Recombinant Geobacter metallireducens ATP synthase subunit c (atpE)
Description
Gene and Functional Context
The atpE gene encodes a subunit essential for the assembly and activity of ATP synthase. In Geobacter metallireducens, ATP synthase operates in reverse under anaerobic conditions, coupling proton influx to ATP hydrolysis or synthesis depending on cellular energy needs .
Key Functions:
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Proton Translocation: Subunit c forms a ring structure with other c-subunits, creating a proton-conductive pathway .
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Energy Coupling: Drives ATP synthesis or hydrolysis via rotational catalysis .
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Electron Transport Linkage: Integrated with Geobacter’s respiratory chains for metal reduction (e.g., Fe³⁺, U⁶⁺) .
Bioremediation and Bioenergy
Geobacter metallireducens is renowned for its ability to oxidize organic acids (e.g., acetate, propionate) and reduce metals . ATP synthase subunit c likely supports energy conservation during these processes. For example:
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Metal-Respiring Biofilms: ATP synthase activity may enhance electron transfer efficiency in microbial fuel cells .
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Autotrophic CO₂ Fixation: Geobacter species use ATP synthase to sustain energy-intensive processes like Calvin-Benson-Bassham cycle activity .
Production and Handling Guidelines
The recombinant atpE is lyophilized and requires careful reconstitution:
| Parameter | Recommendation |
|---|---|
| Reconstitution | 0.1–1.0 mg/mL in deionized sterile water; add 5–50% glycerol for stability |
| Storage | Aliquot at -20°C/-80°C; avoid repeated freeze-thaw cycles |
| Working Aliquot | Store at 4°C for ≤1 week; discard if precipitation occurs |
Comparative Insights from Geobacter Genomes
Comparative genomics highlights Geobacter metallireducens’ unique metabolic adaptations:
These differences suggest atpE may have evolved under distinct selective pressures, potentially affecting its interaction with other respiratory components .
Future Research Directions
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Mechanistic Studies: Cryo-EM or X-ray crystallography to resolve the structure of the F₀ complex in Geobacter.
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Biotechnological Optimization: Engineering atpE for enhanced proton pumping efficiency in bioelectrochemical systems.
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Ecological Relevance: Linking ATP synthase activity to Geobacter’s dominance in subsurface environments .
Product Specs
Note: We will prioritize shipping the format we currently have in stock. However, if you have specific format requirements, please indicate them when placing your order. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: gme:Gmet_3360
STRING: 269799.Gmet_3360
Q&A
What is the function of ATP synthase subunit c in Geobacter metallireducens?
ATP synthase subunit c (atpE) in G. metallireducens is a critical component of the F1F0-ATP synthase complex, which couples the proton gradient generated by the respiratory chain to ATP synthesis. Subunit c forms a cylindrical oligomer in the membrane-embedded F0 portion of the complex and directly cooperates with subunit a in the proton pumping process . In bacterial systems like G. metallireducens, this protein plays an essential role in energy conservation during anaerobic respiration, which is particularly important given this organism's unique ability to respire using metals and electrodes as terminal electron acceptors .
How does ATP synthase subunit c from G. metallireducens differ from other bacterial homologs?
While the search results don't provide specific sequence information for G. metallireducens ATP synthase subunit c, comparative genomic analysis reveals important insights. Unlike mammalian systems that have three isoforms of subunit c (differing only in their targeting peptides) , bacterial ATP synthase subunit c proteins typically exist as a single isoform. G. metallireducens shows significant metabolic versatility compared to closely related species like G. sulfurreducens , suggesting its ATP synthase components may have unique adaptations to support its diverse energy generation pathways. The genomic evidence indicates that metabolism and gene regulation in G. metallireducens may differ dramatically from other Geobacteraceae .
What expression systems are most suitable for recombinant G. metallireducens atpE production?
Expression System Comparison:
| Expression System | Advantages | Limitations | Yield Potential |
|---|---|---|---|
| E. coli BL21(DE3) | Well-established, economical, rapid growth | Potential toxicity, inclusion body formation | Moderate to high |
| E. coli C41/C43 | Engineered for membrane protein expression | Higher cost, slower growth | Moderate to high |
| Cell-free system | Avoids toxicity issues, rapid | Higher cost, complex setup | Low to moderate |
| Native Geobacter host | Proper folding, native environment | Difficult transformation, slow growth | Low |
For membrane proteins like ATP synthase subunit c, using strains specifically designed for membrane protein expression (like C41/C43) and expressing at lower temperatures (16-20°C) can significantly improve functional yield.
What purification strategy yields the highest purity of recombinant G. metallireducens atpE?
Purification of recombinant G. metallireducens ATP synthase subunit c requires specialized approaches due to its hydrophobic nature and membrane association. A multi-step purification strategy is recommended:
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Membrane isolation: After cell lysis by sonication or French press, collect membranes by ultracentrifugation (100,000×g for 1 hour).
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Detergent solubilization: Solubilize membranes using a mild detergent such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) at 1-2% concentration in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl.
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Affinity chromatography: If expressed with a His-tag, use Ni-NTA chromatography with stepwise imidazole elution (50 mM, 100 mM, 250 mM, 500 mM).
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Size exclusion chromatography: For highest purity, perform size exclusion chromatography using Superdex 200 in buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.05% detergent.
This approach typically yields protein with >95% purity as assessed by SDS-PAGE and Western blotting.
How can researchers effectively assess the oligomeric state of recombinant G. metallireducens atpE?
The oligomeric state of ATP synthase subunit c is critical for its function, as it forms a cylindrical c-ring structure. Multiple complementary techniques should be employed:
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Blue Native PAGE: Preserves native protein interactions and can resolve different oligomeric states.
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Analytical ultracentrifugation: Provides accurate determination of the molecular weight of the protein-detergent complex.
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Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): Distinguishes between protein and detergent contributions to determine the absolute molecular weight of the protein complex.
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Negative stain electron microscopy: Allows visualization of the c-ring structure and can provide information about symmetry and subunit arrangement.
Based on studies of other bacterial ATP synthases, the c-ring typically consists of 10-15 subunits, with the exact number being species-specific and potentially related to the bioenergetic requirements of the organism.
How does the unique metabolic versatility of G. metallireducens influence ATP synthase function and structure?
G. metallireducens demonstrates remarkable metabolic versatility compared to related species like G. sulfurreducens, utilizing a much broader range of carbon sources including acetate, benzaldehyde, benzoate, butanol, butyrate, p-cresol, ethanol, and many others . This metabolic flexibility may be reflected in adaptations of its ATP synthase to accommodate diverse bioenergetic requirements.
Comparative Metabolic Analysis:
| Aspect | G. metallireducens | G. sulfurreducens | Potential Impact on ATP Synthase |
|---|---|---|---|
| Carbon source utilization | Diverse (>20 substrates) | Limited (4 substrates) | May require adaptations for varying proton motive force |
| Electron acceptors | Fe(III), Mn(IV), electrodes, humic substances, U(VI) | More limited range | Could influence c-ring stoichiometry |
| Respiratory chain | Complex with multiple pathways | Simpler organization | May affect coupling efficiency |
Research suggests that organisms with more versatile metabolism may have ATP synthases with different c-ring stoichiometries or regulatory mechanisms to accommodate varying energetic demands. Investigating these adaptations could provide insights into how ATP synthase structure relates to metabolic flexibility.
What role might post-translational modifications play in G. metallireducens ATP synthase subunit c function?
While the search results don't specifically address post-translational modifications (PTMs) of G. metallireducens ATP synthase subunit c, this is an important research consideration. In other bacterial systems, ATP synthase subunits can undergo modifications including:
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Phosphorylation: May regulate activity in response to cellular energy status
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Acetylation: Could affect protein-protein interactions within the complex
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Lipid modifications: May influence membrane association and c-ring assembly
To investigate PTMs, researchers should consider:
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Proteomic analysis using mass spectrometry with enrichment techniques specific for phosphorylation, acetylation, or other modifications
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Site-directed mutagenesis of predicted modification sites to assess functional impact
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Comparative analysis with other Geobacter species to identify conserved modification patterns
How do inhibitors of ATP synthase interact with G. metallireducens atpE compared to other species?
ATP synthase inhibitors often target subunit c, making this an important research question. Common inhibitors like oligomycin, venturicidin, and dicyclohexylcarbodiimide (DCCD) bind to the c-ring and disrupt proton translocation.
Researchers should consider:
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Inhibition assay methodology:
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ATP synthesis assays in inverted membrane vesicles
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ATPase activity assays with purified enzyme
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Growth inhibition studies with whole cells
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Comparative inhibition analysis:
| Inhibitor | G. metallireducens IC50 (estimated) | Other Bacteria | Binding Site |
|---|---|---|---|
| DCCD | To be determined | 1-10 μM | Conserved carboxyl residue in subunit c |
| Oligomycin | May be resistant | Variable | Interface between subunits a and c |
| Venturicidin | To be determined | 0.5-5 μM | c-ring |
This comparative analysis could reveal unique structural features of G. metallireducens ATP synthase and potentially identify species-specific inhibitors.
What structural techniques are most appropriate for studying recombinant G. metallireducens atpE?
Structural characterization of ATP synthase subunit c presents significant challenges due to its hydrophobic nature and requirement for a membrane-like environment. Multiple complementary techniques are recommended:
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X-ray crystallography: Challenging but potentially feasible using lipidic cubic phase (LCP) crystallization, which has been successful for other membrane proteins.
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Cryo-electron microscopy (cryo-EM): Increasingly powerful for membrane protein complexes, allowing visualization of the entire ATP synthase complex.
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Solid-state NMR: Particularly valuable for membrane proteins, providing atomic-level details of structure and dynamics in a native-like environment.
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Molecular dynamics simulations: Can provide insights into protein-lipid interactions and conformational changes during the catalytic cycle.
How can researchers accurately measure proton translocation through the G. metallireducens c-ring?
Proton translocation is central to ATP synthase function, and accurate measurement requires specialized techniques:
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pH-sensitive fluorescent probes: Fluorescent indicators like ACMA (9-amino-6-chloro-2-methoxyacridine) can report on proton gradient formation in reconstituted proteoliposomes.
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Patch-clamp electrophysiology: While technically challenging, patch-clamp measurements of isolated c-rings reconstituted into planar lipid bilayers can provide direct measurement of proton conductance.
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Stopped-flow spectroscopy: Allows measurement of rapid proton translocation events using pH-sensitive dyes.
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Isotope exchange measurements: Using deuterium or tritium can provide insights into proton exchange rates.
Experimental Setup Comparison:
| Technique | Time Resolution | Advantages | Limitations |
|---|---|---|---|
| pH-sensitive probes | Seconds | Simple setup, quantitative | Indirect measurement |
| Patch-clamp | Milliseconds | Direct measurement | Technical difficulty |
| Stopped-flow | Milliseconds | Good time resolution | Requires specialized equipment |
| Isotope exchange | N/A | Highly specific | Complex analysis |
How does recombinant G. metallireducens atpE function in bioelectrochemical systems?
G. metallireducens is known for its ability to transfer electrons to electrodes , making the function of its ATP synthase in bioelectrochemical systems particularly interesting. Researchers should consider:
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Electrode-grown cultures: Comparing ATP synthase expression and activity in cells grown with electrodes versus conventional electron acceptors.
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Bioenergetic analysis: Measurement of proton motive force and ATP synthesis rates during electrode respiration.
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Mutational studies: Creating atpE variants to assess their impact on electrode respiration capability.
Research approach matrix:
| Research Question | Methodology | Expected Outcomes | Challenges |
|---|---|---|---|
| ATP synthesis during electrode respiration | Luciferase-based ATP assays in electrode-grown cells | Correlation between current and ATP levels | Maintaining anaerobic conditions |
| atpE expression levels | qRT-PCR, proteomics | Potential upregulation during electrode growth | Normalizing across growth conditions |
| Impact of atpE mutations | Site-directed mutagenesis, bioelectrochemical analysis | Identification of residues critical for electrode respiration | Creating stable mutants |
What insights can we gain from comparing ATP synthase subunit c across different Geobacter species?
Comparative analysis of ATP synthase subunit c across Geobacter species can provide valuable insights into adaptation and evolution:
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Sequence conservation analysis: Identifying highly conserved regions likely critical for function versus variable regions that may relate to species-specific adaptations.
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Correlation with metabolism: Analyzing whether differences in ATP synthase components correlate with metabolic capabilities across species.
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Evolutionary analysis: Investigating whether ATP synthase genes show evidence of horizontal gene transfer or unusual evolutionary patterns.
Combining genomic data with structural predictions could reveal how differences in ATP synthase subunit c contribute to the diverse metabolic capabilities observed across Geobacter species, particularly the greater versatility of G. metallireducens compared to G. sulfurreducens .
