Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_0585 (AF_0585)

What are the optimal conditions for storing and reconstituting recombinant AF_0585?

For optimal storage and reconstitution of recombinant AF_0585:

For maximum protein stability, repeated freezing and thawing should be avoided. Lyophilized powder forms typically have a shelf life of approximately 12 months at -20°C/-80°C, while liquid forms maintain stability for about 6 months under the same conditions .

What expression systems are typically used for recombinant production of AF_0585?

Recombinant AF_0585 has been successfully expressed using various host systems:

E. coli remains the predominant expression system due to its efficiency and cost-effectiveness for producing AF_0585. When using E. coli, the protein is typically fused to an N-terminal His tag to facilitate purification. The resulting recombinant protein demonstrates greater than 90% purity as determined by SDS-PAGE analyses .

What is known about the host organism Archaeoglobus fulgidus and its ecological niche?

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon with the following characteristics:

A. fulgidus can produce biofilms when subjected to environmental stresses such as extreme pH or temperature, high metal concentrations, or exposure to antibiotics, xenobiotics, or oxygen. These archaeons are known to cause corrosion of iron and steel in oil and gas processing systems by producing iron sulfide .

How does AF_0585 compare to other uncharacterized proteins in Archaeoglobus fulgidus?

Several uncharacterized proteins have been identified in the A. fulgidus genome, including:

Unlike AF_0585, AF_0052 and AF_0540 have different structural characteristics suggesting diverse cellular functions. While AF_0585 appears to be membrane-associated, AF_0052 and AF_0540 may have different subcellular localizations and functions. Comparative analysis of these uncharacterized proteins could provide insights into their potential roles in A. fulgidus biology .

What methodologies can be employed to determine the subcellular localization of AF_0585?

To determine the subcellular localization of AF_0585, researchers can employ the following methodologies:

• Computational prediction tools:

  • TMHMM, HMMTOP, or Phobius for transmembrane domain prediction

  • SignalP for signal peptide identification

  • PSORT for general localization prediction

• Experimental approaches:

  • Membrane fractionation: Separate membrane and cytosolic fractions using ultracentrifugation followed by Western blot analysis

  • Immunogold electron microscopy: Using antibodies against AF_0585 or its tags

  • Fluorescence microscopy: Using GFP-fusion constructs in heterologous expression systems

  • Protease accessibility assays: To determine protein topology within membranes

• Protein extraction methodology:

  • Use different detergents (mild to strong) to extract the protein from membranes

  • Compare extraction efficiency under various conditions to confirm membrane association

The hydrophobic nature of the AF_0585 sequence strongly suggests membrane localization, but experimental verification is necessary to determine its precise subcellular localization and membrane topology .

What approaches can be used to investigate potential functions of AF_0585?

To investigate the potential functions of AF_0585, researchers should consider a multi-faceted approach:

Of particular interest would be investigating AF_0585's expression under heat shock conditions. Research on A. fulgidus has identified approximately 350 of its 2,410 open reading frames (14%) that exhibit altered transcript abundance in response to heat shock. These genes span a range of cell functions including energy production, amino acid metabolism, and signal transduction .

How might AF_0585 be involved in A. fulgidus adaptation to extreme environments?

AF_0585 may play a role in A. fulgidus adaptation to extreme environments through the following potential mechanisms:

• Membrane stability: As a putative membrane protein, AF_0585 could contribute to maintaining membrane integrity under extreme temperatures (60-95°C) and pressures (up to 40 MPa).

• Stress response: Similar to the heat shock regulator HSR1 (AF1298) which was identified as part of a heat shock response operon in A. fulgidus, AF_0585 could be involved in stress response pathways .

• Pressure adaptation: Given A. fulgidus' piezotolerant and piezophilic characteristics (growth observed up to 40-50 MPa), AF_0585 might contribute to membrane adaptations required for pressure tolerance .

• Biofilm formation: A. fulgidus produces biofilms under environmental stress, and membrane proteins like AF_0585 could participate in cell-cell communication or adhesion mechanisms necessary for biofilm development .

To investigate these possibilities, researchers should examine AF_0585 expression under various stress conditions and assess the phenotypic effects of gene disruption on stress tolerance .

What expression profiling strategies would be most effective for studying AF_0585 regulation?

For studying AF_0585 regulation, the following expression profiling strategies would be most effective:

For experimental design, researchers should consider:

• Varying environmental conditions: Temperature gradients (60-95°C), pressure ranges (0.3-60 MPa), and different carbon sources to identify condition-specific regulation.

• Time-course experiments: Similar to studies of heat shock response in A. fulgidus, where expression profiles were analyzed at different time points after stress induction.

• Comparative analysis: Examine expression in parallel with known stress-responsive genes like AF1298 (HSR1), which was identified as one of the most highly induced genes at 5 minutes post-heat shock .

How can the structure-function relationship of AF_0585 be investigated?

Investigating the structure-function relationship of AF_0585 requires a multi-dimensional approach:

• Structural prediction and analysis:

  • Use bioinformatics tools (AlphaFold, SWISS-MODEL) to generate structural models

  • Analyze transmembrane topology using specialized prediction algorithms

  • Identify conserved domains and structural motifs

• Experimental structure determination:

  • X-ray crystallography (challenging for membrane proteins)

  • NMR spectroscopy for specific domains

  • Cryo-electron microscopy for membrane protein complexes

• Mutagenesis studies:

  • Site-directed mutagenesis of conserved residues

  • Domain swapping with homologous proteins

  • Truncation analysis to determine functional domains

• Functional validation:

  • Express mutant variants in E. coli or native host

  • Assess changes in localization, interaction partners, or phenotype

  • Complement knockout strains with wild-type or mutant variants

• Comparative analysis with related proteins:

  • Compare with uncharacterized proteins from related Archaeoglobus species

  • Analyze evolutionary conservation patterns to identify functionally important regions

What protocols are most effective for studying protein-protein interactions involving AF_0585?

For studying protein-protein interactions involving AF_0585, the following protocols can be employed, with special considerations for membrane proteins:

When working with membrane proteins like AF_0585, careful optimization of detergent conditions is critical for maintaining protein structure and interactions. For example, studying heat shock proteins in A. fulgidus required specialized approaches for membrane-associated proteins. Research on the AAA protein from A. fulgidus (AMA) has demonstrated that these proteins can form hexameric complexes, and similar quaternary structures might be relevant for AF_0585 .

How do high hydrostatic pressure conditions affect the expression and function of AF_0585?

To investigate the effects of high hydrostatic pressure (HHP) on AF_0585 expression and function, researchers should consider:

• Expression analysis under pressure:

  • Cultivate A. fulgidus under varying pressures (0.3-60 MPa)

  • Measure AF_0585 transcript and protein levels

  • Compare expression patterns between heterotrophic and autotrophic growth conditions

• Experimental considerations:

  • Use specialized high-pressure cultivation vessels

  • Consider the impact of decompression-repressurization cycles on cellular physiology

  • Monitor morphological changes associated with pressure stress

• Functional implications:

  • Assess membrane integrity and cell morphology at different pressures

  • Investigate potential interactions with other pressure-responsive proteins

  • Determine if AF_0585 is involved in the observed morphological changes at pressures above 40 MPa

Research has shown that A. fulgidus exhibits different growth patterns under varying pressures, with heterotrophic growth being moderately piezophilic (optimal at 20 MPa) and autotrophic growth being piezotolerant (similar growth rates from 0.3 to 40 MPa). At pressures above 40 MPa, cells show morphological changes from coccoid to irregular and elongated forms, which could involve membrane proteins like AF_0585 .

What comparative genomics approaches can help infer the function of AF_0585?

Comparative genomics approaches that can help infer the function of AF_0585 include:

• Homology-based analysis:

  • Identify homologs across Archaea and other domains

  • Generate multiple sequence alignments to identify conserved residues

  • Construct phylogenetic trees to understand evolutionary relationships

• Genomic context analysis:

  • Examine gene neighborhood conservation

  • Identify co-occurring genes across genomes

  • Look for operonic structures that include AF_0585

• Domain architecture analysis:

  • Compare domain organization with functionally characterized proteins

  • Identify novel domain combinations that might suggest function

• Comparative expression analysis:

  • Analyze expression patterns across conditions and compare with known genes

  • Identify co-expressed genes across multiple species

• Cross-species complementation:

  • Test functional complementation in related Archaeoglobus species

  • Assess phenotype rescue in heterologous systems

This approach is particularly valuable as comparative genomic studies on archaeal genomes have revealed important evolutionary relationships. For example, research has identified conserved signature proteins that connect Archaeoglobus with methanogens and Thermococci, suggesting shared ancestry or lateral gene transfer events .

What are the challenges and best practices in purifying recombinant AF_0585 for structural studies?

Purifying recombinant AF_0585 for structural studies presents several challenges due to its membrane-associated nature:

Recommended purification protocol:

• Express with N-terminal His-tag in E. coli

• Solubilize membranes with appropriate detergent

• Purify using Ni-NTA affinity chromatography

• Perform size exclusion chromatography

• Assess purity (>95% for structural studies)

• Concentrate to 5-10 mg/mL in appropriate buffer

For structural studies, consider reconstitution into lipid nanodiscs or amphipols to stabilize the protein in a native-like membrane environment while making it amenable to techniques like cryo-EM .

How might AF_0585 contribute to our understanding of extremophile adaptation mechanisms?

Studying AF_0585 can advance our understanding of extremophile adaptation through several avenues:

• Membrane adaptation mechanisms:

  • AF_0585's putative membrane localization may reveal how extremophiles maintain membrane fluidity and integrity at high temperatures (60-95°C) and pressures (up to 40 MPa)

  • Could provide insights into lipid-protein interactions critical for thermostability

• Evolutionary insights:

  • Comparing AF_0585 with homologs in other extremophiles may reveal conserved adaptations

  • Could identify convergent evolution strategies across different extremophile lineages

• Biotechnological applications:

  • Understanding AF_0585's role could inform the development of thermostable membrane systems for industrial applications

  • May guide engineering of pressure-tolerant microorganisms for various biotechnological processes

• Astrobiology implications:

  • A. fulgidus thrives in conditions potentially analogous to extraterrestrial environments

  • Studying its adaptive mechanisms may inform our search for life in extreme environments elsewhere

• Biofilm formation in extreme conditions:

  • If AF_0585 is involved in biofilm formation, it could reveal how multicellular structures provide protection in extreme environments

  • May provide insights into biocorrosion mechanisms relevant to industrial settings

Understanding the function of uncharacterized proteins like AF_0585 is crucial for completing our picture of how extremophiles like A. fulgidus adapt to their challenging environments, with potential implications for evolutionary biology, biotechnology, and astrobiology .