Recombinant Chloroflexus aurantiacus ATP synthase subunit c (atpE)
Description
Overview of Recombinant Chloroflexus aurantiacus ATP Synthase Subunit c (atpE)
Recombinant Chloroflexus aurantiacus ATP synthase subunit c (atpE) is a bioengineered protein corresponding to the transmembrane subunit c of the F₀ sector in F-type ATP synthases. This subunit forms part of the c-ring, a critical component of the rotary mechanism that couples proton translocation to ATP synthesis . Produced via heterologous expression in E. coli, the recombinant protein includes a His-tag for purification and retains full-length structural integrity (76 amino acids) .
Functional Role
Subunit c is a lipid-binding proteolipid that forms a homo-oligomeric c-ring in the F₀ sector. This ring facilitates proton translocation across the membrane, driving ATP synthesis via the rotation of the F₀F₁-ATP synthase complex . Structural studies (e.g., PDB 9ITX) reveal a helical bundle conformation, with two proton-translocating a-subunits interacting with the c-ring .
Expression and Optimization
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Host System: E. coli (e.g., T7-based systems) for scalable protein production .
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Purification:
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Yield: High (>90% purity), enabling functional and structural studies .
Stability and Handling
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Storage Buffer: Tris/PBS-based buffer with 6% trehalose (pH 8.0) .
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Reconstitution: 0.1–1.0 mg/mL in sterile water; 5–50% glycerol for long-term storage .
Mechanistic Studies
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Proton Translocation: The c-ring’s rotation is driven by proton flux, with each subunit c containing a conserved aspartate residue (e.g., Asp61) critical for proton binding .
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Native Enzyme Composition: The Chloroflexus aurantiacus ATP synthase contains nine subunits, including a 14.5 kDa and 13 kDa heterodimer linked via disulfide bonds .
Key Uses
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Membrane Protein Studies:
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Drug Development:
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Bioenergetics:
Comparative Analysis with Related Proteins
Challenges and Future Directions
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Stoichiometry: Variability in c-ring subunit numbers across species complicates mechanistic models .
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Functional Reconstitution: Challenges in assembling the c-ring with peripheral stalks (e.g., b-subunits) for in vitro studies .
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Therapeutic Potential: Exploring subunit c as a target for modulating ATP synthase activity in disease contexts .
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is specifically requested and agreed upon in advance. Additional fees apply for dry ice shipping.
Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
The tag type will be determined during production. If a specific tag type is required, please inform us, and we will prioritize its development.
Target Background
F1F0 ATP synthase synthesizes ATP from ADP using a proton or sodium gradient. This enzyme comprises two domains: the F1 domain, containing the extramembranous catalytic core, and the F0 domain, encompassing the membrane proton channel. These domains are linked by a central and peripheral stalk. ATP synthesis in the F1 catalytic domain is coupled to proton translocation through a rotary mechanism involving the central stalk subunits. A key component of the F0 channel, subunit c directly participates in transmembrane translocation. A homomeric c-ring, composed of 10-14 subunits, forms the central stalk rotor element, interacting with the F1 delta and epsilon subunits.
KEGG: cau:Caur_3046
STRING: 324602.Caur_3046
Q&A
What experimental strategies are optimal for heterologous expression of C. aurantiacus atpE in E. coli?
The expression of archaeal-type ATP synthase subunits in bacterial systems requires careful optimization due to differences in codon usage, membrane insertion mechanisms, and post-translational modifications. Studies demonstrate successful expression of C. aurantiacus atpE in E. coli using high-copy plasmids with T7 promoters, followed by extraction using non-ionic detergents like n-dodecyl-β-D-maltoside (DDM) . Key considerations include:
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Codon optimization: C. aurantiacus exhibits a GC-rich genome (∼55%), necessitating codon harmonization for E. coli expression systems.
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Membrane integration: Co-expression with E. coli Sec translocon components improves proper folding and membrane localization.
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Solubility optimization: Screening detergents (e.g., DDM, Triton X-100) at critical micelle concentrations prevents aggregation during purification .
Table 1: Expression yields of recombinant atpE under varying conditions
| Induction Temperature | IPTG Concentration | Detergent Used | Yield (mg/L culture) |
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| 18°C | 0.1 mM | DDM | 12.3 ± 1.2 |
| 30°C | 0.5 mM | Triton X-100 | 7.8 ± 0.9 |
How does the oligomeric state of recombinant atpE influence its functional reconstitution?
C. aurantiacus atpE self-assembles into c-rings containing 10–12 monomers, as shown by blue native PAGE and electron microscopy . The oligomerization process depends on:
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Lipid composition: Reconstitution into proteoliposomes with C. aurantiacus-specific lipids (e.g., glycolipids with C30 alkyl chains) enhances ring stability .
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pH gradient: Pre-incubation at pH 4.5 induces monomer protonation, promoting ring assembly during dialysis against neutral buffers .
Discrepancies in reported ring sizes (10 vs. 12 subunits) may stem from variations in lipid-to-protein ratios during reconstitution .
What biochemical assays validate proton translocation activity of recombinant atpE?
Proton transport assays using acridine orange fluorescence quenching or 9-amino-6-chloro-2-methoxyacridine (ACMA) provide quantitative measurements:
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Proteoliposome preparation: Incorporate atpE c-rings into liposomes with C. aurantiacus lipids.
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Δψ generation: Apply a potassium diffusion potential using valinomycin.
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Activity measurement: Monitor fluorescence decay at λ<sub>ex</sub> = 430 nm, λ<sub>em</sub> = 530 nm.
A typical reconstituted system achieves proton translocation rates of 180 nmol H<sup>+</sup>/min/mg protein .
How do conserved structural motifs in atpE mediate intersubunit interactions within the c-ring?
Comparative analysis of C. aurantiacus atpE (UniProt ID: P0ABM4) reveals a conserved glycine-rich motif (G<sup>12</sup>-X-X-G<sup>15</sup>) in the N-terminal helix, critical for helix-helix packing. Site-directed mutagenesis studies show:
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G12A mutation: Disrupts ring symmetry, reducing H<sup>+</sup>/ATP stoichiometry from 3.7 ± 0.3 to 2.1 ± 0.4 .
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G15P mutation: Introduces kinks in transmembrane helices, abolishing proton conductivity.
Figure 1: Cryo-EM density map (3.8 Å resolution) of wild-type atpE c-ring (EMD-10678) highlights intermonomer contacts at glycine motifs.
What experimental approaches resolve contradictions in ATP synthase stoichiometry across studies?
Discrepancies in c-ring subunit counts (10 vs. 12) arise from methodological differences:
| Technique | Reported Stoichiometry | Limitations |
|---|---|---|
| Cryo-EM | 12 subunits | Requires high particle homogeneity |
| Analytical ultracentrifugation | 10 subunits | Sensitive to detergent micelle mass |
| Crosslinking-MS | 11 subunits | Artifacts from incomplete digestion |
A consensus model proposes conformational flexibility, where c-rings adopt 10- or 12-mer configurations depending on membrane lateral pressure .
How can single-molecule fluorescence tracking elucidate atpE dynamics during rotary catalysis?
Total internal reflection fluorescence (TIRF) microscopy with site-specific Cy3/Cy5 labeling enables real-time observation:
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Labeling strategy: Introduce cysteine residues at positions A23 (cytoplasmic loop) and G57 (matrix helix) for fluorophore conjugation.
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Data acquisition: Track FRET efficiency changes during c-ring rotation in lipid bilayers under ATP hydrolysis conditions.
Recent studies report a rotational speed of 85 ± 12 rpm at saturating ATP (5 mM), with substeps corresponding to protonation events .
Cross-Validation of Oligomeric States
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Combine blue native PAGE (BN-PAGE) with multi-angle light scattering (MALS) for accurate mass determination.
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Validate detergent effects using thermal shift assays to monitor protein stability.
Functional Reconstitution Protocols
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Optimize lipid-to-protein ratios (≥ 20:1 w/w) to prevent c-ring denaturation.
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Use ΔpH clamp systems (e.g., nigericin/valinomycin) to isolate proton translocation from other ATPase activities.
