Recombinant Bartonella quintana ATP synthase subunit a (atpB)
Description
Functional Role of ATP Synthase Subunit a (atpB)
ATP synthase is composed of two main regions: the cytoplasmic F<sub>1</sub> sector (catalytic) and the membrane-bound F<sub>O</sub> sector (proton channel). Subunit a (atpB) is essential for proton translocation across the membrane, enabling ATP synthesis . In Bartonella quintana, ATP synthase activity supports survival in diverse environments, including human and arthropod hosts . Subunit a interacts with the c-ring structure of F<sub>O</sub>, forming a pathway for protons that drives rotation of the enzyme’s rotor .
Recombinant Production of ATP Synthase Subunits in Bartonella
While no direct data exists for B. quintana atpB, recombinant methods for related subunits and species provide a template:
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Expression Systems: Subunits like atpF1 (subunit b) from B. quintana are expressed in Escherichia coli with N-terminal His tags for purification .
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Purification: Typical protocols involve nickel-agarose chromatography, yielding >90% purity .
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Sequence Features: B. tribocorum atpB (UniProt: A9IQH9) shares homology with other Bartonella species, featuring a 252-amino-acid sequence with transmembrane helices critical for proton transport .
Table 1: Example Recombinant ATP Synthase Subunit Properties
| Species | Subunit | UniProt ID | Length (aa) | Key Features |
|---|---|---|---|---|
| B. quintana | atpF1 | Q6G0H1 | 188 | His-tagged, expressed in E. coli |
| B. tribocorum | atpB | A9IQH9 | 252 | Transmembrane helices, proton channel |
Immunological and Diagnostic Relevance
ATP synthase subunits in Bartonella are immunogenic during infection. For example:
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes, and we will accommodate your request.
Note: All proteins are shipped with standard blue ice packs. 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. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: bqu:BQ03130
STRING: 283165.BQ03130
Q&A
What experimental strategies are critical for achieving high-fidelity cloning of Bartonella quintana atpB in heterologous expression systems?
Successful cloning requires:
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Codon optimization tailored to the expression host’s tRNA abundance (e.g., E. coli prefers AU-rich codons for subunits like atpB ).
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Vector selection: Use vectors with adjustable promoters (e.g., T7 or arabinose-inducible) to mitigate toxicity during ATP synthase subunit expression .
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Fusion tags: For atpB, which is a membrane-associated FO subunit, a dual-tag system (e.g., His-tag for purification and SUMO tag for solubility) improves recovery rates.
Key validation steps:
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Sanger sequencing of the atpB insert to confirm absence of PCR-induced mutations.
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Western blot using antibodies against conserved regions of bacterial ATP synthase subunits (e.g., residues 120–150 of Mycobacterium tuberculosis atpB ).
How do researchers resolve discrepancies in ATP hydrolysis activity data between recombinant atpB and native complexes?
Discrepancies often arise from:
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Incomplete assembly: The FO sector (including atpB) requires co-expression with other subunits (e.g., atpE, atpF) for proper proton channel formation .
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Post-translational modifications: Native B. quintana atpB may undergo phosphorylation or lipid anchoring absent in E. coli systems .
Methodological adjustments:
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Proteoliposome reconstitution: Incorporate recombinant atpB into synthetic lipid bilayers with other FO subunits to assess proton translocation (Fig. 1).
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Single-molecule rotation assays: Compare rotational speeds of chimeric ATP synthases (e.g., B. quintana atpB + Geobacillus stearothermophilus F1 sector) to isolate subunit-specific contributions .
Table 1: Functional Comparison of Recombinant atpB Expressed in Different Systems
What structural biology approaches are prioritized for resolving conformational dynamics of recombinant atpB?
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Cryo-EM single-particle analysis: Resolve atpB’s role in FO rotary mechanics at 3.5–4.0 Å resolution, focusing on helix-helix interactions in the membrane domain .
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Cross-linking mass spectrometry (XL-MS): Identify proximity between atpB and subunits like atpE using DSSO or BS3 cross-linkers.
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Molecular dynamics simulations: Model proton translocation pathways using templates like Mycobacterium smegmatis F-ATP synthase (PDB: 6RZY) .
Case study: Deleting the C-terminal domain of M. tuberculosis atpA increased ATPase activity by 64% , suggesting analogous truncations in atpB could alter proton coupling.
How do researchers validate the physiological relevance of recombinant atpB in non-native ATP synthases?
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Genetic complementation: Introduce recombinant atpB into ATP synthase-deficient E. coli strains (e.g., DK8 ΔatpB) and assess growth on non-fermentable carbon sources .
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Isothermal titration calorimetry (ITC): Measure binding affinity between atpB and subunit a (atp6) to confirm assembly fidelity (expected Kd: 10–50 nM for functional complexes).
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pH-dependent activity profiling: Compare proton-pumping rates at pH 4.5–8.0 to detect anomalies in H+ gating .
What analytical frameworks are used to reconcile conflicting data on atpB’s role in drug resistance?
Contradictions often stem from:
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Species-specific subunit interactions: B. quintana atpB may lack residues critical for bedaquiline binding (e.g., M. tuberculosis atpB Glu61 and Asp28 ).
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In vitro vs. in vivo models: Membrane potential in cell-free assays may not replicate intracellular conditions.
Resolution strategies:
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Alanine scanning mutagenesis: Systematically replace putative drug-binding residues (e.g., polar residues in transmembrane helices).
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Minimum inhibitory concentration (MIC) assays: Compare bedaquiline sensitivity in strains expressing wild-type vs. mutant atpB (Table 2).
Table 2: Bedaquiline Sensitivity in M. smegmatis Expressing atpB Variants
| atpB Variant | MIC (μg/mL) | ATP Synthesis Inhibition (%) |
|---|---|---|
| Wild-type | 0.03 | 92 ± 3 |
| D28A | 0.12 | 45 ± 7 |
| E61A | 0.09 | 58 ± 5 |
| ΔC-terminal (514–548) | 0.04 | 88 ± 4 |
| Data adapted from M. tuberculosis F-ATP synthase studies . |
Which biophysical assays are optimal for quantifying atpB’s role in proton translocation efficiency?
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Fluorescent pH-sensitive dyes: Monitor intra-proteoliposome acidification using ACMA quenching (detection limit: ΔpH 0.1).
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Electrophysiology: Measure single-channel proton currents across atpB-containing lipid bilayers (expected conductance: 5–10 fS ).
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Solid-state NMR: Track conformational changes in atpB’s transmembrane helices during ATP-driven rotation .
Data interpretation: A 10% reduction in H+ pumping in M. smegmatis Δα(514–548) suggests analogous mutations in atpB could decouple synthesis from translocation.
How can researchers address insolubility of recombinant atpB in E. coli?
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Membrane scaffolding proteins: Co-express atpB with E. coli MspA to stabilize membrane protein folding.
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Detergent screening: Test n-dodecyl-β-D-maltopyranoside (DDM) vs. lauryl maltose neopentyl glycol (LMNG) for extraction efficiency (LMNG improves stability by 30% ).
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Chaperone co-expression: Use TF/ GroELS systems to reduce aggregation, increasing soluble yield from 40% to 65% .
What metrics define successful functional reconstitution of atpB into hybrid ATP synthases?
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Coupling efficiency: Ratio of ATP synthesis to hydrolysis activity ≥2.0 indicates proper energy transduction .
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Rotational speed consistency: Single-molecule assays should show 120° stepping at 37°C, matching native bacterial F-ATP synthases .
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Proton-to-ATP stoichiometry: 4 H+/ATP in reconstituted systems vs. 3.3–3.6 in native complexes .
How do post-translational modifications of atpB in eukaryotic systems impact functional studies?
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Phosphorylation: HEK293-expressed atpB shows serine phosphorylation at S112 and S208, reducing proton conductivity by 20% .
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Glycosylation: S. cerevisiae-derived atpB exhibits N-linked glycans at N95, requiring PNGase F treatment for accurate activity assays .
Mitigation: Use CRISPR-edited cell lines (e.g., HEK293 GT1–/–) to eliminate unwanted glycosylation.
What computational tools predict atpB residues critical for intersubunit interactions?
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AlphaFold2 Multimer: Predicts atpB-atpE interface with 85% accuracy (pLDDT >70).
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HADDOCK: Dock atpB’s C-terminal helix (residues 210–240) into F1 sector subunit δ using cryo-EM constraints.
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Conservation scoring: ConSurf identifies evolutionarily invariant residues (e.g., atpB Arg76) as non-negotiable for rotary mechanics .
