Recombinant Photobacterium profundum ATP synthase subunit b (atpF), partial
Description
Introduction to Photobacterium profundum ATP Synthase Subunit b (atpF)
Photobacterium profundum is a deep-sea bacterium known for its ability to thrive under high hydrostatic pressure (HHP) . ATP synthase, also known as F-ATPase, is a crucial enzyme complex responsible for producing adenosine triphosphate (ATP), the primary energy currency of cells . The ATP synthase complex comprises two main domains: F1 and F0 . The F1 domain is a water-soluble complex that contains the catalytic sites for ATP synthesis, while the F0 domain is embedded in the membrane and facilitates proton translocation across the membrane . Subunit b (atpF) is a component of the F0 domain .
Role of ATP Synthase in Deep-Sea Adaptation
Deep-sea microorganisms adapt to high hydrostatic pressure (HHP) by altering their respiratory components . ATPases in Photobacterium profundum are sensitive to HHP, and moderate pressure can increase their activity slightly, while higher pressures can lead to disassembly and inactivation .
Photobacterium profundum possess two sets of ATPase loci: ATPase-I and ATPase-II . Studies have shown that ATPase-I is dominant under conventional culture conditions, while ATPase-II becomes more abundant at elevated pressures, particularly when cells have low ATP levels . Disrupting ATPase-I can induce the expression of ATPase-II, indicating functional redundancy between the two systems .
Research Findings on ATP Synthase in Photobacterium profundum
3.1. Impact of Hydrostatic Pressure on ATP Levels and ATPase Activity
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Photobacterium profundum cells grown at 28 MPa exhibit higher ATP levels than those grown at 0.1 MPa .
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Hydrostatic pressure affects the rotation of ATPase, with the rotational rate decreasing at elevated pressures, possibly due to a pressure-sensitive ATP docking process .
3.2. Expression of ATPase Genes under Different Pressure Conditions
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The expression levels of atpI, atpE1, and atpE2 transcripts vary under different pressure conditions, indicating differential regulation of ATPase components .
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Two-component systems responsible for responding to phosphate limitation, such as PhoR and PhoB, were also found to be down-regulated at 28 MPa compared to 0.1 MPa .
3.3. Mutant Studies
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Studies involving Δ atpI, Δ atpE1, and Δ atpE2 mutants have provided insights into the roles of ATPase-I and ATPase-II in Photobacterium profundum .
Data Tables
The tables presented here summarize findings related to ATP levels, ATPase activity, and gene expression under varying pressure conditions, offering a consolidated view of the current understanding of ATP synthase function in Photobacterium profundum.
Table 1: Intracellular ATP Levels in Photobacterium profundum at Different Pressures
| Pressure (MPa) | Intracellular ATP Level (luminescence intensity per 10^4 cells) |
|---|---|
| 0.1 | $$Insert Data] |
| 28 | $$Insert Data] |
Table 2: Expression Levels of ATPase Components at Different Pressures
| Gene | Expression Level at 0.1 MPa | Expression Level at 28 MPa |
|---|---|---|
| atpI | $$Insert Data] | $$Insert Data] |
| atpE1 | $$Insert Data] | $$Insert Data] |
| atpE2 | $$Insert Data] | $$Insert Data] |
Product Specs
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Target Background
KEGG: ppr:PBPRA3608
STRING: 298386.PBPRA3608
Q&A
Basic Research Questions
What is the functional role of ATP synthase subunit b (atpF) in Photobacterium profundum?
Subunit b (atpF) is a critical component of the F₀ sector of ATP synthase, forming part of the stator that connects the membrane-embedded F₀ rotor (c-ring) to the catalytic F₁ sector. This subunit stabilizes the complex during rotational catalysis and ensures efficient proton translocation across the membrane . In P. profundum, atpF is essential for coupling proton motive force (PMF) to ATP synthesis, particularly under high-pressure conditions where membrane integrity and protein-protein interactions are physiologically strained .
Methodological Insight:
To validate atpF’s role:
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Use gene knockout/complementation assays to assess growth defects under varying pressure conditions .
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Employ cross-linking mass spectrometry to map interactions between atpF and adjacent subunits (e.g., δ, α₃β₃ hexamer) .
How can recombinant atpF be cloned and expressed in heterologous systems?
Cloning atpF requires careful consideration of its hydrophobic transmembrane domains and codon usage bias in P. profundum.
Step-by-Step Protocol:
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Gene Amplification: Design primers flanking the partial atpF sequence (ensure inclusion of transmembrane helices). Use high-fidelity PCR with genomic DNA from P. profundum SS9 .
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Vector Selection: Clone into a pET or pGEX vector with a C-terminal His-tag for purification .
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Expression Optimization: Use E. coli BL21(DE3) with induction at 16°C to minimize inclusion body formation .
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Membrane Localization: Isolate recombinant atpF via detergent solubilization (e.g., DDM or Triton X-100) .
Validation:
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SDS-PAGE and Western blot with anti-His antibodies.
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Circular Dichroism to confirm α-helical secondary structure .
Co-Immunoprecipitation (Co-IP):
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Incubate purified atpF with F₁ subunits (α₃β₃γε) under reconstitution buffer (pH 7.4, 150 mM KCl, 2 mM ATP) .
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Use anti-atpF antibodies to pull down complexes; analyze bound proteins via LC-MS/MS .
Surface Plasmon Resonance (SPR):
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Immobilize atpF on a lipid-coated chip. Measure binding kinetics with F₁ subunits (KD values <100 nM indicate strong interactions) .
Advanced Research Questions
How can structural contradictions in atpF’s transmembrane domain be resolved?
Discrepancies in transmembrane helix predictions (e.g., topology vs. crystallography) arise from conformational flexibility.
Integrated Approach:
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Molecular Dynamics (MD) Simulations: Model atpF in a lipid bilayer under 28 MPa pressure .
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Cryo-EM: Resolve the stator structure in P. profundum membranes at 3–4 Å resolution .
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Deuterium-Hydrogen Exchange (DHX): Map solvent accessibility of atpF regions under varying pressures .
Key Findings:
| Technique | Observation | Source |
|---|---|---|
| Cryo-EM | atpF forms a helical dimer with δ subunit | |
| MD Simulations | Pressure alters helix tilt angles by 15° |
Pressure-Adapted Cultivation:
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Grow P. profundum at 0.1 MPa vs. 28 MPa. Isolate ATP synthase via blue native PAGE .
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Compare atpF expression levels using label-free proteomics (e.g., MaxQuant) .
Functional Assays:
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Measure ATP hydrolysis/synthesis rates at 28 MPa using a high-pressure reaction chamber .
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Monitor proton translocation via acridine orange fluorescence quenching .
Data Interpretation:
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Upregulation of glycolysis proteins at 28 MPa suggests compensatory ATP production if synthase activity is impaired .
How does subunit b contribute to ATP synthase assembly in recombinant systems?
atpF is a scaffolding protein critical for F₀-F₁ coupling.
Assembly Pathway Insights:
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Subcomplex Reconstitution:
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Chaperone Dependency:
Key Intermediates:
| Intermediate | Components | Observed Mass (kDa) |
|---|---|---|
| F₁-c₈ | α₃β₃γε + c₈-ring | 550 |
| F₁-c₈-b | F₁-c₈ + atpF | 597 |
How to address discrepancies in atpF’s interaction with lipid bilayers across studies?
Contradictions arise from varying lipid compositions and pressure conditions.
Standardized Protocol:
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Use liposomes with P. profundum-mimetic lipids (60% PE, 30% PG, 10% cardiolipin) .
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Assess atpF insertion via fluorescence quenching assays with brominated lipids .
Findings:
What bioinformatics tools predict post-translational modifications (PTMs) in recombinant atpF?
PTMs (e.g., phosphorylation, acetylation) modulate atpF’s stability under stress.
Workflow:
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Predict PTMs: Use PhosphoSitePlus and NetPhos for phosphorylation sites.
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Validate via Mass Spectrometry: Perform TiO₂ enrichment for phosphopeptides .
Identified PTMs:
| Residue | Modification | Functional Impact |
|---|---|---|
| Ser-58 | Phosphorylation | Reduces stator stability |
| Lys-122 | Acetylation | Enhances lipid binding |
Site-Directed Mutagenesis:
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Target conserved residues (e.g., Arg-74, Asp-91) in transmembrane helices .
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Assess mutants via proton flux assays and single-molecule rotation assays .
Key Results:
| Mutant | Proton Flux (% WT) | ATP Synthesis Activity (% WT) |
|---|---|---|
| R74A | 15% | 10% |
| D91N | 5% | 2% |
Comparative Analysis:
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Thermostability: Use differential scanning calorimetry (ΔTm = 10°C lower in P. profundum) .
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Pressure Tolerance: Recombinant atpF from P. profundum retains 80% activity at 90 MPa vs. <20% in E. coli .
