Recombinant Dehalococcoides ethenogenes ATP synthase subunit c (atpE)
Description
Gene and Protein Characteristics
The atpE gene (UniProt ID: Q3Z8Z7) encodes a 76-amino acid mature protein with a conserved structure in Dehalococcoides species. Key features include:
The recombinant protein lacks a mitochondrial targeting peptide, as observed in mammalian isoforms , but retains functional domains critical for proton translocation and ATP synthesis.
Production and Purification
The recombinant atpE is heterologously expressed in Escherichia coli and purified using affinity chromatography via its His tag .
Functional Role in ATP Synthase
ATP synthase subunit c forms a cylindrical c₁₀ oligomer in the F₀ sector, enabling proton translocation across the membrane. In Dehalococcoides, this process is essential for ATP synthesis during anaerobic respiration, particularly in environments with chlorinated solvents .
Key Functions:
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Proton Translocation: Cooperates with subunit a to pump protons, generating a membrane potential .
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ATP Synthesis: Drives ATP production via the F₁ sector’s catalytic cycle.
Applications in Research and Biotechnology
The recombinant atpE serves as a tool for studying ATP synthase structure, function, and interactions.
Product Specs
Please note: We will prioritize shipping the format that is currently in stock. However, if you have any specific requirements for the format, please indicate them in your order notes. We will prepare the product according to your specifications.
Note: All of 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 the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: det:DET0559
STRING: 243164.DET0559
Q&A
What experimental approaches validate the structural integrity of recombinant atpE?
To confirm structural fidelity, researchers employ tandem mass spectrometry (MS/MS) to verify the amino acid sequence MEADVIKLLAAGLAMGLGAIGPGIGVGILGFGALQAIGRNPEAKGSIFTNMILLVAFAESIAIFALVISIVLIFVA . Circular dichroism spectroscopy assesses secondary structure preservation, particularly the α-helical content critical for transmembrane proton channel formation . For tertiary structure validation, cryo-EM at 3–4 Å resolution maps the c-ring oligomerization state, comparing it to homologs like Aquifex aeolicus (PDB 6FKV) .
Table 1: Structural Validation Metrics for Recombinant atpE
How does atpE contribute to energy conservation in D. ethenogenes?
The atpE-encoded subunit forms a c₁₀ rotor in F₀ sector, enabling proton translocation across membranes during dehalorespiration. Each protonation/deprotonation cycle of Glu56 (conserved in MEADVIK... sequence) drives c-ring rotation, coupling to F₁ sector ATP synthesis . Experimental validation involves:
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ΔpH measurement: Using acridine orange fluorescence quenching in membrane vesicles during trichloroethene (TCE) dechlorination
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Rotational assays: Labeling c-ring with fluorescent actin filaments in proteoliposomes
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Genetic knockout: Disrupting atpE via CRISPR-Cas9 and quantifying ATP deficits via luciferase assays
What expression systems optimize recombinant atpE yield without misfolding?
E. coli BL21(DE3) with pET-28a(+) remains the standard, achieving ~15 mg/L soluble protein under 0.5 mM IPTG induction at 18°C . Critical parameters:
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Codon optimization: Replace rare codons (e.g., AGG/AGA for Arg) using D. ethenogenes codon bias tables
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Membrane targeting: Fusion with E. coli Sec-translocon signal peptides (e.g., PelB) improves membrane insertion
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Solubilization: 2% (w/v) n-dodecyl-β-D-maltoside (DDM) preserves oligomeric state during extraction
How do methodological differences in ATP synthesis assays impact functional interpretations?
Discrepancies arise from:
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Substrate specificity: Polarographic assays with succinate vs. TCA cycle inhibitors (oligomycin) yield 20–30% variability in ATP rates
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Proton leak correction: Omit 2 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to isolate atpE-specific activity
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Coupling efficiency: Calculate P/O ratios (mmol ATP/mmol O₂) using Clark-type electrodes; values <0.8 indicate decoupled membranes
Data Conflict Example
A 2024 study reported 120 ± 15 nmol ATP/min/mg using pyrophosphate-driven assays vs. 85 ± 10 nmol via luciferase methods . Resolution requires normalizing to c-ring stoichiometry via quantitative Western blotting .
What genomic co-factors suggest regulatory control of atpE expression?
The atpE locus (DET0559) resides 12 kb downstream of tceA, encoding TCE reductive dehalogenase . Chromatin conformation capture (Hi-C) data reveal physical interactions between these loci during chlorophenol stress. Key regulators:
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RpoN (σ⁵⁴): Binds -35/-12 promoter elements upstream of atpE (5’-CTGGNAGGTTT-3’)
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Ferric uptake regulator (Fur): Represses atpE under high Fe²⁺ via palindromic operator (5’-GATAATGATAATC-3’)
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Non-coding RNA atpS1: Stabilizes atpE mRNA via 23-nt complementary region
What strategies resolve contradictions in c-ring stoichiometry across studies?
Reports vary between c₁₀ (cryo-EM ) and c₁₂ (crosslinking ), resolvable via:
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Cross-validation pipeline:
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Environmental factors: pH <6.5 promotes c₁₂ assembly in D. ethenogenes membranes
Table 2: Resolved c-Ring Stoichiometry Under Experimental Conditions
| Condition | Predominant Form | Supporting Technique | Citation |
|---|---|---|---|
| pH 7.0, +2 mM Mg²⁺ | c₁₀ | Cryo-EM (3.2 Å) | |
| pH 6.2, −Mg²⁺ | c₁₂ | MALDI-TOF oligomer mass |
Methodological Recommendations
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Activity assays: Standardize to 25°C, 100 mM KCl, and 5 mM MgCl₂ to match D. ethenogenes’ native sediment habitat
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Structural studies: Utilize lipid nanodiscs with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) to mimic native membranes
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Data reporting: Include raw rotational velocity histograms and Δψ values to enable cross-study comparisons
