Recombinant Photobacterium profundum UPF0234 protein PBPRA2024 (PBPRA2024)

What is PBPRA2024 and why is it significant for deep-sea microbiology research?

PBPRA2024 is an UPF0234 family protein from the piezophilic (pressure-loving) bacterium Photobacterium profundum strain SS9. This protein is significant because it comes from an organism that serves as an established model for studying high-pressure adaptation mechanisms in deep-sea environments . P. profundum SS9 was isolated from a depth of 2500m and has an optimal growth pressure of 28 MPa, while still being able to grow at atmospheric pressure (0.1 MPa), making it an excellent model organism for studying pressure adaptation .

The protein belongs to the UPF (Uncharacterized Protein Family) class, specifically UPF0234, indicating that while its sequence is known, its precise biological function remains to be fully characterized. Studying this protein can provide insights into molecular adaptations to extreme environments.

What are the basic structural characteristics of recombinant PBPRA2024?

Based on the available data, recombinant PBPRA2024 has the following characteristics:

This recombinant protein represents the complete sequence of the native PBPRA2024 protein from P. profundum SS9 .

How should recombinant PBPRA2024 be stored for optimal stability?

For optimal stability of recombinant PBPRA2024, follow these evidence-based storage guidelines:

• Short-term storage: Working aliquots can be stored at 4°C for up to one week .

• Long-term storage:

  • Liquid form: Store at -20°C/-80°C with a shelf life of approximately 6 months

  • Lyophilized form: Store at -20°C/-80°C with a shelf life of approximately 12 months

• Important precautions:

  • Avoid repeated freeze-thaw cycles as this significantly reduces protein stability

  • For long-term storage, it is recommended to add glycerol to a final concentration of 50%

  • Aliquot the protein solution to minimize freeze-thaw cycles

These storage conditions are critical as protein stability is affected by multiple factors including buffer ingredients, storage temperature, and intrinsic protein stability .

What is the recommended protocol for reconstituting lyophilized PBPRA2024?

For optimal reconstitution of lyophilized PBPRA2024, follow this methodological approach:

• Centrifuge the vial briefly before opening to bring the contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (default recommendation is 50%) for long-term storage

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted protein according to the storage guidelines (working aliquots at 4°C for up to one week, long-term storage at -20°C/-80°C)

For specific experimental applications, optimization of buffer conditions may be required based on your specific assay requirements.

How does the expression of PBPRA2024 compare between high-pressure and atmospheric conditions?

While the search results don't provide specific expression data for PBPRA2024 itself, research on P. profundum SS9 has shown that many proteins exhibit differential expression patterns between high-pressure (28 MPa) and atmospheric pressure (0.1 MPa) conditions .

Proteomic analyses have revealed:

• Proteins involved in glycolysis/gluconeogenesis pathways are typically up-regulated at high pressure (28 MPa)

• Several proteins involved in oxidative phosphorylation pathways are up-regulated at atmospheric pressure (0.1 MPa)

• Ribosomal proteins show significant differential expression, with 25 ribosomal proteins being up-regulated at high pressure, representing one of the highest enrichment factors observed in pressure-related proteomic studies

As UPF0234 belongs to an uncharacterized protein family, determining its specific expression pattern under different pressure conditions would require targeted experiments using techniques such as RNA-seq or quantitative proteomics as employed in previous studies on P. profundum .

What methodologies are most effective for studying protein-protein interactions involving PBPRA2024?

For investigating protein-protein interactions involving PBPRA2024, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against PBPRA2024 or potential interacting partners to pull down protein complexes from P. profundum lysates

• Bacterial two-hybrid system: This would be particularly useful for screening potential interaction partners when expressed in a surrogate host like E. coli

• Pull-down assays with recombinant protein: Using tagged recombinant PBPRA2024 as bait to identify interaction partners

• Cross-linking mass spectrometry: This approach can identify transient or weak interactions that may be particularly relevant for proteins functioning under extreme pressure conditions

• Pressure-modulated interaction studies: Given that P. profundum is a piezophile, interactions should be studied under both atmospheric and high-pressure conditions (28 MPa), as some interactions may only occur under specific pressure conditions

For proteins from piezophilic organisms, it's important to consider that protein-protein interactions may be pressure-dependent, and experimental designs should account for this unique characteristic.

How might researchers investigate the functional role of PBPRA2024 in pressure adaptation mechanisms?

To investigate the functional role of PBPRA2024 in pressure adaptation, a comprehensive research strategy should include:

• Gene knockout/knockdown studies:

  • Create a PBPRA2024 deletion mutant in P. profundum SS9

  • Compare growth phenotypes at various pressures (0.1 MPa, 28 MPa, 45 MPa)

  • Examine morphological changes under pressure stress, similar to approaches used for other genes in P. profundum

• Complementation experiments:

  • Express PBPRA2024 in the knockout strain using a plasmid-based system

  • Test if pressure sensitivity is rescued, similar to approaches used with other proteins like DiaA and SeqA

• Transcriptome and proteome analysis:

  • Compare global gene expression changes in wild-type vs. PBPRA2024 mutant strains under different pressure conditions

  • Identify pathways affected by PBPRA2024 deletion using RNA-seq methodology as demonstrated in previous studies

• Structural biology approaches:

  • Determine the 3D structure of PBPRA2024 at different pressures

  • Investigate pressure-induced conformational changes using techniques like high-pressure NMR or crystallography

• Heterologous expression studies:

  • Express PBPRA2024 in non-piezophilic bacteria and evaluate changes in pressure tolerance

  • Test if PBPRA2024 confers any pressure-adaptive advantages to host organisms

This multi-faceted approach would help elucidate whether PBPRA2024 plays a direct role in pressure adaptation mechanisms in P. profundum SS9.

What challenges exist in correlating transcriptomic and proteomic data for proteins like PBPRA2024 in piezophilic bacteria?

Several significant challenges exist when correlating transcriptomic and proteomic data for piezophilic bacteria like P. profundum, particularly for proteins like PBPRA2024:

• Pressure-related methodological limitations:

  • Depressurization during sample collection can trigger stress responses that alter gene expression within minutes, potentially masking true in situ expression patterns

  • As noted in proteomic studies: "All care was taken to harvest and freeze cells as quickly as possible, [but] it may be that some stress response signals were activated as soon as the cell cultures were de-pressurized"

• Anti-correlation between transcriptomic and proteomic data:

  • Previous studies have documented anti-correlation between transcript and protein levels for stress-response proteins in P. profundum

  • For example, GroEL and DnaK were found up-regulated at 28 MPa in proteomic studies but down-regulated in transcriptomic studies

• Temporal dynamics of expression:

  • Proteins like DnaK and DnaJ are involved in early phases of cellular stress responses

  • The timing of sample collection can significantly impact observed expression patterns

• Specific challenges for UPF proteins:

  • As uncharacterized proteins, basic information about regulation, turnover rates, and post-translational modifications is often lacking

  • Limited availability of specific antibodies for detection and quantification

• Chromosomal location effects:

  • P. profundum has two chromosomes with different gene expression patterns

  • The primary chromosome (chr. 1) has 30.6-32.9% of genes in operons, while the secondary chromosome (chr. 2) has only 7.7-10.9%

  • This chromosomal organization affects co-regulation patterns and complicates expression analysis

These challenges underscore the need for integrated approaches and careful experimental design when studying piezophilic bacteria and their proteins.

How can structural studies of PBPRA2024 inform our understanding of protein adaptation to high-pressure environments?

Structural studies of PBPRA2024 can provide critical insights into protein adaptation to high-pressure environments through several research approaches:

• Comparative structural analysis under different pressures:

  • Determine the 3D structure of PBPRA2024 at atmospheric pressure (0.1 MPa) and high pressure (28 MPa)

  • Identify pressure-induced conformational changes and evaluate their reversibility

  • Analyze the volume changes of the protein's hydration shell and internal cavities, which are critical factors in pressure adaptation

• Investigation of protein flexibility and compressibility:

  • Measure the compressibility of PBPRA2024 using techniques like pressure perturbation calorimetry

  • Analyze how pressure affects the dynamics of different protein regions using hydrogen-deuterium exchange mass spectrometry

  • Compare flexibility parameters with homologous proteins from non-piezophilic organisms

• Analysis of amino acid composition and distribution:

  • Evaluate the amino acid composition of PBPRA2024 compared to homologs from non-piezophilic bacteria

  • Analyze the distribution of charged, hydrophobic, and flexible residues, as these features often differ in pressure-adapted proteins

  • Identify potential pressure-sensing domains or motifs

• In silico molecular dynamics simulations:

  • Perform molecular dynamics simulations at different pressures to predict conformational changes

  • Calculate the volume change of the protein upon pressurization

  • Identify key residues involved in pressure sensing or adaptation

• Structure-guided mutagenesis:

  • Design mutations based on structural insights to test hypotheses about pressure adaptation mechanisms

  • Express mutant versions in P. profundum and evaluate their functionality under different pressure conditions

These structural studies would significantly advance our understanding of how proteins like PBPRA2024 contribute to the piezophilic lifestyle of deep-sea bacteria.

What experimental design would best address the potential role of PBPRA2024 in the ToxR regulatory network of P. profundum?

To investigate the potential role of PBPRA2024 in the ToxR regulatory network of P. profundum, the following comprehensive experimental design is recommended:

Phase 1: Establishing Regulatory Relationships

• Transcriptional profiling:

  • Compare wild-type and toxR mutant (TW30) strains at different pressures (0.1 MPa and 28 MPa)

  • Use RNA-seq to determine if PBPRA2024 expression is altered in the toxR mutant compared to wild-type

  • Group PBPRA2024 with other genes showing similar expression patterns (e.g., similar to OmpH which is known to be regulated by ToxR)

• Promoter analysis:

  • Identify the PBPRA2024 promoter region

  • Search for potential ToxR binding motifs using computational approaches

  • Perform electrophoretic mobility shift assays (EMSA) to test direct binding of ToxR to the PBPRA2024 promoter

  • Construct reporter gene fusions to measure PBPRA2024 promoter activity in different genetic backgrounds

Phase 2: Functional Analysis

• Double mutant studies:

  • Create a PBPRA2024 knockout strain

  • Create a PBPRA2024/toxR double mutant

  • Compare growth phenotypes at different pressures

  • Evaluate if the phenotype of a toxR mutant is exacerbated or suppressed by the absence of PBPRA2024

• Complementation experiments:

  • Express PBPRA2024 in the toxR mutant using plasmid pFL190 under the control of an arabinose-inducible promoter, similar to approaches used for other genes

  • Determine if any toxR mutant phenotypes are suppressed by PBPRA2024 overexpression

Phase 3: Integration into the ToxR Regulon

• Transcriptome and proteome comparison:

  • Compare global gene expression changes in PBPRA2024 mutant, toxR mutant, and wild-type strains

  • Identify overlapping sets of differentially expressed genes

  • Construct a regulatory network model including ToxR, PBPRA2024, and other components

• High-pressure phenotypic assays:

  • Compare cellular morphology of wild-type, PBPRA2024 mutant, and toxR mutant at different pressures, as morphological changes have been observed in pressure-sensitive mutants

  • Assess membrane composition changes, as ToxR is known to regulate outer membrane proteins like OmpH and OmpL

This experimental design would comprehensively address whether PBPRA2024 functions within the ToxR regulatory network and provide insights into its role in pressure adaptation mechanisms in P. profundum.

How does the operon structure and genomic context of PBPRA2024 compare between different Photobacterium species, and what evolutionary insights might this provide?

Analyzing the operon structure and genomic context of PBPRA2024 across different Photobacterium species can provide valuable evolutionary insights into adaptation to varied pressure environments:

Methodological approach:

• Comparative genomic analysis:

  • Identify PBPRA2024 homologs in different Photobacterium species through BLAST searches

  • Compare species living at different depths (surface vs. deep sea)

  • Extend comparison to other Vibrionaceae family members

• Operon structure determination:

  • Analyze transcriptomic data to identify if PBPRA2024 is expressed as part of a polycistronic transcript

  • Compare operon predictions across species using both computational methods and RNA-seq data

  • Determine if PBPRA2024 is located on chromosome 1 or 2, as these chromosomes show different patterns of operon organization in P. profundum (30.6-32.9% of genes in operons on chr. 1 vs. 7.7-10.9% on chr. 2)

• Synteny analysis:

  • Compare the conservation of gene order around PBPRA2024 across species

  • Identify conserved gene clusters that may indicate functional relationships

  • Detect genomic rearrangements that might affect regulation

Potential evolutionary insights:

The analysis could reveal:

• Pressure adaptation signatures:

  • Whether PBPRA2024 shows sequence divergence patterns correlating with depth habitat

  • If operon structure is more conserved in deep-sea species compared to shallow-water relatives

  • Whether the genomic location (chromosome 1 vs. 2) is consistent across species

• Functional inferences:

  • If PBPRA2024 is consistently co-expressed with specific pathways across species

  • Whether horizontal gene transfer events have influenced its evolution

  • If selective pressure has maintained certain genomic arrangements

• Regulatory evolution:

  • Changes in promoter regions that might reflect adaptation to different environmental pressures

  • Conservation of regulatory elements potentially involved in pressure-responsive expression

  • Differences in 5'-UTR length, as P. profundum shows an unexpectedly high number of genes (992) with large 5'-UTRs that could harbor cis-regulatory RNA structures

This comparative approach would provide crucial context for understanding how PBPRA2024 may have evolved specialized functions related to pressure adaptation in deep-sea Photobacterium species.