Recombinant ATP synthase subunit b, organellar chromatophore (atpF)
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Target Background
F1F0 ATP synthase synthesizes ATP from ADP using a proton or sodium gradient. This enzyme comprises two domains: F1, containing the extramembranous catalytic core, and F0, containing the membrane proton channel. These domains are linked by a central and peripheral stalk. ATP synthesis in the F1 catalytic domain is coupled, via a rotary mechanism of the central stalk subunits, to proton translocation. This protein is a component of the F0 channel, forming part of the peripheral stalk which connects F1 and F0.
Q&A
What is the role of ATP synthase subunit b (atpF) in organellar chromatophores?
ATP synthase subunit b (atpF) is a critical component of the F₀ sector in ATP synthase, anchoring the F₁ catalytic head to the membrane-embedded rotor. In bacterial chromatophores (e.g., Rhodobacter sphaeroides), atpF facilitates proton translocation across the membrane, coupling proton motive force to ATP synthesis . Unlike subunit c, which forms the rotating rotor ring, subunit b stabilizes the stator structure and ensures efficient energy transduction. Researchers studying atpF often focus on its interactions with other subunits (e.g., subunit a) and lipid environments to elucidate its role in maintaining proton channel integrity .
Table 1: Key Functional Attributes of atpF
What are the standard methodologies for recombinant atpF purification?
Recombinant atpF purification typically involves heterologous expression in E. coli or yeast systems, followed by affinity chromatography and detergent solubilization. Key steps include:
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Vector design: Use pET or pGEX vectors with His-tags for IMAC purification .
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Membrane extraction: Isolate atpF-containing membranes via ultracentrifugation (100,000 × g, 1 hour).
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Detergent screening: Test n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) for optimal solubilization .
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Chromatography: Apply size-exclusion chromatography (SEC) to remove aggregates and isolate monodisperse protein .
Critical quality checks include SDS-PAGE for purity (>95%), circular dichroism for secondary structure integrity, and functional assays measuring proton translocation rates .
How can structural characteristics of atpF be analyzed experimentally?
High-resolution techniques such as atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) are pivotal. For example, AFM imaging of intact chromatophores revealed atpF’s spatial organization relative to LH2 and cyt bc₁ complexes . To minimize artifacts:
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Sample preparation: Use gentle detergent-free buffers to preserve native curvature .
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Imaging parameters: Optimize tapping mode AFM with soft cantilevers (0.1 N/m spring constant) to reduce tip-sample forces to <50 pN .
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Data validation: Cross-reference AFM topographs with crystallographic data to confirm subunit orientations .
How should researchers design assays to quantify atpF’s proton translocation efficiency?
Proton translocation assays require reconstituting atpF into proteoliposomes and measuring pH gradients using fluorescent probes (e.g., ACMA). A validated protocol includes:
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Proteoliposome preparation: Mix purified atpF with E. coli polar lipids (3:1 ratio) and dialyze to remove detergents .
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pH gradient initiation: Add ascorbate/TMPD to generate a proton motive force.
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Fluorescence quenching: Monitor ACMA fluorescence decay (excitation 410 nm, emission 490 nm) upon ATP addition .
Table 2: Common Pitfalls in Proton Translocation Assays
| Issue | Solution | Citation |
|---|---|---|
| Dye leakage | Use larger liposomes (200 nm diameter) | |
| Non-specific binding | Include control liposomes without atpF | |
| Signal drift | Calibrate pH electrodes every 15 minutes |
What strategies resolve contradictions in experimental data on atpF’s oligomeric state?
Data inconsistencies often arise from variations in detergent use or crystallization conditions. A systematic approach involves:
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Cross-validation: Compare SEC-MALS (multi-angle light scattering) and analytical ultracentrifugation data .
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Discretization criteria: Apply consistent thresholds for classifying oligomers (e.g., ±5% mass tolerance) .
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Outlier analysis: Use decision trees to identify methodological outliers (e.g., improper buffer pH) .
For example, rough set theory (RST) successfully classified 85% of contradictory atpF oligomerization data by evaluating attribute dependencies in discretized datasets .
How can atpF be integrated into chromatophore structural models?
Integrating atpF into chromatophore vesicle models requires combining AFM, crystallography, and stoichiometric data:
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Stoichiometric mapping: Assign 2 ATP synthases per vesicle based on mass spectrometry .
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Orientation constraints: Align atpF’s stator domain perpendicular to the membrane using AFM height profiles .
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Energy landscape modeling: Simulate proton paths using GROMACS or NAMD to validate electrostatic interactions .
What evolutionary insights can be gained from comparative studies of atpF homologs?
Phylogenetic analysis of atpF across phototrophic bacteria reveals conserved glycine-rich motifs critical for stator flexibility. Researchers should:
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Sequence alignment: Use Clustal Omega to identify conserved residues (e.g., GxGxG motifs).
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Functional convergence testing: Express homologs in atpF-knockout strains and measure growth rates under varying light intensities .
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Positive selection analysis: Apply PAML’s site models to detect diversifying selection in residues facing the lipid bilayer .
