Recombinant Photobacterium profundum Flavohemoprotein (hmp)

What is Photobacterium profundum and why is it significant as a research model?

Photobacterium profundum SS9 is a gram-negative bacterium originally isolated from the Sulu Sea. Its genome consists of two chromosomes and an 80 kb plasmid. What makes P. profundum particularly valuable as a research model is its ability to grow under a wide range of pressures, with optimal growth occurring at 28 MPa and 15°C. Crucially, it can also grow at atmospheric pressure, which enables easy genetic manipulation and culturing in standard laboratory conditions. This unique characteristic has established P. profundum as a model organism for studying piezophily (adaptation to high pressure environments) .

The dual ability to thrive at both high pressure and atmospheric conditions allows researchers to investigate pressure-dependent changes in gene expression, protein structure, and metabolic pathways through comparative studies. This makes P. profundum an ideal system for understanding deep-sea microbial adaptations and the biochemical mechanisms that enable survival in extreme environments.

What are the structural characteristics of flavohemoproteins like Hmp?

Flavohemoproteins (Hmp) are two-domain proteins consisting of a heme domain (HD) and a flavin domain (FD). In the well-studied Escherichia coli Hmp, which shares functional similarities with P. profundum Hmp, the heme domain contains a globin fold that can bind gases like O₂, CO, and NO, while the flavin domain houses binding sites for FAD and NAD(P)H .

The structural organization of these domains is crucial for the protein's function. Studies using CO-difference spectra have shown that the isolated heme domain can bind CO, though with a slightly blue-shifted peak compared to the full-length protein. This indicates that the domain interaction affects the heme pocket environment . Both domains must work in concert for full functional activity, as separately expressed domains show reduced functionality compared to the complete protein in protection against nitric oxide toxicity.

How does pressure affect protein expression in Photobacterium profundum?

Photobacterium profundum displays differential protein expression patterns depending on hydrostatic pressure conditions. Proteomic analysis using label-free quantitation and mass spectrometry has revealed distinct expression profiles between atmospheric (0.1 MPa) and high pressure (28 MPa) growth conditions .

Key findings on pressure-dependent protein expression include:

These expression differences suggest that P. profundum may switch between fermentation metabolism at high pressure (typical of low-oxygen deep-sea environments) and respiratory metabolism at atmospheric pressure (where oxygen is more available) . The ability to modulate these pathways enables the organism to adapt to different pressure environments.

What methods are used to culture Photobacterium profundum for protein studies?

Culturing P. profundum for comparative protein studies requires specific methodologies to maintain consistent growth conditions while varying only the pressure parameter. Based on established protocols, the following approach is recommended:

• Start with stock cultures from -80°C freezer stocks inoculated into marine broth (28 g/liter 2216 medium) supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5).

• Grow the initial culture at 17°C to an OD of 1.5 at 600 nm.

• For experimental cultures, inoculate 50 mL of marine broth with 100 μl of the stock culture, then aliquot into sterile plastic Pasteur pipettes (6 ml each), excluding air to ensure anaerobic conditions and even pressure distribution.

• Seal the pipettes using a Bunsen burner and bag sealer.

• For atmospheric pressure (0.1 MPa) growth, wrap pipettes in aluminum foil and incubate at 17°C.

• For high pressure growth, incubate pipettes at 28 MPa in a water-cooled pressure vessel at 17°C.

• Grow cultures to stationary phase (approximately 5 days) before harvesting by centrifugation at 800×g for 10 minutes .

This methodology ensures that pressure is the only variable while maintaining all other growth parameters constant, which is essential for accurate comparative studies.

What are the best expression systems for producing recombinant Photobacterium profundum Hmp?

Based on research with similar proteins, E. coli expression systems have proven effective for producing recombinant flavohemoproteins. For P. profundum cytochrome P450, successful expression has been achieved using the following approach:

• Amplify the target gene from P. profundum chromosomal DNA using appropriate primers containing restriction enzyme sites (e.g., NdeI and NotI).

• Clone the purified PCR product into an expression vector such as pET29a (Novagen) that incorporates a C-terminal hexahistidine tag for purification.

• Transform the resulting plasmid into an appropriate E. coli expression strain .

How can researchers optimize heterologous protein expression using Hmp fusion technology?

Recent research has demonstrated that translational fusion of E. coli Hmp to the N-terminus of heterologous proteins significantly increases their expression. This approach could be valuable for researchers working with difficult-to-express proteins from P. profundum or using P. profundum Hmp as an expression tag .

Key optimization strategies include:

• Design constructs where the target protein is fused to the C-terminus of Hmp or Hmp fragments.

• As little as the first 100 amino acids of Hmp are sufficient to boost protein production, so truncated versions can be used to minimize tag size.

• The expression enhancement is independent of Hmp's O₂-binding and catalytic activity, meaning that even catalytically inactive versions can be used as fusion tags.

• The effect operates at the translational level, likely downstream of initiation, suggesting that codon optimization of the Hmp portion may further enhance expression .

This approach may be particularly valuable for expressing challenging piezophilic proteins that are typically difficult to produce in conventional systems.

What methodologies are most effective for studying pressure-dependent conformational changes in P. profundum proteins?

Studying pressure-dependent conformational changes in proteins from piezophilic organisms requires specialized techniques. Based on approaches used with P. profundum cytochrome P450, the following methodologies are recommended:

• High-pressure spectroscopy: Using specialized equipment capable of maintaining samples under variable pressure conditions while performing spectroscopic measurements. This approach has been effective in studying the transition between open and closed conformational states in P. profundum cytochrome P450 .

• Pressure perturbation studies: Applying controlled pressure changes to protein samples to probe transitions between conformational states. This technique has revealed that P. profundum proteins often exhibit equilibrium between open and closed states that is shifted toward the closed state even in ligand-free conditions, representing an adaptation to high hydrostatic pressure environments .

• Comparative structural analysis: Comparing protein structures from piezophilic and non-piezophilic organisms to identify pressure-adaptive features. This approach has shown that proteins from P. profundum often feature adaptations that maintain functionality under high pressure.

• Molecular dynamics simulations: Computational approaches can complement experimental methods by modeling protein behavior under different pressure conditions, offering insights into water accessibility and conformational flexibility.

These methodologies provide complementary data for understanding how proteins from deep-sea organisms maintain functionality under extreme pressure conditions.

How do the domains of flavohemoproteins contribute to nitric oxide detoxification?

Understanding the contribution of individual domains to flavohemoprotein function has been investigated through domain engineering studies. Research on E. coli Hmp, which shares functional similarities with P. profundum Hmp, has provided valuable insights into domain-specific contributions to nitric oxide detoxification .

Experimental findings from domain separation studies show:

These results demonstrate that while the heme domain can provide some protection against NO toxicity (similar to the single-domain Vitreoscilla hemoglobin), the maximal protection requires both domains working in concert. This suggests that the flavin domain plays a crucial role in the electron transfer necessary for complete NO detoxification .

What are the challenges in maintaining protein stability during pressure-shift experiments with P. profundum proteins?

Pressure-shift experiments with proteins from piezophilic organisms present several methodological challenges that researchers must address:

• Pressure equipment limitations: Standard laboratory equipment is typically designed for atmospheric pressure operation. Specialized high-pressure vessels with appropriate monitoring and control systems are required for accurate pressure manipulation.

• Protein denaturation risk: Rapid pressure changes can cause irreversible denaturation in proteins not adapted to pressure fluctuations. P. profundum proteins, while pressure-adapted, may still exhibit sensitivity to the rate of pressure change rather than just the absolute pressure value.

• Buffer considerations: Buffer systems can be affected by pressure changes, with certain buffers exhibiting significant pH shifts under pressure (pressure-dependent ionization). Researchers should select buffers with minimal pressure sensitivity or account for these changes in experimental design.

• Temperature control: Pressure application generates heat through adiabatic compression, potentially confounding experimental results if not properly controlled. Temperature must be carefully monitored and regulated during pressure-shift experiments.

• Reversibility verification: To ensure observed changes are due to reversible conformational shifts rather than denaturation, proteins should be tested for activity recovery after pressure is released. This control is essential for validating pressure-dependent conformational change studies.

Addressing these challenges requires specialized equipment, careful experimental design, and appropriate controls to verify that observed changes represent physiologically relevant adaptations rather than experimental artifacts.

What proteomic approaches are most effective for studying P. profundum protein expression under different pressure conditions?

Label-free quantitative proteomic analysis has proven effective for studying pressure-dependent protein expression in P. profundum. The following methodology has been successfully employed:

• Sample preparation: Grow P. profundum cultures under defined pressure conditions (e.g., 0.1 MPa vs. 28 MPa), harvest cells, and lyse using appropriate buffers containing protease inhibitors.

• Protein digestion: Perform tryptic digestion of extracted proteins following standard protocols.

• LC-MS/MS analysis: Utilize shotgun proteomic approaches with liquid chromatography coupled to tandem mass spectrometry.

• Quantification: Employ label-free quantitation using software such as Progenesis (Nonlinear Dynamics) for comparative analysis.

• Data processing: For protein quantification, sum the unique peptide ions associated with each protein to generate abundance values. Transform data using appropriate statistical methods (e.g., ArcSinH transformation) to handle near-zero measurements.

• Statistical analysis: Calculate fold changes using within-group means and determine p-values using one-way ANOVA on transformed data.

• Differential expression criteria: Consider proteins as differentially expressed when detected by two or more peptides, with an absolute ratio of at least 1.5 (fold change) and p<0.05 .

This approach has successfully identified numerous differentially expressed proteins involved in P. profundum's adaptation to different pressure environments, including metabolic enzymes, transporters, and stress response proteins.

How can researchers effectively clone and express P. profundum Hmp for functional studies?

Based on successful approaches with similar proteins from P. profundum, the following protocol is recommended for cloning and expressing the Hmp gene:

• Primer design: Design PCR primers that include appropriate restriction sites (e.g., NdeI at the 5' end and NotI at the 3' end) compatible with your chosen expression vector. Example primers might follow this pattern:

  • Forward: 5'-GCGGAATTCCATATGATG(gene-specific sequence)-3'

  • Reverse: 5'-GCGGAGCTCGCGGCCGC(gene-specific sequence)-3'

• PCR amplification: Amplify the Hmp gene from P. profundum SS9 chromosomal DNA using high-fidelity polymerase.

• Cloning: Purify the PCR product, digest with appropriate restriction enzymes, and ligate into a suitable expression vector (e.g., pET29a) that provides a C-terminal histidine tag for purification.

• Verification: Confirm the sequence of the cloned gene to ensure no mutations were introduced during PCR or cloning steps.

• Expression optimization: Transform the construct into an appropriate E. coli expression strain and optimize expression conditions, considering:

  • Lower induction temperatures (15-20°C) to enhance folding

  • Supplementation with heme precursors (δ-aminolevulinic acid)

  • Iron supplementation to enhance heme incorporation

  • Anaerobic or microaerobic conditions if oxygen sensitivity is observed .

• Purification: Harvest cells, lyse using appropriate methods, and purify using nickel affinity chromatography followed by size exclusion chromatography if needed.

This approach provides a starting point that can be optimized based on specific experimental requirements and observed protein behavior.

What analytical techniques are most informative for characterizing the functional properties of recombinant P. profundum Hmp?

Several complementary analytical techniques provide comprehensive characterization of recombinant flavohemoproteins:

• UV-visible spectroscopy: Characterize the heme environment through:

  • Absorbance spectra of oxidized and reduced forms

  • CO-difference spectra to confirm functional heme incorporation

  • Ligand binding studies through spectral shifts

  • NO binding and reduction kinetics measurements

• Oxygen consumption assays: Measure NO dioxygenase activity using:

  • Clark-type oxygen electrodes

  • Fiber optic oxygen sensors

  • Coupled assay systems with oxygen-sensitive dyes

• NO consumption assays: Directly measure NO detoxification using:

  • NO-selective electrodes

  • Chemiluminescence NO analyzers

  • DAF-FM or other fluorescent NO indicators

• Electron transfer measurements: Characterize the flavin domain function through:

  • NAD(P)H oxidation rates

  • Cytochrome c reduction assays

  • Stopped-flow kinetic measurements

• Pressure-dependent functional analysis: Assess pressure effects on activity using:

  • High-pressure spectroscopic cells

  • Activity measurements at variable pressures

  • Pressure-jump relaxation kinetics

These techniques provide complementary data on different aspects of flavohemoprotein function, allowing researchers to fully characterize both the individual domains and their coordinated activity in the intact protein.

How can recombinant P. profundum Hmp be used to enhance heterologous protein expression?

Flavohemoproteins have shown significant potential as fusion tags to enhance heterologous protein expression. Research with E. coli Hmp has demonstrated that N-terminal fusion to target proteins can substantially increase expression levels, and similar approaches may be effective with P. profundum Hmp .

Potential applications include:

• Expression enhancement: Fusion of P. profundum Hmp to difficult-to-express proteins may improve yields in heterologous expression systems. The enhancement effect is independent of Hmp's catalytic activity, suggesting that even modified versions can serve as effective tags.

• Cold-adapted expression systems: Since P. profundum is adapted to lower temperatures (optimal growth at 15°C), its Hmp may function effectively as a fusion tag in cold-expression systems, which can improve folding of challenging proteins.

• Pressure-tolerant expression: For proteins that require high-pressure folding or assembly, P. profundum Hmp may provide a pressure-adapted fusion partner that maintains functionality under these conditions.

• Solubility enhancement: The solubility properties of piezophilic proteins like those from P. profundum may help improve the solubility of fusion partners, potentially reducing inclusion body formation.

To implement this approach, researchers should design constructs where the target protein is fused to the C-terminus of the Hmp protein, with an appropriate linker and potentially a protease cleavage site for tag removal if needed .

What insights does P. profundum Hmp provide about protein adaptation to extreme environments?

Proteins from piezophilic organisms like P. profundum offer valuable insights into biochemical adaptations to extreme pressure environments. Several key adaptations have been observed in P. profundum proteins that may apply to its flavohemoprotein:

• Conformational equilibrium shifts: P. profundum proteins often show a shift in conformational equilibrium toward more compact, "closed" states even in the absence of ligands. This may represent an adaptation to counteract the tendency of high pressure to favor water penetration into protein cores .

• Modified hydration patterns: Proteins adapted to high pressure often feature altered patterns of surface hydration and internal water molecules, which may help maintain structural integrity under pressure.

• Pressure-sensing mechanisms: Some P. profundum proteins appear to use pressure directly as a sensing mechanism to regulate their activity, suggesting that Hmp might show pressure-dependent regulation of its NO detoxification or oxygen-binding functions.

• Metabolic pathway switching: Proteomic studies have shown that P. profundum switches between fermentative metabolism at high pressure and respiratory metabolism at atmospheric pressure. This suggests that proteins involved in these pathways, potentially including Hmp, have evolved to function optimally under specific pressure regimes .

These adaptations provide insights into the general principles of protein adaptation to extreme environments and may inspire biomimetic approaches for designing pressure-resistant proteins for biotechnological applications.

How do the functional mechanisms of P. profundum Hmp compare with flavohemoproteins from non-piezophilic organisms?

Comparative analysis of flavohemoproteins from piezophilic and non-piezophilic organisms reveals important functional adaptations:

While E. coli Hmp has been extensively characterized as an NO dioxygenase that protects against nitrosative stress , the P. profundum Hmp likely maintains this function while incorporating adaptations that allow it to function efficiently under high hydrostatic pressure. These adaptations may include modified domain interfaces, altered ligand access channels, and pressure-resistant electron transfer mechanisms.

Understanding these differences provides valuable insights into how proteins evolve to maintain the same fundamental function while adapting to extreme environmental conditions.

What are common challenges in expressing and purifying recombinant P. profundum Hmp, and how can they be addressed?

Expression and purification of recombinant proteins from piezophilic organisms present several challenges:

• Codon bias issues:

  • Challenge: P. profundum has different codon usage patterns than common expression hosts like E. coli.

  • Solution: Use codon-optimized synthetic genes or express in strains with rare codon tRNAs (e.g., Rosetta, CodonPlus).

• Temperature sensitivity:

  • Challenge: P. profundum proteins are adapted to lower temperatures (15°C) and may misfold at standard induction temperatures.

  • Solution: Induce expression at lower temperatures (15-18°C) for extended periods (24-48 hours).

• Improper heme incorporation:

  • Challenge: Insufficient heme incorporation leads to inactive apoprotein.

  • Solution: Supplement growth media with δ-aminolevulinic acid (50-100 μg/mL) and iron sources; consider co-expression with heme transport systems.

• Protein aggregation:

  • Challenge: Pressure-adapted proteins may aggregate at atmospheric pressure.

  • Solution: Include stabilizing agents (glycerol, arginine, trehalose) in buffers; consider purification under pressure if specialized equipment is available.

• Oxidative sensitivity:

  • Challenge: Flavohemoproteins can be sensitive to oxidative damage during purification.

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) in buffers and work under nitrogen atmosphere when possible.

• Protein instability:

  • Challenge: Multi-domain proteins may show domain dissociation or unfolding during purification.

  • Solution: Minimize purification steps; consider on-column refolding approaches; use stabilizing buffers with osmolytes.

These strategies can be adapted based on specific experimental observations and the particular properties of the target protein.

How can researchers accurately assess the effects of pressure on P. profundum Hmp structure and function?

Accurate assessment of pressure effects on protein structure and function requires specialized approaches:

• High-pressure spectroscopic cells:

  • Use specialized cells capable of maintaining samples under pressure while performing spectroscopic measurements

  • Conduct parallel measurements at atmospheric and high pressure (28 MPa) to identify pressure-dependent changes

  • Measure absorbance spectra, fluorescence, and circular dichroism under pressure to monitor structural changes

• Pressure-jump experiments:

  • Apply rapid pressure changes while monitoring spectroscopic signals

  • Measure relaxation kinetics to identify pressure-sensitive conformational transitions

  • Verify reversibility by returning to starting pressure

• Activity assays under pressure:

  • Develop assays compatible with high-pressure equipment (e.g., fluorescence-based assays)

  • Compare reaction rates and substrate affinities at different pressures

  • Account for pressure effects on assay components (substrates, cofactors)

• Controls for pressure effects:

  • Include non-piezophilic protein controls (e.g., E. coli Hmp) to distinguish adaptation from general pressure effects

  • Account for pressure effects on buffer systems (pH shifts)

  • Monitor protein stability under pressure to ensure observed effects are not due to denaturation

• Data analysis considerations:

  • Use appropriate thermodynamic models that incorporate pressure terms

  • Calculate volume changes (ΔV) associated with conformational transitions

  • Consider pressure-temperature phase diagrams for comprehensive characterization

These approaches provide complementary information about how pressure affects protein structure, dynamics, and function, allowing researchers to identify specific adaptations in P. profundum Hmp.