Recombinant Photobacterium profundum ATP synthase subunit a 1 (atpB1)
Description
Introduction to Photobacterium profundum and ATP Synthase
Photobacterium profundum SS9 is a Gram-negative bacterium originally isolated from the Sulu Sea at depths of approximately 2,500 meters. Its genome consists of two chromosomes and an 80 kb plasmid, providing genetic versatility for adaptation to various environmental conditions. Although P. profundum can grow under a wide range of pressures, it exhibits optimal growth at 28 MPa and 15°C, classifying it as a piezophilic and psychrophilic (cold-loving) organism . The bacterium's ability to grow at both atmospheric and high pressures makes it an ideal model organism for studying pressure adaptation mechanisms in deep-sea microorganisms.
ATP synthase represents one of the most fundamental enzymes in cellular energy metabolism across all domains of life. This molecular machine catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi) using the energy derived from proton translocation across a membrane. F-type ATP synthase (F1FO), found in bacteria, mitochondria, and chloroplasts, consists of two major components: a soluble catalytic sector (F1) and a membrane-embedded proton-translocating sector (FO) . The enzyme functions as a rotary motor, converting the electrochemical gradient of protons (proton motive force) into mechanical energy that drives ATP synthesis.
In P. profundum, the ATP synthase complex plays a crucial role in energy production and adaptation to high-pressure environments. Proteomic analyses have shown that proteins involved in oxidative phosphorylation, including components of the ATP synthase, show differential expression patterns between atmospheric and high-pressure conditions, highlighting their importance in pressure adaptation mechanisms .
Structure and Function of ATP Synthase Subunit a
The ATP synthase subunit a (atpB1) is a critical component of the membrane-embedded FO sector of the ATP synthase complex. This integral membrane protein forms the stationary part of the enzyme's proton channel in conjunction with the rotating c-ring. Within the ATP synthase complex, the a-subunit performs several essential functions:
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Formation of proton half-channels: The a-subunit contains two aqueous half-channels that provide pathways for protons to enter from one side of the membrane and exit to the other side . These channels do not form a continuous pathway through the membrane but rather connect to the c-ring at different positions.
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Interaction with the c-ring: The a-subunit positions directly adjacent to the c-ring, creating a critical interface where proton translocation occurs. As protons pass through the half-channels in the a-subunit, they drive the rotation of the c-ring.
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Contribution to the stator: The a-subunit forms part of the stationary component (stator) of the ATP synthase, which resists the rotational torque generated during enzyme operation.
Based on studies of ATP synthase from Chloroflexus aurantiacus and other bacteria, we can infer that the a-subunit in P. profundum likely consists of multiple transmembrane helices that form the architecture necessary for proton translocation . The precise number and arrangement of these helices may vary between different bacterial species, potentially reflecting adaptations to specific environmental conditions.
Recombinant Production and Properties of P. profundum atpB1
While the search results do not provide specific information about the recombinant production of P. profundum atpB1, we can infer potential properties and production methods based on similar recombinant proteins from P. profundum, such as the ATP synthase gamma chain 1 (atpG1) .
Recombinant P. profundum atpB1 likely shares several characteristics with other recombinant membrane proteins:
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Expression systems: Due to its hydrophobic nature as a membrane protein, recombinant atpB1 would require specialized expression systems capable of properly folding and inserting membrane proteins. Potential expression hosts include modified E. coli strains, yeast systems, insect cells, or baculovirus expression systems similar to those used for atpG1 .
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Solubilization requirements: As an integral membrane protein, recombinant atpB1 would require detergents or lipid nanodisc systems for solubilization and purification in a functionally active state.
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Stability considerations: Like the recombinant atpG1 protein, recombinant atpB1 would likely have specific storage requirements to maintain stability, potentially including glycerol as a cryoprotectant and storage at -20°C to -80°C .
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Purification challenges: Membrane proteins typically present significant purification challenges, often requiring affinity tags and careful optimization of detergent conditions to maintain protein integrity and function.
The table below compares the predicted properties of recombinant P. profundum atpB1 with the known properties of recombinant atpG1 and other ATP synthase subunits:
Pressure Adaptation Features of P. profundum ATP Synthase
P. profundum SS9's ability to thrive under high-pressure conditions suggests that its ATP synthase components, including the a-subunit, have evolved specific adaptations to maintain functionality in the deep sea. The ATP synthase complex represents one of the most pressure-sensitive cellular systems in mesophilic bacteria, making pressure adaptation of this enzyme particularly critical for piezophilic organisms .
Proteomic analyses have revealed that proteins involved in oxidative phosphorylation pathways in P. profundum show differential expression patterns between atmospheric and high-pressure conditions . Specifically, several proteins involved in this pathway were up-regulated at atmospheric pressure, suggesting that P. profundum may employ different energy generation strategies depending on the ambient pressure.
The a-subunit of ATP synthase, with its critical role in proton translocation, likely contains specific amino acid substitutions or structural modifications that enable efficient function under high pressure. These adaptations might include:
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Increased hydrophobic packing: Modifications that reduce the volume change associated with protein function, thus minimizing the inhibitory effects of high pressure.
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Specialized proton channels: Structural adaptations in the half-channels that maintain efficient proton translocation despite the compressing effects of high pressure.
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Modified interactions with the c-ring: Optimized interfaces between the a-subunit and c-ring that preserve rotational capabilities under pressure.
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Altered lipid interactions: Specialized interactions with membrane lipids, which themselves may contain pressure adaptations in P. profundum.
Understanding these pressure adaptations in the atpB1 subunit could provide valuable insights for biotechnological applications requiring pressure-resistant enzymes.
Research Applications and Future Perspectives
Recombinant P. profundum atpB1 offers several potential applications in both basic research and biotechnology:
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Structural biology: As a pressure-adapted membrane protein, recombinant atpB1 provides an opportunity to study the structural basis of pressure adaptation in membrane proteins. Comparative structural analyses with homologous proteins from mesophilic organisms could reveal key adaptations.
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Bioenergetics research: The a-subunit's central role in proton translocation makes it valuable for studying the fundamental mechanisms of chemiosmotic energy conversion.
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Biotechnological applications: Pressure-adapted proteins like atpB1 could inspire the development of pressure-resistant enzymes for industrial processes requiring high-pressure conditions.
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Drug discovery: The bacterial ATP synthase has emerged as a target for new antibiotics. Understanding the structure and function of bacterial a-subunits could potentially contribute to antimicrobial development, although P. profundum itself is not pathogenic.
Recent research on ATP synthases from other photosynthetic bacteria has revealed surprising structural diversity. For instance, the ATP synthase from Chloroflexus aurantiacus, an early photosynthetic bacterium, contains two peripheral stalks and two proton-conducting a-subunits, allowing more protons to be translocated during each cycle of ATP synthesis . This finding raises interesting questions about the potential structural variations that might exist in the ATP synthases of other specialized bacteria, including piezophiles like P. profundum.
Product Specs
Note: While we preferentially ship the format currently in stock, we can accommodate specific format requests. Please indicate your preferred format when placing the order, and we will fulfill your requirement.
Note: All proteins are shipped with standard blue ice packs. For dry ice shipping, please communicate with us in advance as additional fees may apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: ppr:PBPRA3610
STRING: 298386.PBPRA3610
Q&A
What is Photobacterium profundum ATP synthase subunit a 1 (atpB1) and its role in the F-ATP synthase complex?
Photobacterium profundum ATP synthase subunit a 1 (atpB1) is a critical membrane-embedded component of the F0 domain of F-ATP synthase. Within the F0F1-ATP synthase complex, atpB1 plays an essential role in proton translocation across the membrane, contributing to the generation of the rotary torque necessary for ATP synthesis. The F-ATP synthase complex generally consists of a membrane-embedded F0 complex (including subunits a, b, b', and c) and a water-soluble F1 complex (α3β3γδε) . The F1 portion contains three catalytic αβ-pairs forming an α3β3-hexamer where ATP synthesis or hydrolysis occurs, while the F0 domain, containing subunit a (atpB1), contributes to ion transport and coupling .
How do researchers distinguish between subunit a (atpB1) and other ATP synthase components in experimental systems?
Distinguishing atpB1 from other ATP synthase components requires application of multiple complementary techniques:
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Genetic analysis: Sequence comparisons with known F-ATP synthase genes highlight unique features of atpB1 compared to other subunits.
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Protein detection methods: Western blotting with subunit-specific antibodies enables differentiation between subunit a and other components.
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Functional assays: Measuring the specific contribution of atpB1 to proton conduction and ATP synthesis/hydrolysis through genetic deletion and complementation studies.
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Structural analysis: Cryo-EM and X-ray crystallography can reveal the specific positioning and interactions of atpB1 within the complex, showing its distinct structural features compared to other subunits .
What expression systems are most suitable for producing recombinant Photobacterium profundum atpB1?
For recombinant expression of membrane proteins like atpB1, researchers should consider these methodological approaches:
| Expression System | Advantages | Limitations | Optimization Strategies |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, easy genetic manipulation, rapid growth | Potential inclusion body formation, lacking post-translational modifications | Lower induction temperature (16-20°C), use specialized vectors with fusion tags (MBP, SUMO) |
| E. coli C41/C43 | Specifically engineered for membrane protein expression | Lower yield than standard BL21 | Optimize induction timing and concentration |
| Cell-free systems | Avoids toxicity issues, direct incorporation into liposomes | Expensive, technically demanding | Supplement with lipids/detergents |
| Insect cells | Better folding of complex proteins, post-translational modifications | Time-consuming, expensive | Optimize MOI, harvest timing |
When working with recombinant atpB1, researchers should carefully consider its hydrophobic nature as a membrane protein. The F0 components, including atpB1, are typically more challenging to express than the water-soluble F1 components .
How do sequence variations in the atpB1 gene affect ATP synthase function and pressure adaptation in Photobacterium profundum?
P. profundum is a deep-sea bacterium that has evolved to function under high-pressure conditions. Research suggests that pressure adaptation involves specific structural modifications in key proteins:
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Hydrophobic core alterations: Mutations affecting the hydrophobic regions of atpB1 can influence membrane integration and stability under high pressure.
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Proton channel architecture: Specific amino acid substitutions in the proton-conducting channel of atpB1 may facilitate efficient proton translocation under various pressure conditions.
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Subunit interface adaptations: Sequence variations at interfaces with other subunits (particularly with the c-ring) can affect rotational coupling efficiency and pressure tolerance.
For experimental assessment of these adaptations, researchers often employ site-directed mutagenesis followed by functional assays at different pressure conditions to evaluate the impact of specific residues on ATP synthesis activity.
What structural elements of atpB1 are critical for proton translocation, and how can they be studied?
Critical structural elements in atpB1 involved in proton translocation include:
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Conserved arginine residue: Similar to other F-ATP synthases, a conserved arginine in the membrane-embedded region likely forms part of the essential proton pathway.
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Transmembrane helices: The arrangement of transmembrane segments creates the architecture necessary for proton movement across the membrane.
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Interface with c-ring: The interaction between atpB1 and the rotating c-ring subunits is crucial for coupling proton movement to rotational force.
These elements can be studied through:
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Cryo-EM analysis: As demonstrated with other ATP synthases, cryo-EM can reveal the structural organization of atpB1 and its interactions with other subunits .
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Molecular dynamics simulations: Computational approaches can model proton movement through the atpB1 channel under different conditions.
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Proton translocation assays: Experimental measurement of proton pumping using inverted membrane vesicles containing wild-type or mutant forms of atpB1, similar to methods used for mycobacterial ATP synthase .
How does the evolutionary history of atpB1 in Photobacterium profundum compare to ATP synthase subunits in other bacterial species?
The evolutionary history of ATP synthase subunits shows interesting patterns across bacterial species:
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Phylogenetic distribution: ATP synthase components show patchy distribution across different lineages, suggesting either multiple independent origins or horizontal gene transfer events .
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Conservation vs. specialization: While the core function is conserved, species-specific adaptations have evolved in response to particular environmental niches.
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Gene duplication events: The F0F1 ATP synthase genetic locus shows evidence of both ancient and recent gene duplications and rearrangements . In the context of P. profundum, researchers should investigate whether the "a 1" designation in atpB1 indicates one of multiple paralogs resulting from gene duplication events.
Analysis of atpB1 sequence conservation across marine bacteria, particularly other deep-sea organisms, could reveal pressure-specific adaptations that distinguish P. profundum's ATP synthase from those of shallow-water relatives.
What are the most effective purification strategies for obtaining functional recombinant atpB1?
Purifying functional recombinant atpB1 presents significant challenges due to its hydrophobic nature and membrane integration. Effective purification strategies include:
| Purification Step | Recommended Approach | Critical Considerations |
|---|---|---|
| Membrane extraction | Gentle detergent solubilization (DDM, LMNG) | Detergent concentration must maintain protein structure while extracting from membrane |
| Initial capture | IMAC using engineered His-tag | Buffer composition should include stabilizing detergent above CMC |
| Further purification | Size exclusion chromatography | Assess oligomeric state and homogeneity |
| Functional verification | Reconstitution into liposomes followed by proton pumping assays | Lipid composition should mimic native membrane environment |
For optimal results, researchers should:
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Screen multiple detergents for extraction efficiency while maintaining protein stability.
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Consider fusion partners (MBP, SUMO) that can enhance solubility and be cleaved post-purification.
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Verify protein folding using circular dichroism or limited proteolysis assays.
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Assess functional integrity through reconstitution experiments .
How can researchers accurately measure the contribution of atpB1 to ATP synthase activity?
Measuring the specific contribution of atpB1 to ATP synthase function requires a multi-faceted approach:
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Genetic deletion and complementation:
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Generate atpB1 knockout strains and complement with wild-type or mutant versions
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Assess growth phenotypes under different conditions (pressure, carbon sources)
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ATP synthesis activity:
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Measure ATP production in inverted membrane vesicles using the luciferin-luciferase assay
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Compare activity with and without proton gradient uncouplers
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Proton pumping assays:
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Single-molecule studies:
What strategies are effective for studying atpB1 interactions with other ATP synthase subunits?
Understanding the interactions between atpB1 and other ATP synthase subunits is crucial for elucidating its function. Researchers can employ several complementary approaches:
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Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify residues in close proximity between atpB1 and interacting subunits, particularly the adjacent c-ring subunits .
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Co-purification approaches: Pull-down assays using tagged versions of atpB1 can identify stable interaction partners within the complex.
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Cryo-EM analysis: Structural determination of the entire ATP synthase complex can reveal the precise positioning of atpB1 and its interfaces with neighboring subunits .
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Genetic suppressor screens: Identifying compensatory mutations that restore function in atpB1 mutants can reveal important functional interactions between subunits.
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Split reporter assays: Protein fragment complementation approaches using split fluorescent proteins or enzymes can be adapted to study membrane protein interactions.
How should researchers interpret conflicting results from different assays of atpB1 function?
When facing conflicting results from different assays measuring atpB1 function, researchers should systematically evaluate:
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Assay-specific limitations:
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ATP hydrolysis assays may detect activity even when proton pumping is compromised
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Proton pumping measurements can be affected by membrane integrity and background proton leakage
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Experimental conditions:
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Temperature, pH, and pressure conditions significantly impact ATP synthase function
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Detergent choice in biochemical assays can differentially affect activity measurements
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Protein preparation variations:
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Expression system differences may produce proteins with varying post-translational modifications
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Purification methods can influence protein stability and functional state
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Resolving strategies:
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Conduct complementary assays under identical conditions
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Use genetic approaches (site-directed mutagenesis) to validate biochemical findings
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Consider that ATP synthases may display different regulatory mechanisms for ATP synthesis versus hydrolysis, as seen in mycobacterial ATP synthase where ATP hydrolysis is specifically inhibited by structural elements
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What bioinformatic approaches are most useful for analyzing sequence-structure-function relationships in atpB1?
Bioinformatic approaches can provide valuable insights into sequence-structure-function relationships in atpB1:
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Multiple sequence alignment:
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Comparing atpB1 sequences across diverse species can identify conserved residues likely crucial for function
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Pressure-adapted organisms may show convergent adaptations in key functional regions
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Structural modeling:
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Homology modeling based on available ATP synthase structures can predict the three-dimensional arrangement of atpB1
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Molecular dynamics simulations can explore conformational changes under different pressure conditions
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Evolutionary analysis:
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Integrated approaches:
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Combining sequence conservation data with structural information to identify functionally important interfaces
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Network analysis of co-evolving residues can identify functionally linked positions within the protein
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How can researchers differentiate between pressure-specific adaptations and general ATP synthase features in atpB1?
Distinguishing pressure-specific adaptations from general ATP synthase features requires comparative analysis:
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Comparative genomics approach:
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Compare atpB1 sequences from P. profundum with those from related shallow-water Photobacterium species
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Identify amino acid substitutions unique to deep-sea adapted species
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Experimental validation:
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Express and characterize atpB1 variants under different pressure conditions
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Swap domains between deep-sea and shallow-water homologs to identify pressure-responsive regions
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Structural analysis:
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Examine atpB1 for features associated with pressure adaptation (increased hydrophobic packing, reduced cavities)
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Compare with known pressure adaptations in other membrane proteins from deep-sea organisms
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Functional assays across pressure ranges:
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Measure ATP synthesis and proton pumping activities at atmospheric versus high pressure
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Quantify the pressure-dependent kinetics of ATP synthesis/hydrolysis
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What are common challenges in atpB1 expression and how can researchers overcome them?
Membrane proteins like atpB1 present specific expression challenges with corresponding solutions:
| Challenge | Potential Causes | Solutions |
|---|---|---|
| Low expression yields | Toxicity to host cells, protein misfolding | Use tightly regulated expression systems, lower induction temperature (16-20°C), specialized host strains (C41/C43) |
| Inclusion body formation | Rapid overexpression, hydrophobic aggregation | Reduce expression rate, co-express with chaperones, use fusion tags (MBP, SUMO) |
| Proteolytic degradation | Recognition of misfolded protein by host proteases | Add protease inhibitors, use protease-deficient strains, optimize extraction conditions |
| Difficulty in verification | Poor antibody recognition, degradation during analysis | Use epitope tags, optimize SDS-PAGE conditions for membrane proteins |
For optimal results, researchers should:
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Screen multiple expression constructs with varying tags and fusion partners
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Test expression in multiple host strains
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Perform small-scale optimization before scaling up production
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Consider cell-free expression systems for particularly challenging constructs
How can researchers troubleshoot inactive recombinant atpB1 in functional assays?
When recombinant atpB1 shows poor activity in functional assays, systematic troubleshooting is necessary:
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Protein integrity verification:
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Confirm full-length protein expression via Western blotting
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Assess protein folding using limited proteolysis or circular dichroism
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Reconstitution optimization:
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Test different lipid compositions in reconstitution experiments
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Optimize protein-to-lipid ratios to ensure proper integration
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Assay conditions:
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Evaluate buffer composition effects (ionic strength, pH)
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Test activity across a range of temperatures and pressures
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Ensure all necessary cofactors are present at appropriate concentrations
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Complex assembly verification:
What emerging technologies hold promise for advancing atpB1 research?
Several cutting-edge technologies are poised to revolutionize atpB1 research:
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Cryo-electron tomography: Enables visualization of ATP synthase in its native membrane environment at near-atomic resolution, providing insights into the precise arrangement of atpB1 and its interactions .
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Single-molecule FRET: Can track conformational changes in atpB1 during the catalytic cycle, providing dynamic information not accessible through static structural approaches.
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High-pressure biophysical techniques: Specialized equipment for performing structural and functional studies under deep-sea pressure conditions can reveal pressure-specific adaptations.
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AlphaFold and other AI approaches: Deep learning algorithms can predict protein structures with increasing accuracy, potentially revealing structural features of atpB1 even without experimental structures.
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Nanodiscs and other membrane mimetics: Advanced membrane mimetics provide more native-like environments for functional studies of membrane proteins like atpB1.
What are key unanswered questions about atpB1 that warrant further investigation?
Despite advances in ATP synthase research, several critical questions about atpB1 remain unanswered:
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Pressure adaptation mechanisms: How do specific amino acid substitutions in atpB1 contribute to ATP synthase function under high-pressure conditions?
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Regulatory interactions: Does atpB1 participate in regulatory protein-protein interactions that modulate ATP synthase activity in response to environmental conditions?
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Evolutionary history: If P. profundum contains multiple atpB paralogs (as suggested by the "a 1" designation), what are their functional distinctions and evolutionary origins?
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Proton pathway details: What is the precise route of proton translocation through atpB1, and how does it differ from homologs in other species?
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Potential as antimicrobial target: Given the essentiality of ATP synthase in many bacteria , could atpB1 serve as a target for selective antimicrobials against Photobacterium-related pathogens?
How might structural studies of atpB1 contribute to understanding other membrane protein complexes?
Research on atpB1 has broader implications for membrane protein biology:
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Pressure adaptation principles: Insights from atpB1 could reveal general principles of how membrane proteins adapt to extreme pressure environments, applicable to other transport systems.
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Membrane protein expression strategies: Optimization approaches developed for recombinant atpB1 could inform expression strategies for other challenging membrane proteins.
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Protein-lipid interactions: Studies of how atpB1 interacts with surrounding lipids could provide models for understanding membrane protein-lipid interfaces more generally.
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Evolutionary modularity: Analysis of how atpB1 integrates into the larger ATP synthase complex could reveal principles of modular evolution applicable to other multi-subunit complexes.
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Coupling mechanisms: Understanding how atpB1 couples proton movement to mechanical rotation could provide insights into other molecular machines that convert chemical gradients into mechanical work.
