organisms. For AF_1455, consider the following methodological approach to storage:

• Evaluate multiple storage buffers, typically containing:

  • 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 100-300 mM NaCl

  • 1-5 mM reducing agent (DTT or β-mercaptoethanol)

  • Optional: 10% glycerol or 0.5 mM EDTA

• Perform stability testing at different temperatures:

  • 4°C for short-term storage (1-2 weeks)

  • -20°C with 30-50% glycerol for medium-term storage (1-3 months)

  • -80°C for long-term storage (>3 months)

  • Liquid nitrogen for archival storage

• Assess the impact of multiple freeze-thaw cycles on protein activity

The following table summarizes stability data typically observed for thermostable archaeal proteins under various storage conditions:

For AF_1455 specifically, aliquoting the protein into single-use volumes before freezing is recommended to avoid repeated freeze-thaw cycles, which can significantly reduce activity.

How does AF_1455 compare structurally and functionally to other proteins in Archaeoglobus fulgidus?

Comparative analysis of AF_1455 with other A. fulgidus proteins reveals important insights into its potential structure and function. When examining similarities with the well-characterized AfAgo protein, researchers should consider the following methodological approach:

• Perform sequence alignment using BLAST, HHpred, and HMMER to identify conserved domains

• Compare predicted secondary structures using programs like PSIPRED and JPred

• Analyze conserved residues and motifs that might indicate functional sites

• Examine potential structural homology with proteins of known function using I-TASSER or Phyre2

The Archaeoglobus fulgidus Argonaute protein (AfAgo) provides an interesting comparative case, as it was initially classified as a long-B pAgo but contains only MID and catalytically inactive PIWI domains, similar to short pAgos . Further research revealed that AfAgo forms a heterodimeric complex with another protein encoded upstream in the same operon, which functionally complements its structure . This finding suggests that AF_1455 might similarly function as part of a protein complex rather than as an isolated entity.

Researchers should investigate the genomic context of AF_1455 to identify potential interaction partners encoded in proximity, as observed with AfAgo. When analyzing potential functions, consider the prevalence of nucleic acid-binding proteins in Archaeoglobus fulgidus and their roles in defense mechanisms against mobile genetic elements.

What computational approaches can predict AF_1455 function based on sequence homology?

Predicting the function of uncharacterized proteins like AF_1455 requires sophisticated computational approaches. The following methodological workflow is recommended:

• Perform sensitive sequence homology searches using PSI-BLAST and HHpred to identify remote homologs

• Apply protein fold recognition methods (threading) using programs like LOMETS and Phyre2

• Analyze conserved functional domains using InterProScan and SMART

• Perform genomic context analysis to identify conserved gene neighborhoods

• Apply machine learning approaches (DeepFRI, ESM-1b) that integrate sequence, structure, and evolutionary information

When interpreting computational predictions, researchers should consider multiple lines of evidence rather than relying on a single approach. The genomic context analysis is particularly important for archaeal proteins, as functionally related genes are often co-located in operons. Phylogenetic profiling can provide additional insights by identifying proteins with similar patterns of presence/absence across species, suggesting functional relationships.

For AF_1455, special attention should be paid to structural predictions that indicate potential nucleic acid binding capabilities, similar to the Argonaute proteins in A. fulgidus that use small oligonucleotide guides to bind complementary nucleic acid targets . The presence of specific structural motifs might indicate involvement in gene expression regulation, mobile genetic element silencing, or defense against viruses and plasmids.

How can site-directed mutagenesis be used to study AF_1455 function?

Site-directed mutagenesis represents a powerful approach to elucidate the functional significance of specific residues within AF_1455. The following methodological strategy should be employed:

• Identify candidate residues for mutagenesis based on:

  • Conservation analysis across homologous proteins

  • Structural predictions identifying potential active sites

  • Charged or hydrophobic surface patches that might mediate interactions

• Design a systematic mutagenesis approach:

  • Alanine scanning of conserved residues

  • Conservative substitutions to retain chemical properties

  • Non-conservative substitutions to dramatically alter properties

  • Deletion or truncation of specific domains

• Evaluate the impact of mutations on:

  • Protein stability and folding (using CD spectroscopy and thermal shift assays)

  • Interaction with potential binding partners (using pull-down assays or SPR)

  • Catalytic activity (if enzymatic function is identified)

  • Oligomerization state (using SEC-MALS)

For thermostable proteins like AF_1455, additional consideration should be given to mutations that might affect thermostability. Researchers should design mutations that specifically target residues predicted to contribute to thermostability (such as proline residues in loops, salt bridges, and hydrophobic core packing) to understand the structural basis of this property.

The table below outlines a potential site-directed mutagenesis experimental design for AF_1455:

What are potential interaction partners of AF_1455 within cellular processes?

Identifying interaction partners is crucial for understanding the biological role of uncharacterized proteins like AF_1455. Based on studies of other A. fulgidus proteins, the following methodological approach is recommended:

• Perform co-immunoprecipitation (Co-IP) experiments using tagged recombinant AF_1455 with A. fulgidus cell lysate

• Employ yeast two-hybrid or bacterial two-hybrid systems for targeted interaction screening

• Conduct pull-down assays using purified AF_1455 as bait

• Apply proximity-dependent biotin identification (BioID) for in vivo interaction mapping

• Perform analytical size exclusion chromatography to identify stable protein complexes

When designing interaction studies, researchers should consider that AF_1455 might interact with proteins encoded in proximity within the genome, similar to the AfAgo protein that forms a heterodimeric complex with a protein encoded upstream in the same operon . This heterodimeric complex enhances the performance of AfAgo in guide RNA-mediated target DNA binding, suggesting a functional complementation mechanism .

For archaeal proteins, it's particularly important to perform interaction studies under conditions that mimic the native environment, including elevated temperatures (75-85°C) and appropriate salt concentrations. Crosslinking approaches may be necessary to capture transient interactions that occur under these extreme conditions.

How can researchers address experimental challenges when working with recombinant AF_1455?

Working with recombinant proteins from extremophiles presents unique challenges that require specialized approaches. For AF_1455, researchers should consider the following methodological strategies:

• Addressing solubility issues:

  • Test multiple solubility tags (MBP, SUMO, TrxA)

  • Employ on-column refolding techniques

  • Adjust buffer conditions (pH, salt concentration, additives)

  • Consider co-expression with potential binding partners

• Ensuring proper folding:

  • Validate secondary structure using CD spectroscopy

  • Compare thermal stability with native protein using DSC

  • Assess native-like oligomerization using SEC-MALS

• Optimizing functional assays:

  • Perform assays at physiologically relevant temperatures (75-85°C)

  • Include appropriate cofactors and metal ions

  • Consider the impact of buffer components on activity

  • Validate activity using multiple complementary assay formats

• Addressing protein degradation:

  • Add protease inhibitors during purification

  • Remove proteolytically sensitive regions

  • Identify and mutate protease recognition sites

For experimental design in fractional factorial experiments, researchers should carefully select factors to test simultaneously while being mindful of potential interaction effects3. This approach is particularly useful when optimizing multiple parameters for expression, purification, or crystallization of challenging proteins like AF_1455. The resolution of the design indicates how well the experimental setup can distinguish between different effects3, with higher resolutions providing more detailed information at the cost of increased experimental complexity.

How can cryo-EM contribute to structural studies of AF_1455 and its complexes?

Cryo-electron microscopy (cryo-EM) offers powerful advantages for studying thermophilic proteins like AF_1455, particularly when examining potential complexes. The following methodological approach leverages recent advances in this technique:

• Sample preparation considerations:

  • Optimize protein concentration (typically 0.5-5 mg/mL)

  • Test multiple grid types and freezing conditions

  • Consider mild crosslinking to stabilize complexes

  • Evaluate detergent or amphipol addition if membrane association is suspected

• Data collection strategy:

  • Collect data at different defocus values

  • Implement beam-tilt data collection for aberration correction

  • Consider energy filtering to improve contrast

  • Implement motion correction algorithms

• Image processing workflow:

  • Perform 2D classification to identify homogeneous particles

  • Generate ab initio models for 3D reconstruction

  • Implement focused refinement on domains of interest

  • Validate maps using independent half-sets (FSC)

For AF_1455, cryo-EM may be particularly valuable if the protein forms higher-order complexes similar to other archaeal proteins. The ability to visualize proteins without crystallization is advantageous for thermophilic proteins that may resist crystallization due to flexible regions or heterogeneous conformations.

What insights can integrative structural biology provide about AF_1455 function?

Integrative structural biology combines multiple experimental and computational techniques to generate comprehensive structural models. For AF_1455, the following methodological framework is recommended:

This integrative approach is particularly valuable for proteins like AF_1455 that may form complexes or contain flexible regions challenging to characterize by a single technique. The combination of structural data at different resolutions provides complementary insights, enabling researchers to develop more complete models of protein structure and function.