Recombinant Photobacterium profundum UPF0758 protein PBPRA0202 (PBPRA0202)

What is PBPRA0202 and why is it significant for deep-sea microbiology research?

PBPRA0202 is a UPF0758 domain-containing protein from Photobacterium profundum strain SS9, a model organism for studying high-pressure adaptation. The significance lies in understanding how proteins from deep-sea bacteria function under extreme conditions, given that oceans cover 70% of Earth's surface with an average depth of 3800 meters . The protein belongs to a family of uncharacterized proteins (UPF - Uncharacterized Protein Family), suggesting its function remains to be fully elucidated, presenting valuable research opportunities for exploring novel biochemical adaptations to high-pressure environments.

What expression systems are available for producing recombinant PBPRA0202, and how do they affect downstream applications?

Recombinant PBPRA0202 can be produced using multiple expression systems, each providing different advantages depending on research objectives:

The choice depends on whether you need to prioritize yield, post-translational modifications, or structural integrity. For initial biochemical characterization, E. coli-expressed protein is typically sufficient, while functional studies examining potential high-pressure adaptations may benefit from baculovirus-expressed protein to ensure proper folding.

How should recombinant PBPRA0202 be reconstituted and stored to maintain optimal activity?

For optimal reconstitution and storage of PBPRA0202:

• Centrifuge the vial briefly before opening to collect contents at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For long-term storage, add 5-50% glycerol (final concentration)

• Aliquot into small volumes to minimize freeze-thaw cycles

• Store at -20°C/-80°C for up to 12 months (lyophilized) or 6 months (in solution)

Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they lead to protein denaturation and activity loss. This is particularly important for proteins from extremophiles, as they may have unusual stability properties compared to mesophilic proteins.

What are the recommended methods for assessing PBPRA0202 protein purity and integrity?

Multiple complementary techniques should be employed to comprehensively assess PBPRA0202 purity and integrity:

For PBPRA0202, standard SDS-PAGE typically shows >85% purity , but this represents only one dimension of quality assessment. For high-precision structural or functional studies, mass spectrometry is essential to verify sequence integrity, especially since proteins from extremophiles may exhibit unusual migration patterns on gels due to their unique amino acid compositions and structural properties.

What methodological approaches can be used to identify potential interaction partners of PBPRA0202?

Several complementary approaches can be employed to identify interaction partners:

• Pull-down assays: Using tagged recombinant PBPRA0202 as bait to capture interacting proteins from P. profundum lysates.

• Yeast two-hybrid screening: Particularly useful for detecting direct protein-protein interactions, though may require optimization for proteins from extremophiles.

• Proximity-dependent biotin identification (BioID): Fusing PBPRA0202 to a biotin ligase to biotinylate proximal proteins in vivo, allowing for subsequent purification and identification.

• Co-immunoprecipitation followed by mass spectrometry: Using antibodies against PBPRA0202 to isolate protein complexes from native environments.

• In silico prediction: Using computational approaches to predict functional partners based on:

  • Genomic context (gene neighborhood)

  • Co-expression patterns under different pressure conditions

  • Structural homology with characterized protein complexes

For P. profundum proteins, context-specific considerations include performing interaction studies under varying pressure conditions, as protein-protein interactions may be pressure-dependent, as observed with other piezophilic bacterial proteins .

What bioinformatic approaches can help predict the function of PBPRA0202 given its classification as an "uncharacterized protein"?

Given PBPRA0202's status as an uncharacterized protein, multiple bioinformatic approaches should be integrated:

• Sequence homology analysis: Compare PBPRA0202 sequence against characterized proteins using PSI-BLAST or HHpred to identify distant homologs.

• Domain architecture analysis: The UPF0758 domain architecture may provide functional clues when compared with other domain organizations.

• Structural prediction: Use AlphaFold2 or RoseTTAFold to generate structural models, followed by structural alignment against characterized proteins to identify potential functional similarities not evident from sequence alone.

• Genomic context analysis: Examine genes adjacent to PBPRA0202 in the P. profundum genome, as functionally related genes are often clustered together.

• Gene expression correlation: Analyze transcriptomic data from P. profundum under different pressure conditions to identify co-expressed genes with known functions .

• Phylogenetic profiling: Compare the distribution of PBPRA0202 homologs across species to identify organisms that have gained or lost this gene, potentially revealing its functional context.

The protein sequence MSLKLLPEESRPREKLLTRG AKALSDAELL AIFLRTGIKG MNAVELATHL LAEFGSLRAL FAADQTLFCL HKGLGPAKYA QLQAIIEMSQ RHLEETLKEG DVLTSPQHTR HYLSQLLRDR QREVFYVLFL DNQHRVIAGE VLFEGTINSA AVYPREIVKR SLEFNAVALI LAHNHPSGVA EPSQSDLRIT RTISDALALV DIRVLDHFIV GDGEIVSFSE QGW contains motifs that may be analyzed through specialized tools like ELM (Eukaryotic Linear Motif) resource or MEME Suite to identify functional elements.

How does the amino acid composition of PBPRA0202 compare to homologous proteins from non-piezophilic bacteria?

Comparative analysis of amino acid composition between PBPRA0202 and homologs from non-piezophilic bacteria may reveal adaptations to high pressure. The following table presents a hypothetical comparison based on known piezophilic adaptations:

• Identify homologs using BLAST against non-piezophilic bacterial genomes

• Perform multiple sequence alignment

• Calculate amino acid composition percentages for each protein

• Apply statistical tests to identify significant differences

• Map differences onto structural models to identify potential functional implications

This approach has revealed characteristic adaptations in other P. profundum proteins that contribute to their function under high pressure conditions .

How does PBPRA0202 relate to the ToxR regulon and pressure-responsive gene expression in P. profundum?

The relationship between PBPRA0202 and the ToxR regulon can be analyzed through several approaches:

• Promoter analysis: Examine the PBPRA0202 promoter region for ToxR binding sites, which typically contain direct repeats separated by 7-8 nucleotides.

• Comparative transcriptomics: Analyze RNA-seq data from wild-type P. profundum and toxR mutant strains under various pressure conditions. Search result indicates that RNA-seq analysis of ToxR mutant (TW30) and parental strain (DB110) has been performed, which could reveal if PBPRA0202 expression is ToxR-dependent.

• ChIP-seq analysis: Perform chromatin immunoprecipitation followed by sequencing to directly identify if ToxR binds to the PBPRA0202 promoter region.

• Expression correlation analysis: Determine if PBPRA0202 expression patterns cluster with known ToxR-regulated genes like OmpH, which responds to high hydrostatic pressure. Research has identified 22 genes with expression profiles similar to OmpH .

If PBPRA0202 is part of the ToxR regulon, this would suggest it plays a role in pressure adaptation. The toxR gene encodes a transmembrane DNA-binding protein that regulates genes in a pressure-dependent manner in P. profundum , functioning as a pressure sensor that controls the expression of genes involved in environmental adaptation.

What approaches can be used to determine if PBPRA0202 is involved in DNA repair mechanisms similar to RecD in P. profundum?

Given that RecD function is essential for high-pressure growth in P. profundum , investigating potential relationships between PBPRA0202 and DNA repair mechanisms requires:

• Co-expression analysis: Determine if PBPRA0202 is co-expressed with known DNA repair genes under stress conditions, particularly high pressure.

• Phenotypic analysis of gene knockouts: Create PBPRA0202 knockout strains and assess:

  • Growth rates under various pressure conditions

  • Sensitivity to DNA-damaging agents (UV radiation, methyl methanesulfonate)

  • Plasmid stability (a phenotype observed in recD mutants)

  • Cell morphology under high pressure

• Biochemical assays: Test purified PBPRA0202 for activities associated with DNA repair:

  • DNA binding capability

  • Nuclease activity

  • Helicase activity

  • Interaction with known DNA repair proteins

• Complementation studies: Attempt to complement recD mutants with PBPRA0202 to determine if it can restore high-pressure growth capability.

• Localization studies: Determine if PBPRA0202 localizes to the nucleoid under DNA damage or high-pressure conditions.

A comprehensive approach combining genetic and biochemical methods will provide the most reliable evidence for potential involvement in DNA repair pathways adaptations to high-pressure environments.

How does PBPRA0202 compare with the photolyase (Phr) system in P. profundum for potential roles in stress response?

Comparing PBPRA0202 with the photolyase (Phr) system requires systematic analysis:

• Structural comparison: Analyze whether PBPRA0202 contains features similar to photolyases, such as FAD or MTHF binding domains.

• Functional assays: Test if PBPRA0202 exhibits any photolyase-like activity:

  • In vitro DNA repair assays using UV-damaged DNA

  • Photoreactivation experiments comparing wild-type and PBPRA0202 knockout strains

• Expression analysis: Compare expression patterns of PBPRA0202 and phr genes under:

  • Various light conditions

  • UV stress

  • Different pressure conditions

• Co-localization studies: Determine if PBPRA0202 co-localizes with Phr proteins or UV-damaged DNA.

According to search result , P. profundum possesses a deoxyribodipyrimidine photolyase (Phr) system that repairs UV-damaged DNA through photoreactivation. This system includes genes like P3TCK_10663 (phr) and is studied through techniques such as:

• Constructing deletion mutants (e.g., pFL304 with Δ22 deletion of phr gene cluster)

• In vivo photoreactivation experiments using defined UV exposure (253.7 nm at 220 μW/cm²) followed by blue light recovery (350-400 nm at 20 μW/cm²)

These established methodologies for studying the Phr system can be adapted to investigate potential functional relationships with PBPRA0202.

What are the major technical challenges in working with recombinant proteins from piezophilic bacteria?

Several technical challenges must be addressed when working with proteins from piezophilic bacteria:

• Expression optimization: Proteins from deep-sea bacteria may not fold properly when expressed in conventional systems at atmospheric pressure. Consider:

  • Low-temperature expression to slow folding

  • Co-expression with chaperones from P. profundum

  • Expression under moderate pressure conditions if specialized equipment is available

• Buffer composition: Proteins evolved for high-pressure environments may require special buffer considerations:

  • Higher salt concentrations to mimic deep-sea conditions

  • Compatible solutes that provide pressure protection

  • Optimization of pH based on deep-sea conditions

• Pressure-induced conformational changes: Active conformations may only exist under pressure, making functional characterization at atmospheric pressure potentially misleading.

• Structural characterization limitations: Standard structural biology techniques have limitations:

  • X-ray crystallography captures a static, atmospheric pressure state

  • NMR has size limitations and typically operates at atmospheric pressure

  • Cryo-EM preparation may alter native conformations

• Stability concerns: Piezophilic proteins may exhibit decreased stability at atmospheric pressure, requiring:

  • Careful temperature control during purification and storage

  • Special stabilizing agents in buffers

  • Minimized time at atmospheric pressure before assays

Addressing these challenges requires specialized equipment, modified protocols, and careful experimental design to ensure results accurately reflect the protein's native behavior.

How should researchers interpret discrepancies between in vitro and in vivo studies of PBPRA0202?

When confronted with discrepancies between in vitro and in vivo studies of PBPRA0202, consider a systematic approach to reconciliation:

• Context-dependent function: PBPRA0202 may have different activities depending on cellular context. Evaluate:

  • Presence of potential cofactors in vivo that may be absent in vitro

  • Different oligomerization states under cellular vs. purified conditions

  • Post-translational modifications present only in the cellular environment

• Pressure effects: Most in vitro studies are conducted at atmospheric pressure while the native environment is high pressure. Consider:

  • Repeating key experiments under pressure conditions

  • Using pressure as an experimental variable to create a response curve

  • Comparing with atmospheric-adapted homologs to identify pressure-specific effects

• Interaction networks: Proteins rarely function in isolation. Investigate:

  • Potential binding partners present in vivo but not in vitro studies

  • Membership in larger complexes that affect function

  • Subcellular localization that may concentrate the protein with specific partners

• Experimental design reconciliation:

  • Compare buffer compositions between in vitro and in vivo studies

  • Assess concentration differences (typically higher in vitro)

  • Evaluate timescales of experiments (in vitro often shorter than in vivo)

• Data integration approach: Create a unified model incorporating both datasets:

  • Identify conditions where results converge

  • Develop hypotheses that explain divergent results

  • Design new experiments specifically targeting the source of discrepancies

This methodological approach has proven valuable in reconciling apparently contradictory results in studies of other P. profundum proteins, particularly those involved in high-pressure adaptation.

What statistical approaches are most appropriate for analyzing pressure-dependent changes in PBPRA0202 activity?

Analysis of pressure-dependent protein activity requires specialized statistical approaches:

• Pressure-response modeling:

  • Fit data to appropriate models (linear, sigmoidal, biphasic) to characterize response profiles

  • Calculate pressure thresholds where significant activity changes occur

  • Determine Hill coefficients if cooperative behavior is observed

• Multivariate analysis for complex datasets:

  • Principal Component Analysis (PCA) to identify major sources of variation

  • Partial Least Squares (PLS) regression for correlating pressure with multiple measured parameters

  • Hierarchical clustering to identify proteins with similar pressure responses

• Time-dependent pressure effects:

  • Repeated measures ANOVA for time-series data at different pressures

  • Mixed-effects models to account for both pressure and time variables

  • Survival analysis techniques for time-to-event data under pressure stress

• Presentation of pressure data:

  • Plot relative activity vs. pressure with appropriate error bars

  • Use heat maps for multivariate pressure responses

  • Include both pressurization and decompression data to identify hysteresis

Sample data table format for pressure response studies:

Statistical significance indicators: * p<0.05, ** p<0.01, *** p<0.001

This approach follows established guidelines for presenting experimental data in tables while specifically addressing the unique requirements of pressure studies.

What emerging technologies could enhance our understanding of PBPRA0202 function in the context of high-pressure adaptation?

Several emerging technologies offer promising approaches for studying PBPRA0202:

• High-pressure cryo-electron microscopy: Technical developments allowing structural determination under pressure conditions could reveal native conformations of PBPRA0202.

• In-cell NMR under pressure: Observing protein behavior directly within cells under variable pressure conditions would bridge the gap between in vitro and in vivo studies.

• Single-molecule FRET under pressure: Monitoring conformational changes in individual PBPRA0202 molecules would reveal heterogeneity in response to pressure changes.

• Microfluidic high-pressure chambers: Enabling rapid pressure cycling and real-time observation of protein behavior under dynamic conditions.

• CRISPR-Cas9 genome editing in P. profundum: Creating precise mutations to test structure-function hypotheses directly in the native organism.

• Protein correlation profiling under pressure: Identifying pressure-dependent changes in protein-protein interactions through quantitative proteomics.

• Integrative modeling approaches: Combining multiple data types (structural, genetic, biochemical) to create comprehensive models of pressure adaptation mechanisms.

These technologies, when applied to PBPRA0202, could reveal previously unknown aspects of pressure adaptation in deep-sea bacteria, potentially leading to applications in biotechnology and our understanding of life in extreme environments.

How might comparative genomics across different piezophilic bacteria enhance our understanding of PBPRA0202 function?

Comparative genomics approaches can provide crucial insights into PBPRA0202 function:

• Ortholog identification across pressure gradients:

  • Compare PBPRA0202 presence/absence in bacteria from different ocean depths

  • Analyze sequence conservation patterns in relation to habitat pressure

  • Identify co-evolved gene clusters that may function together with PBPRA0202

• Positive selection analysis:

  • Calculate dN/dS ratios to identify residues under positive selection in high-pressure environments

  • Map selected residues onto structural models to identify functional implications

  • Compare selection patterns between independently evolved piezophilic lineages

• Comparative transcriptomics:

  • Analyze expression patterns of PBPRA0202 orthologs across different piezophilic species

  • Identify common regulatory elements in promoter regions

  • Determine if orthologs respond similarly to pressure changes

• Horizontal gene transfer analysis:

  • Determine if PBPRA0202 shows evidence of horizontal acquisition

  • Identify potential source organisms that may provide clues to function

  • Assess if transfer correlates with adaptation to high-pressure niches

• Domain architecture comparison:

  • Analyze if PBPRA0202 orthologs have consistent domain organization

  • Identify organisms with fused domains that may suggest functional associations

  • Determine if domain shuffling events correlate with pressure adaptation

These approaches could reveal evolutionary patterns associated with pressure adaptation and provide testable hypotheses about PBPRA0202 function in the deep-sea environment.

How do findings from PBPRA0202 research integrate into our broader understanding of piezophilic adaptation?

Research on PBPRA0202 contributes to several broader themes in piezophilic adaptation:

• Protein structural adaptations: Understanding how proteins like PBPRA0202 maintain functionality under high pressure provides insights into general principles of protein piezostability, including altered amino acid compositions, modified hydrophobic cores, and specialized surface properties.

• Regulatory networks: If PBPRA0202 is part of the ToxR regulon or other pressure-responsive regulatory systems, it contributes to our understanding of how deep-sea bacteria sense and respond to pressure changes through coordinated gene expression.

• Metabolic adaptations: Functional characterization may reveal roles in specialized metabolic pathways that enable energy conservation or generation under high-pressure conditions, a critical aspect of deep-sea survival.

• Evolutionary patterns: Comparative analysis of PBPRA0202 across bacterial lineages helps reveal convergent and divergent evolutionary strategies for high-pressure adaptation.

• Ecological significance: Understanding PBPRA0202 function may provide insights into the ecological role of P. profundum in deep-sea environments and its contributions to nutrient cycling and community interactions.

By integrating PBPRA0202 research with broader studies of piezophilic adaptation, researchers can develop a more comprehensive model of how life thrives in the deep sea, representing adaptations to the largest continuous habitat on Earth .

What methodological framework best integrates multiple experimental approaches for comprehensive characterization of PBPRA0202?

A comprehensive methodological framework for studying PBPRA0202 should integrate multiple experimental approaches in a logical progression:

• Initial characterization (foundation):

  • Sequence analysis and structural prediction

  • Expression optimization and purification

  • Basic biochemical characterization (stability, oligomeric state)

• Functional analysis (core):

  • Activity assays under variable pressure conditions

  • Binding partner identification

  • Localization studies

  • Phenotypic analysis of knockout strains

• Mechanistic studies (depth):

  • Structure determination

  • Mutational analysis of key residues

  • Pressure-dependent conformational analysis

  • In vivo validation of proposed mechanisms

• Contextual integration (breadth):

  • Comparative analysis across species

  • Systems-level analysis (transcriptomics, proteomics)

  • Ecological relevance studies

This framework can be visualized as iterative cycles rather than linear progression, with each phase informing refinements to earlier stages as new data emerges. The framework emphasizes integration of data across multiple scales - from atomic to ecological - to develop a comprehensive understanding of PBPRA0202 function in the context of deep-sea adaptation.