Recombinant Picrophilus torridus UPF0095 protein PTO0617 (PTO0617)

What is Picrophilus torridus and why is it significant for protein research?

Picrophilus torridus is a thermoacidophilic euryarchaeon that thrives optimally at 60°C and pH 0.7. Strains of this species were first isolated from a dry solfataric field in northern Japan. The organism belongs to the Picrophilaceae family, which includes the most acidophilic organisms known, capable of growth at negative pH values and even adapting to conditions such as those in 1.2 M sulfuric acid. Unlike other thermoacidophilic organisms that maintain internal pH values close to neutral, P. torridus has an unusually low intracellular pH of 4.6. This extreme adaptation makes P. torridus and its proteins valuable models for studying thermoacidophilic adaptations in biological systems .

What is known about the genomic context of PTO0617 in P. torridus?

PTO0617 is one of the 1,535 ORFs identified in the 1,545,900-bp circular chromosome of P. torridus. The genome exhibits the highest coding density (92%) among thermoacidophiles, with approximately 74% of all ORFs having assigned functions. While specific information about the genomic neighborhood of PTO0617 is limited in the available data, it is located within a genome that contains numerous adaptation mechanisms for extreme pH and temperature conditions. Researchers should examine the gene's location relative to potential operons or functionally related genes to understand its regulation and potential role in cellular processes .

How does the UPF0095 protein family classification inform research on PTO0617?

The UPF (Uncharacterized Protein Family) designation indicates that PTO0617 belongs to a protein family with conserved sequence and structure but unclear function. When researching PTO0617, it's methodologically sound to analyze other characterized proteins in the UPF0095 family across different species to generate hypotheses about its function. Sequence alignment tools like BLAST and structure prediction algorithms can help identify conserved domains and motifs that might indicate functional roles. Additionally, phylogenetic analysis of UPF0095 proteins across archaea can provide evolutionary context about potential divergence or conservation of function in extremophiles.

What are the optimal conditions for culturing P. torridus for protein extraction?

P. torridus DSM 9790 can be cultured using ATCC 2011 medium consisting of (NH₄)₂SO₄ (0.2 g L⁻¹), MgSO₄ (0.5 g L⁻¹), CaCl₂·2H₂O (0.25 g L⁻¹), KH₂PO₄ (3.0 g L⁻¹), and yeast extract (2.0 g L⁻¹). The medium should be adjusted to pH 0.7 with concentrated H₂SO₄. For optimal growth, cultures should be incubated at 58°C with gentle shaking (75 rpm). Growth can be monitored by measuring absorbance at 600 nm or by direct cell counting using a Petroff-Hauser counting chamber. Researchers should note that growth phase significantly affects cellular composition, so harvesting cells at a consistent growth phase is crucial for reproducible protein isolation .

What purification challenges are specific to recombinant PTO0617, and how can they be addressed?

Purifying recombinant proteins from acidophilic organisms presents unique challenges. A methodological approach should include:

• Buffer selection: Using acidic buffers (pH 4-5) during initial purification steps to maintain protein stability, then gradually adjusting to experimental conditions

• Denaturation risk: Avoiding rapid pH or temperature changes that could denature the acid-adapted protein structure

• Contaminant removal: Implementing heat treatment steps (60°C) to leverage the thermostability of PTO0617 while denaturing E. coli proteins if expressed heterologously

• Chromatography selection: Utilizing ion exchange chromatography at acidic pH followed by size exclusion under carefully controlled conditions

• Activity preservation: Including stabilizing agents like glycerol (10-20%) or specific ions found in the P. torridus cytoplasm to maintain native conformation

What structural features distinguish PTO0617 from mesophilic homologs?

Proteins from extremophiles like P. torridus typically exhibit structural adaptations that enable function in harsh conditions. For PTO0617, researchers should examine:

• Increased surface negative charge to maintain protein solubility at low pH

• Higher proportion of acidic residues (Asp, Glu) in surface-exposed regions

• Reduced number of salt bridges that would be disrupted at extreme acidity

• Enhanced hydrophobic core packing for thermostability

• Lower proportion of thermolabile residues (Asn, Gln, Cys, Met)

• Potential disulfide bonds that contribute to stability in oxidizing acidic environments
  Comparative structural analysis using homology modeling based on crystallized homologs, followed by molecular dynamics simulations under varying pH conditions, can reveal these adaptations. Circular dichroism spectroscopy at different pH values (0.7-7.0) would provide experimental validation of predicted structural stability.

How does the amino acid composition of PTO0617 reflect adaptation to acidic environments?

The amino acid composition of proteins from acidophiles often shows distinctive patterns reflecting adaptation to low pH environments. While specific data for PTO0617 is not provided in the search results, proteins from P. torridus typically exhibit increased proportions of acidic amino acids like aspartic acid and glutamic acid in surface-exposed regions, which maintain negative charge even at very low pH, preventing protonation and aggregation. There is typically a decreased frequency of lysine residues (which are more susceptible to acid denaturation) and increased usage of arginine (which maintains positive charge at low pH due to its higher pKa). Methodologically, researchers can perform comparative amino acid frequency analysis between PTO0617 and homologs from neutrophilic organisms to identify these acid-adaptation signatures.

What experimental approaches are most effective for determining the function of uncharacterized proteins like PTO0617?

For uncharacterized proteins like PTO0617, a multi-faceted approach is recommended:

• Bioinformatic analysis: Employ tools like InterProScan, HMMER, and structure prediction (AlphaFold) to identify functional domains and structural motifs

• Transcriptomic profiling: Analyze expression patterns of PTO0617 under different conditions to identify co-regulated genes

• Gene knockout/knockdown: Create deletion mutants in P. torridus (if genetic systems exist) to observe phenotypic effects

• Protein-protein interaction studies: Use pull-down assays or bacterial/yeast two-hybrid systems adapted to acidic conditions to identify interaction partners

• Enzymatic activity screening: Test purified protein against substrate libraries under varying pH and temperature conditions

• Localization studies: Generate fluorescently tagged versions to determine subcellular localization

• Heterologous expression: Express PTO0617 in model organisms under stress conditions to observe potential protective effects
  This systematic approach can gradually narrow down potential functions, particularly in the context of acid adaptation mechanisms.

How might PTO0617 contribute to acid tolerance in P. torridus?

Based on P. torridus' extraordinary acid tolerance and its unique internal pH of 4.6 (unlike other acidophiles that maintain near-neutral internal pH), PTO0617 could potentially contribute to acid tolerance through several mechanisms:

• Membrane association: It may function as part of the membrane proteome that controls proton flux

• Proton pumping: It could be involved in active proton extrusion systems

• Cytoplasmic buffering: It might participate in cytoplasmic pH homeostasis mechanisms

• Protein protection: It could function as a chaperone that prevents acid-induced protein denaturation

• DNA/RNA protection: It may bind to and stabilize nucleic acids under acidic stress
  To methodologically investigate these possibilities, researchers should conduct subcellular fractionation studies, pH-dependent activity assays, and interaction studies with known acid stress response elements in the P. torridus proteome .

What does comparative genomic analysis reveal about the conservation of PTO0617 across archaeal species?

Comparative genomic analysis of proteins from the UPF0095 family across archaea can provide insights into the evolutionary conservation and potential functional importance of PTO0617. Methodologically, researchers should:

• Perform reciprocal BLAST searches against archaeal genomes to identify true orthologs

• Analyze synteny (gene neighborhood conservation) across related species

• Examine conservation patterns in relation to phylogenetic distance and ecological niche

• Identify co-evolution with other proteins that might indicate functional relationships

• Compare selection pressure (dN/dS ratios) on PTO0617 orthologs to estimate functional constraints
  Given that P. torridus contains 397 hypothetical ORFs, of which 79 are unique to the organism, determining whether PTO0617 belongs to the core archaeal genome or represents a lineage-specific adaptation is crucial for understanding its significance .

How do the genomic features of P. torridus inform research on PTO0617?

The genomic analysis of P. torridus reveals several features that provide context for PTO0617 research:

How can structural data on PTO0617 inform protein engineering for industrial applications?

Understanding the structural basis of PTO0617's acid and thermal stability could inform protein engineering strategies for industrial enzymes. Methodologically, researchers should:

• Determine high-resolution structure through X-ray crystallography or cryo-EM under acidic conditions

• Identify stability-enhancing motifs through computational analysis of electrostatic interactions

• Perform alanine scanning mutagenesis to identify critical residues for stability

• Design chimeric proteins incorporating acid-stability domains from PTO0617 into industrially relevant enzymes

• Test engineered proteins under industrial conditions (high temperature, low pH, presence of organic solvents)
  This approach could yield valuable insights for designing enzymes for biocatalysis in acidic environments, such as biomass hydrolysis or food processing applications.

What methods can be employed to study the adaptation of PTO0617 to temperature variations?

The adaptation of P. torridus proteins to temperature variations can be studied through both genomic and lipidomic approaches. Research on P. torridus has shown that temperature affects the abundance and composition of tetraether lipids, with the abundance of core and polar GDGTs per cell decreasing with increasing incubation temperature. The polar GDGT ring index was positively correlated (Pearson R² = 0.97) with incubation temperature .
For studying PTO0617 specifically, methodologies should include:

• Temperature-dependent activity assays (if enzymatic function is determined)

• Thermal shift assays to determine melting temperatures at different pH values

• Circular dichroism spectroscopy to monitor secondary structure changes with temperature

• Comparative expression studies at various temperatures (53°C, 58°C, 63°C) to determine if PTO0617 is differentially regulated

• Molecular dynamics simulations at different temperatures to identify flexible regions and stabilizing interactions

How might replication origin analysis in P. torridus genome inform studies of PTO0617 regulation?

The Z-curve analysis method has been used to identify replication origins in archaeal genomes, including those of other extremophiles. Understanding the location of PTO0617 relative to replication origins could provide insights into its regulation and expression patterns. Methodologically, researchers should:

• Apply Z-curve analysis to locate replication origins in the P. torridus genome

• Determine the position of PTO0617 relative to these origins

• Analyze gene expression patterns based on distance from origins

• Identify potential DNA binding motifs in the promoter region of PTO0617

• Perform chromatin immunoprecipitation experiments to identify proteins binding to the PTO0617 promoter under different growth conditions
  This approach could reveal whether PTO0617 expression is linked to DNA replication or cell cycle progression, providing additional functional context .

What are the main technical challenges in expressing PTO0617 in heterologous systems?

Expressing proteins from extremophiles like P. torridus in heterologous systems presents several challenges:

• Codon usage bias: P. torridus has a G+C content of 36%, which differs significantly from common expression hosts

• Protein folding: The acidophilic nature of P. torridus proteins may require specialized chaperones absent in heterologous hosts

• Post-translational modifications: Any archaea-specific modifications would be absent in bacterial expression systems

• Toxicity: Membrane or regulatory proteins may be toxic when expressed in heterologous hosts

• Solubility: Proteins adapted to extreme conditions may aggregate in mesophilic expression systems
  To address these challenges, researchers should consider codon optimization, co-expression with chaperones, fusion with solubility tags, and testing multiple expression systems (bacterial, yeast, and cell-free systems) .

How can researchers optimize growth conditions for P. torridus to maximize PTO0617 expression?

To optimize growth conditions specifically for studying PTO0617, researchers should consider:

• Growth phase effects: The composition of P. torridus cells varies significantly throughout the growth cycle. Core and polar lipid fractions showed distinct patterns between lag/stationary phases versus logarithmic growth phases, suggesting comprehensive proteomic changes during growth. Researchers should determine in which growth phase PTO0617 is maximally expressed .

• Temperature effects: P. torridus growth at different temperatures (53°C, 58°C, 63°C) affects cellular composition. The positive correlation between temperature and the polar GDGT ring index (Pearson R² = 0.97) indicates systematic cellular adaptations to temperature. Testing PTO0617 expression at these temperature points would reveal its role in thermal adaptation .

• pH optimization: While P. torridus grows optimally at pH 0.7, testing expression levels across a range of acidic conditions (pH 0-2) could reveal whether PTO0617 is differentially regulated in response to varying acidity.