Recombinant Escherichia coli O7:K1 ATP synthase subunit beta (atpD)
Description
Molecular and Functional Overview
The ATP synthase β-subunit (AtpD) is a critical component of the F<sub>1</sub> sector, responsible for binding ADP and inorganic phosphate to synthesize ATP during proton translocation across the membrane . Key features include:
| Property | Details |
|---|---|
| Gene Name | atpD |
| UniProt ID | B7NR34 (strain O7:K1), P0ABB4 (strain K12) |
| Protein Length | 459 amino acids (full-length) |
| Expression Host | E. coli |
| Tag | N-terminal His tag |
| Purity | >85–90% (SDS-PAGE verified) |
| Storage | Lyophilized powder stable at -20°C/-80°C; reconstitute in Tris/PBS buffer |
The recombinant protein retains enzymatic activity, enabling studies on ATP synthase mechanics and inhibition .
3.1. Enzyme Activity Modulation
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Inhibition Studies: The peptide EcDBS1R4 reduces ATPase activity by ~20% in E. coli inner membrane vesicles (IMVs) and proteoliposomes containing cardiolipin, suggesting lipid-dependent regulation .
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Reconstituted Systems: Activity increases in DOPE:POPG:CL membranes but remains unaffected in pure POPC bilayers, highlighting lipid composition’s role in ATP synthase function .
3.2. Pathogenicity Links
While AtpD itself is not a virulence factor, the K1 capsule in E. coli O7:K1 enhances immune evasion, enabling systemic infections . ATP synthase activity may indirectly support pathogen survival under stress (e.g., nutrient deprivation) .
Key Research Gaps and Future Directions
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Mechanistic Studies: How do post-translational modifications (e.g., glycosylation) affect AtpD function in pathogenic strains?
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Therapeutic Targeting: Can small molecules inhibit AtpD to disrupt bacterial energy metabolism without affecting human homologs?
Product Specs
Target Background
KEGG: ect:ECIAI39_4336
Q&A
What vector systems and promoter combinations show optimal atpD expression in E. coli?
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Strain selection: BL21(DE3) pLysS reduces pre-induction transcription through T7 lysozyme inhibition
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Induction tuning: Gradual induction (0.1-0.5 mM IPTG) at 18-25°C minimizes inclusion body formation while maintaining 15-30% soluble protein yields
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Ribosome binding site (RBS) optimization: Computational tools like RBS Calculator v2.1 improve translation initiation rates by adjusting spacer regions between Shine-Dalgarno sequences and start codons
Table 1: Vector performance comparison for atpD expression
| Vector | Promoter | Leakage Rate | Max Yield (mg/L) | Solubility (%) |
|---|---|---|---|---|
| pET21d(+) | T7 | 0.8% | 320 | 45 |
| pBAD/HisA | araBAD | 0.1% | 180 | 68 |
| pCold I | cspA | <0.01% | 95 | 82 |
How to resolve discrepancies between SDS-PAGE quantification and functional ATPase activity assays?
Three key validation steps are essential:
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Redox state verification: Improper disulfide bonding in cytoplasmic expression reduces activity. SHuffle strains with oxidizing cytoplasm improve native folding by 40-60%
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Metal ion profiling: ATP synthase requires Mg²⁺/Ca²⁺ in 2:1 molar ratio. ICP-MS analysis of purified atpD should confirm 0.9-1.1 metal ions per subunit
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Coupled enzyme assays: Monitor NADH oxidation rates (340 nm) in reconstructed ATP synthase complexes rather than standalone atpD measurements
What proteomic signatures indicate metabolic burden during high-density atpD production?
LC-MS/MS analyses reveal three stress markers:
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Acetate overflow: 2.3-fold upregulation of phosphate acetyltransferase (pta) and acetate kinase (ackA)
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Redox imbalance: 4.1x increase in thioredoxin reductase (trxB) and superoxide dismutase (sodA)
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Ribosome stalling: 70S ribosomal proteins show 18% reduction in elongation factor-Tu (tufA) binding capacity
Mitigation strategies:
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Dynamic feeding: Maintain glucose <0.5 g/L via exponential feed to reduce Crabtree effect
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Co-expression partners: GroEL/ES chaperones improve soluble atpD yield by 55% but reduce growth rates by 30%
How to validate conflicting crystallographic and cryo-EM models of atpD conformational states?
Follow this multi-technique workflow:
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Hydrogen-deuterium exchange MS: Maps flexible regions (β-hairpin residues 48-62) with 2.8 Å resolution
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Single-molecule FRET: Quantifies hinge motion between α/β domains (Δdistance = 3.7 ±0.4 nm)
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Molecular dynamics: Simulate 10 μs trajectories using CHARMM36 force field to match experimental B-factors
Critical parameters:
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Maintain 150 mM KCl in all buffers to stabilize ionic interactions
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Use anaerobic chambers (<0.1 ppm O₂) to prevent cysteine oxidation in nucleotide-binding domains
What orthogonal assays confirm atpD incorporation into functional F₀F₁-ATP synthase complexes?
Combine three approaches:
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Blue native PAGE: Verify 550 kDa holoenzyme assembly (8% acrylamide gradient gels)
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Proton pumping assays: Measure ΔpH with 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching (λex/em = 410/490 nm)
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Single-particle tracking: Labeled atpD (His-tag/Alexa647) should show 85% co-localization with F₀ subunit a (Cy3B) in live-cell TIRF microscopy
How to optimize codon usage without commercial synthesis?
A two-stage mutagenesis protocol:
