Recombinant Photobacterium profundum ATP synthase subunit beta 2 (atpD2)

What is Photobacterium profundum ATP synthase and why is it significant for deep-sea adaptation research?

Photobacterium profundum ATP synthase is a crucial enzyme complex in this deep-sea bacterium that catalyzes ATP synthesis from ADP and inorganic phosphate using the energy from transmembrane proton motive force. The significance of this enzyme lies in its adaptation to high hydrostatic pressure (HHP) environments. P. profundum possesses two distinct ATPase systems that exhibit pressure-dependent expression patterns, making it an excellent model organism for studying piezophilic (pressure-loving) adaptations in energy metabolism . The structural and functional modifications of ATP synthase in P. profundum represent evolutionary adaptations that enable efficient energy production under extreme deep-sea conditions, providing valuable insights into biochemical adaptations to high-pressure environments.

How do the two ATPase systems in P. profundum differ functionally?

P. profundum SS9 possesses two distinct ATPase systems (ATPase-I and ATPase-II) that show significant differences in their expression patterns and functional roles depending on growth conditions:

Research has demonstrated that disrupting ATPase-I (via ΔatpI mutation) induces expression of ATPase-II, suggesting a compensatory relationship. Interestingly, the two systems appear functionally redundant when cells are grown in MB2216 complex medium . This dual-system architecture likely represents an adaptive strategy that allows P. profundum to maintain energy production efficiency across varying environmental conditions encountered in deep-sea habitats.

What are the optimal expression and purification protocols for recombinant P. profundum ATP synthase subunits?

For optimal expression and purification of recombinant P. profundum ATP synthase subunits, researchers typically employ the following protocol:

• Expression System: E. coli is the preferred heterologous expression system for P. profundum ATP synthase subunits, as demonstrated by successful expression of the atpB2 subunit .

• Vector Construction:

  • Clone the target gene (e.g., atpB2) into an expression vector with an N-terminal His-tag

  • Ensure the vector contains an appropriate promoter (typically T7 or tac)

  • Include antibiotic resistance markers for selection

• Expression Conditions:

  • Culture E. coli transformants in LB or similar medium at 37°C

  • Induce protein expression with IPTG (0.5-1 mM) when OD600 reaches 0.6-0.8

  • Continue cultivation at lower temperature (16-25°C) for 4-16 hours to enhance soluble protein yield

• Purification Process:

  • Harvest cells by centrifugation and lyse using sonication or pressure-based methods

  • Perform affinity chromatography using Ni-NTA resins to capture His-tagged proteins

  • Wash extensively to remove non-specifically bound proteins

  • Elute with imidazole gradient (50-300 mM)

  • Further purify by size exclusion chromatography if higher purity is required

• Storage and Handling:

  • Store at -20°C/-80°C upon receipt

  • Aliquot for multiple use to avoid repeated freeze-thaw cycles

  • For short-term use, working aliquots can be stored at 4°C for up to one week

• Reconstitution:

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

  • Add glycerol (final concentration 5-50%) for long-term storage

  • A 50% glycerol concentration is typically recommended

This methodology has been successfully applied to produce recombinant P. profundum ATP synthase subunit a 2 with greater than 90% purity as determined by SDS-PAGE analysis .

How can researchers effectively measure ATP synthase activity under varying pressure conditions?

Measuring ATP synthase activity under varying pressure conditions requires specialized equipment and methodological considerations:

• High-Pressure Vessels: Custom high-pressure vessels equipped with optical windows for spectrophotometric measurements or electrical connections for electrochemical assays are essential for studying pressure effects on enzyme activity.

• Activity Measurement Approaches:

  • ATP Synthesis Assay: Monitor the formation of ATP from ADP and Pi under varying pressure conditions using luciferase-based luminescence assays

  • ATP Hydrolysis Assay: Measure the release of inorganic phosphate using colorimetric methods (e.g., malachite green assay)

  • Proton Translocation Assay: Use pH-sensitive fluorescent probes to monitor proton movement across membranes

• Sample Preparation Options:

  • Purified enzyme in detergent micelles

  • Reconstituted proteoliposomes (preferred for functional studies)

  • Inverted membrane vesicles

  • Intact cells (for in vivo measurements)

• Experimental Design Considerations:

  • Establish baseline activity at atmospheric pressure

  • Apply incremental pressure increases (e.g., 0.1 MPa, 10 MPa, 28 MPa, 45 MPa)

  • Maintain constant temperature (critical, as pressure changes can alter temperature)

  • Include appropriate controls (pressure-insensitive enzymes)

  • Measure activity after pressure release to assess reversibility

• Data Analysis:

  • Calculate specific activity (μmol ATP/min/mg protein)

  • Determine pressure coefficients (effect of pressure on reaction rates)

  • Apply appropriate kinetic models to analyze pressure effects on enzyme parameters (Km, Vmax)

Research has demonstrated that intracellular ATP levels in P. profundum vary with pressure, but with opposite trends depending on growth media. For instance, in MG medium, ATP levels decrease with increasing pressure, while in MB2216 medium, ATP levels increase with pressure . These observations highlight the importance of considering growth conditions when designing experiments to assess pressure effects on ATP synthase activity.

How does pressure adaptation affect ATP synthase structure and function in P. profundum?

Pressure adaptation in P. profundum ATP synthase involves complex structural and functional modifications that enable efficient energy production under high hydrostatic pressure conditions:

These adaptations collectively represent an evolutionary strategy that enables P. profundum to maintain energy homeostasis in the deep-sea environment where high hydrostatic pressure is a constant challenge.

What role does inorganic phosphate binding play in P. profundum ATP synthase function?

Inorganic phosphate (Pi) binding is fundamental to ATP synthase function, including in piezophilic organisms like P. profundum:

Understanding Pi binding in P. profundum ATP synthase could provide valuable insights into how this crucial enzymatic step has adapted to function efficiently under high hydrostatic pressure conditions.

How do mutations in ATP synthase subunits affect pressure adaptation in P. profundum?

Mutations in ATP synthase subunits significantly impact pressure adaptation in P. profundum, revealing key insights into the molecular mechanisms of piezophilic adaptation:

• Effects of ATPase System Disruption:

  • ΔatpI mutants (disrupted ATPase-I) show induced expression of ATPase-II, indicating a compensatory relationship

  • ΔatpE1 and ΔatpE2 mutants (disrupted subunits of ATPase-I and ATPase-II, respectively) demonstrate that the two systems are functionally redundant in complex media (MB2216)

  • These findings suggest a sophisticated regulatory network that maintains energy homeostasis under varying pressure conditions

• Growth Phenotypes of Mutants:

  • Mutations affecting ATPase-II show more pronounced growth defects at elevated pressures in minimal glucose medium

  • This supports the hypothesis that ATPase-II plays a more critical role in pressure adaptation

  • The severity of growth defects correlates with pressure levels, with more severe phenotypes observed at higher pressures

• ATP Production in Mutants:

  • Mutants with disrupted ATPase systems show altered ATP levels compared to wild-type strains

  • The pressure-dependent trends in ATP production are affected differently depending on which ATPase system is disrupted

  • These observations highlight the specialized roles of each ATPase system in energy production under different pressure regimes

• Structural Implications:

  • Mutations in membrane-spanning regions likely affect proton translocation efficiency

  • Alterations in catalytic subunits influence ATP synthesis/hydrolysis rates

  • Mutations in regulatory subunits may impact the pressure-dependent switching between ATPase systems

• Evolutionary Significance:

  • The presence of two ATPase systems that can be differentially regulated represents an evolutionary adaptation to the variable pressure conditions encountered in deep-sea environments

  • This redundancy provides P. profundum with metabolic flexibility that contributes to its ecological success in deep-sea habitats

These findings collectively demonstrate that P. profundum has evolved sophisticated mechanisms to maintain energy production under high pressure conditions, with mutations in ATP synthase subunits revealing the importance of specific structural and regulatory elements in pressure adaptation.

What experimental designs are most effective for comparing the two ATPase systems in P. profundum?

To effectively compare the two ATPase systems in P. profundum, researchers should implement comprehensive experimental designs that address multiple aspects of these systems:

• Growth Condition Matrices:

  • Cultivate P. profundum under a matrix of conditions including:

    • Pressure levels (0.1 MPa, 10 MPa, 28 MPa, 45 MPa)

    • Media types (minimal glucose medium, complex MB2216 medium)

    • Carbon sources (glucose, amino acids, other substrates)

    • Temperature variations (4°C, 15°C, 20°C)

  • This approach enables systematic analysis of ATPase system expression and function across environmental variables

• Genetic Manipulation Strategies:

  • Generate single and double knockout mutants:

    • ΔatpI (ATPase-I disruption)

    • ΔatpE1 (ATPase-I subunit disruption)

    • ΔatpE2 (ATPase-II subunit disruption)

    • Combination mutants with selective deletions

  • Create tagged variants for expression monitoring (e.g., GFP fusions, His-tagged constructs)

  • Develop inducible expression systems for controlled expression of each ATPase system

• Analytical Methods Combination:

  • Protein Expression Analysis:

    • Quantitative proteomics to determine relative abundances

    • Western blotting with system-specific antibodies

    • RNA-seq for transcriptional analysis

  • Functional Assays:

    • ATP synthesis/hydrolysis rates under varying pressure

    • Proton translocation efficiency measurements

    • Membrane potential determination

  • Structural Studies:

    • Cryo-EM analysis under different pressure conditions

    • Cross-linking studies to capture pressure-dependent conformational changes

• Experimental Controls and Validations:

  • Include appropriate controls (pressure-insensitive strains, non-piezophilic bacteria)

  • Perform complementation experiments to verify phenotype specificity

  • Use multiple independent biological replicates (minimum n=3)

  • Conduct statistical analyses appropriate for multi-factorial experiments

Based on previous research, this comprehensive approach has revealed that ATPase-I plays a dominant role when cultivated in MB2216 complex medium, whereas ATPase-II is more abundant in minimal glucose medium, especially at elevated pressure . These findings highlight the importance of considering multiple growth conditions when studying the differential roles of the two ATPase systems.

How should researchers interpret conflicting data on ATP production and pressure adaptation?

Interpreting conflicting data on ATP production and pressure adaptation in P. profundum requires careful consideration of multiple factors:

• Growth Medium Effects:

  • Research has shown opposite trends in intracellular ATP levels with increasing pressure depending on the growth medium used

  • In MB2216 complex medium, ATP levels increase with pressure

  • In minimal glucose (MG) medium, ATP levels decrease with pressure

  • This fundamental contradiction highlights the critical importance of standardizing growth conditions when comparing results across studies

• Methodological Considerations:

  • ATP Measurement Techniques: Different methods (luminescence-based, HPLC, enzymatic assays) may yield varying results

  • Sampling Timing: ATP levels fluctuate with growth phase; sampling at different points can produce conflicting data

  • Cell Handling: Pressure changes during sampling can trigger rapid metabolic responses, potentially altering results

  • Normalization Approaches: Results normalized to cell number versus protein content may lead to different interpretations

• Biological Explanations for Conflicting Data:

  • Metabolic Adaptation: Cells may employ different metabolic strategies under different nutrient conditions

  • ATPase System Switching: The dominance of ATPase-I versus ATPase-II depends on growth conditions

  • Energy Allocation Shifts: Under pressure stress, energy may be redirected from growth to maintenance functions

  • Compensatory Mechanisms: Alternative ATP-generating pathways may be activated under certain conditions

• Analytical Framework for Resolving Conflicts:

  • Multi-factorial Analysis: Apply statistical methods suitable for complex datasets (ANOVA, principal component analysis)

  • Metadata Integration: Consider all experimental variables when comparing across studies

  • Mechanistic Modeling: Develop mathematical models incorporating known regulatory pathways

  • Validation Experiments: Design experiments specifically to test hypotheses explaining observed contradictions

• Case Study Resolution Example:
  The apparent contradiction in ATP levels between different media can be explained by considering that in complex media, P. profundum primarily uses ATPase-I, which may be more efficient at ATP production under pressure. Conversely, in minimal glucose medium, the shift to predominantly ATPase-II, while beneficial for growth, may result in initially lower ATP production efficiency under pressure . This represents an adaptation strategy where short-term energy efficiency is traded for long-term growth benefits.

By systematically addressing these factors, researchers can reconcile seemingly contradictory findings and develop a more comprehensive understanding of the complex relationship between pressure, energy metabolism, and adaptation in P. profundum.

What are the most promising research directions for understanding P. profundum ATP synthase pressure adaptation?

Several high-potential research directions could significantly advance our understanding of P. profundum ATP synthase pressure adaptation:

• Structural Biology Approaches:

  • High-resolution cryo-EM studies of intact P. profundum ATP synthase under varying pressure conditions

  • Comparative structural analysis between ATPase-I and ATPase-II to identify pressure-adaptive features

  • Development of pressure-resistant crystallization techniques to capture pressure-induced conformational changes

  • Molecular dynamics simulations to model pressure effects on enzyme dynamics

• Systems Biology Integration:

  • Multi-omics analysis (proteomics, transcriptomics, metabolomics) across pressure gradients

  • Flux balance analysis to model energy metabolism under pressure

  • Network analysis to identify regulatory pathways controlling the switch between ATPase systems

  • Comparative genomics across piezophilic and non-piezophilic bacteria to identify evolutionary signatures

• Synthetic Biology Applications:

  • Engineering chimeric ATP synthases with components from both ATPase systems

  • Development of pressure-sensitive cellular biosensors based on ATP synthase components

  • Creation of minimal ATP synthase models to identify essential pressure-adaptive features

  • Engineering pressure-resistant ATP synthases for biotechnological applications

• Advanced Functional Studies:

  • Single-molecule analysis of ATP synthase rotation under pressure

  • Real-time monitoring of ATP production in living cells under pressure

  • Investigation of pressure effects on proton translocation efficiency

  • Analysis of pressure-dependent protein-protein interactions within the ATP synthase complex

• Evolutionary and Ecological Perspectives:

  • Studying ATP synthase diversity across depth gradients in marine environments

  • Investigating horizontal gene transfer patterns of ATP synthase genes

  • Analyzing the co-evolution of ATP synthase components with other pressure-adapted cellular systems

  • Examining ATP synthase adaptation in extremophiles that face multiple stresses including pressure

These research directions would build upon current knowledge that P. profundum exhibits a more pronounced piezophilic phenotype in minimal medium with glucose than in complex medium, with differential expression and function of its two ATPase systems . By pursuing these avenues, researchers could develop a comprehensive understanding of how ATP synthase adapts to high-pressure environments, with potential applications in biotechnology, bioenergy, and astrobiology.

What technical advances are needed to better study recombinant P. profundum ATP synthase components?

Advancing research on recombinant P. profundum ATP synthase components requires several key technical innovations:

• Expression System Optimization:

  • Development of pressure-adaptable expression hosts capable of proper membrane protein folding

  • Engineering of expression vectors with pressure-inducible promoters

  • Creation of chaperone co-expression systems specialized for ATP synthase assembly

  • Establishment of cell-free expression systems that can operate under high pressure

• Purification Technology Enhancements:

  • Design of high-pressure compatible chromatography systems

  • Development of specialized detergents that maintain protein stability under pressure

  • Creation of affinity tags specifically optimized for membrane protein purification

  • Implementation of automated systems for handling pressure-sensitive proteins

• Structural Analysis Innovations:

  • High-pressure cryo-EM sample preparation techniques

  • Development of pressure cells for in situ X-ray diffraction

  • Advanced NMR methodologies for membrane protein analysis under pressure

  • Integration of mass spectrometry with high-pressure protein chemistry

• Functional Assay Advancements:

  • Miniaturized high-pressure vessels compatible with plate readers

  • Development of fluorescent probes specific for ATP synthase activity under pressure

  • Real-time monitoring systems for proton translocation under pressure

  • Microfluidic platforms for single-molecule studies under pressure conditions

• Computational Tool Development:

  • Specialized software for modeling membrane protein behavior under pressure

  • Machine learning approaches to predict pressure effects on protein structure

  • Improved algorithms for molecular dynamics simulations incorporating pressure parameters

  • Integrative modeling platforms combining data from multiple experimental sources

Current methods for handling recombinant P. profundum ATP synthase components include expression in E. coli, purification via His-tag affinity chromatography, and storage as lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . While these methods have enabled basic characterization, they fall short of facilitating comprehensive functional and structural studies under genuine high-pressure conditions. The technical advances outlined above would bridge this gap, enabling more authentic recreation of the deep-sea environment and providing deeper insights into the pressure adaptation mechanisms of this remarkable enzyme complex.