Recombinant Shewanella loihica ATP synthase subunit c (atpE)
Description
Structure and Biochemical Properties
The Shewanella loihica ATP synthase subunit c is a hydrophobic, membrane-embedded protein. Key structural features include:
This subunit belongs to the F₀ sector of the ATP synthase complex, forming a c-ring that facilitates proton translocation across the membrane. Its lipid-binding properties are essential for embedding into the membrane and coordinating rotational motion during ATP synthesis .
Production and Purification
The recombinant protein is produced in E. coli using optimized expression systems:
Commercial suppliers like CUSABIO and Creative Biomart offer this protein in lyophilized powder form, with quantities starting at 50 µg .
Functional Role in ATP Synthesis
Subunit c forms a ring structure (cₙ) within the ATP synthase F₀ sector. Its primary functions include:
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Proton Translocation: Each c-subunit contains a conserved acidic residue (e.g., aspartate) that binds protons, enabling rotational motion during oxidative phosphorylation .
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ATP Synthesis Regulation: The c-ring stoichiometry (e.g., c₁₂ vs. c₁₃) determines the ion-to-ATP ratio, influencing cellular energy efficiency .
In Shewanella loihica, this subunit is critical for adapting to environmental stressors, such as alkaline pH, by modulating proton flux .
Biotechnological Uses
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Reference Standard: Used in ELISA and SDS-PAGE assays to study ATP synthase dynamics .
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Structural Reconstitution: Recombinant c-subunits enable in vitro assembly of c-rings for cryo-EM or X-ray crystallography studies .
Challenges and Future Directions
Product Specs
Please note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order notes, and we will fulfill your request.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please notify us in advance, and additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: slo:Shew_3850
STRING: 323850.Shew_3850
Q&A
What is the function of ATP synthase subunit c (atpE) in Shewanella loihica?
ATP synthase subunit c (atpE) in Shewanella loihica serves as a critical component of the F0 portion of ATP synthase, forming an oligomeric ring structure within the membrane. This c-ring plays an essential role in energy conservation by facilitating proton translocation across the membrane, which drives the rotation of the ATP synthase complex. Each c-subunit binds and transports one H+ across the membrane as the ring rotates, converting the proton motive force into mechanical energy that ultimately drives ATP synthesis .
The c-subunit is particularly important in electrochemically active bacteria (EAB) like Shewanella species, which can utilize various electron acceptors including electrodes. The ATP synthase complex, including the c-subunit, is involved in energy conservation when these organisms grow using electrodes as terminal electron acceptors .
What expression systems are most effective for recombinant production of Shewanella loihica atpE?
For recombinant production of Shewanella loihica ATP synthase subunit c (atpE), an Escherichia coli expression system with codon optimization is highly recommended. Based on successful approaches with similar proteins, expression as a fusion protein with maltose binding protein (MBP) has proven effective for improving solubility and yield .
Methodological approach:
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Clone the codon-optimized atpE gene into an expression vector with an MBP fusion tag
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Transform into an E. coli expression strain (BL21(DE3) or similar)
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Induce expression at lower temperatures (16-25°C) to improve proper folding
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Include detergents during cell lysis and protein purification to maintain stability of this membrane protein
This fusion protein approach addresses the hydrophobic nature of the c-subunit, which otherwise tends to form inclusion bodies when expressed alone. The MBP tag enhances solubility while allowing for affinity purification .
What purification methods are recommended for recombinant Shewanella loihica atpE?
Purification of recombinant Shewanella loihica ATP synthase subunit c requires a specialized approach due to its hydrophobic nature as a membrane protein. A multi-step purification protocol is recommended:
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Initial purification: Affinity chromatography using amylose resin to capture the MBP-tagged fusion protein
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Protease cleavage: Release the c-subunit from the fusion tag using a specific protease (TEV or Factor Xa) in the presence of an appropriate detergent
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Final purification: Reversed phase column chromatography using ethanol as an eluent, which has been successfully employed for similar c-subunits
Table 1: Recommended Detergents for atpE Purification
| Detergent | Concentration | Advantages | Limitations |
|---|---|---|---|
| n-Dodecyl β-D-maltoside (DDM) | 0.02-0.05% | Mild, maintains protein structure | Higher cost |
| Triton X-100 | 0.1-0.5% | Effective solubilization | Difficult to remove |
| CHAPS | 0.5-1.0% | Compatible with many assays | Less effective for highly hydrophobic regions |
| SDS | 0.1% | Highly effective solubilization | May denature protein |
After purification, circular dichroism spectroscopy can be used to confirm that the recombinant protein maintains its native alpha-helical secondary structure .
How can I confirm the proper folding and oligomerization of purified recombinant atpE?
Confirming proper folding and oligomerization of purified recombinant Shewanella loihica ATP synthase subunit c requires multiple analytical techniques:
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Secondary structure analysis: Circular dichroism (CD) spectroscopy should show characteristic alpha-helical peaks, indicating proper folding of the protein with its native secondary structure .
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Oligomeric state analysis: Blue native polyacrylamide gel electrophoresis (BN-PAGE) can be used to assess the formation of the c-ring complex.
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Functional reconstitution: Incorporate the purified c-subunit into liposomes and assess proton translocation capability using pH-sensitive fluorescent dyes.
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Electron microscopy: Negative staining or cryo-electron microscopy can visualize the ring structure formed by the c-subunits.
Research has shown that properly folded monomeric c-subunits can spontaneously form oligomeric rings similar to their native form when reconstituted in liposomes, which is a critical indicator of functional integrity .
How does electrode potential affect atpE expression and function in Shewanella loihica?
Electrode potential significantly influences the expression and function of ATP synthase components, including the c-subunit (atpE), in electrochemically active bacteria like Shewanella species. Research with the related organism Shewanella oneidensis MR-1 provides valuable insights:
Higher electrode potentials (+0.5V vs. standard hydrogen electrode) upregulate genes encoding ATP synthase subunits, including those in the atp operon. This response is part of a broader metabolic shift where NADH-dependent catabolic pathways are activated . The Arc regulatory system plays a crucial role in sensing electrode potentials and regulating the expression of these genes .
Experimental data from S. oneidensis MR-1 showing effects of electrode potential:
Table 2: Effects of Electrode Potential on Growth and Energy Conservation
| Electrode Potential | Current Density | Protein Yield (per lactate) | ATP Synthase Expression |
|---|---|---|---|
| +0.5V (High) | High | High | Upregulated |
| +0.2V (Middle) | Medium | Medium | Moderately expressed |
| 0V (Low) | Low | Low | Baseline expression |
These findings suggest that Shewanella loihica likely modulates atpE expression in response to electrode potential, optimizing energy conservation by adjusting the expression of ATP synthase components, including the c-subunit .
What is the stoichiometry of the c-ring in Shewanella loihica ATP synthase and how does it affect bioenergetics?
This stoichiometry has profound implications for cellular bioenergetics, as it directly determines the H+/ATP ratio. Each c-subunit binds and transports one H+ across the membrane during ring rotation, while a complete rotation drives the synthesis of 3 ATP molecules .
Table 3: Theoretical H+/ATP Ratios Based on c-Ring Stoichiometry
| c-Ring Stoichiometry | H+ Transported per 360° Rotation | ATP Synthesized per Rotation | H+/ATP Ratio |
|---|---|---|---|
| 8 | 8 | 3 | 2.67 |
| 10 | 10 | 3 | 3.33 |
| 12 | 12 | 3 | 4.00 |
| 14 | 14 | 3 | 4.67 |
| 15 | 15 | 3 | 5.00 |
For electrochemically active bacteria like Shewanella loihica, which must adapt to varying redox environments, the c-ring stoichiometry may represent an evolutionary adaptation to optimize energy conservation under specific environmental conditions .
How can recombinant Shewanella loihica atpE be used in microbial fuel cell research?
Recombinant Shewanella loihica ATP synthase subunit c (atpE) offers several valuable applications in microbial fuel cell (MFC) research:
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Mechanistic studies of energy conservation:
Purified and reconstituted c-subunits can be used to investigate how electrode potential influences ATP synthesis in electrochemically active bacteria. By manipulating the c-subunit structure or expression, researchers can better understand the energy conservation mechanisms in MFCs . -
Comparative analysis with other Shewanella species:
Shewanella loihica PV-4 has been compared with Shewanella oneidensis MR-1 for current-generating capabilities in MFCs . Recombinant atpE can be used in structural and functional studies to understand species-specific adaptations. -
Engineering improved MFC performance:
Knowledge of how ATP synthase components respond to electrode potentials can inform genetic engineering approaches to optimize energy conservation and current generation in MFCs.
Experimental approach for MFC studies using recombinant atpE:
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Generate atpE variants with site-directed mutagenesis
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Express and purify the variants
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Reconstitute in liposomes or proteoliposomes
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Measure ATP synthesis rates under varying electrode potentials
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Correlate structural features with functional performance
These studies could potentially reveal how Shewanella loihica ATP synthase has evolved to function optimally in its native deep-sea hydrothermal vent environment, which may have unique implications for MFC applications .
