Recombinant Cyanothece sp. ATP synthase subunit b 2 (atpF2)
Description
Introduction to Recombinant Cyanothece sp. ATP Synthase Subunit b 2 (atpF2)
Recombinant Cyanothece sp. ATP synthase subunit b 2 (atpF2) is a protein component of the ATP synthase complex, which plays a crucial role in the production of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate. This process is driven by a proton electrochemical gradient across the thylakoid membranes in photosynthetic organisms like cyanobacteria. The atpF2 subunit is part of the F0 sector of the ATP synthase, which is embedded in the membrane and acts as a proton channel.
Structure and Function
The ATP synthase complex consists of two main parts: the F1 sector, which is the catalytic core responsible for ATP synthesis, and the F0 sector, which is the membrane-bound proton channel. The atpF2 subunit is integral to the F0 sector, facilitating the translocation of protons across the membrane, which drives the rotation of the stalk subunits and ultimately leads to ATP synthesis in the F1 sector.
| Component | Function | Location |
|---|---|---|
| F1 Sector | Catalytic core for ATP synthesis | Soluble portion |
| F0 Sector | Proton channel | Membrane-bound |
| atpF2 Subunit | Part of F0 sector, involved in proton translocation | Membrane-bound |
Recombinant Expression
Recombinant expression of the atpF2 subunit involves the use of a host organism, typically Escherichia coli (E. coli), to produce the protein. This is achieved by inserting the gene encoding the atpF2 subunit into an expression vector, which is then introduced into E. coli. The expressed protein is often tagged with a His-tag to facilitate purification.
| Expression Details | Description |
|---|---|
| Host Organism | Escherichia coli (E. coli) |
| Tag | N-terminal His-tag |
| Protein Length | Full-length (typically around 175 amino acids) |
| Form | Lyophilized powder |
| Purity | Greater than 90% as determined by SDS-PAGE |
Research Findings
Research on ATP synthase subunits, including atpF2, has highlighted their importance in photosynthetic organisms. In cyanobacteria, ATP synthase activity is regulated by a small protein known as AtpΘ, unlike in chloroplasts where redox regulation plays a role . The efficiency of ATP synthesis can vary based on the number of c subunits in the c ring, affecting the ATP/NADPH ratio in photosynthesis .
Significance and Applications
Understanding the structure and function of ATP synthase subunits like atpF2 is crucial for improving photosynthetic efficiency and for developing biotechnological applications. Enhancing ATP synthase activity can lead to increased biomass production and stress tolerance in photosynthetic organisms . Additionally, insights into ATP synthase regulation can inform strategies for improving crop resilience and productivity.
Product Specs
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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, containing the membrane proton channel. These domains are linked by a central and a peripheral stalk. ATP synthesis in the F1 catalytic domain is coupled to proton translocation via the rotary mechanism of the central stalk subunits.
This protein is a component of the F0 channel, forming part of the peripheral stalk and linking F1 to F0.
KEGG: cyt:cce_4486
STRING: 43989.cce_4486
Q&A
What experimental approaches are recommended for studying the functional role of atpF2 in ATP synthase activity?
To elucidate the role of atpF2 in ATP synthase, researchers should prioritize biochemical assays alongside complementary genetic and biophysical methods. Key strategies include:
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Recombinant protein expression: Use heterologous systems (e.g., E. coli or Synechocystis) to produce atpF2, ensuring proper folding and membrane integration. Validate via Western blot or ELISA (e.g., analogous to Cyanothece sp. ATP synthase subunit a 2 assays ).
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ATPase activity assays: Measure ATP hydrolysis rates in presence/absence of atpF2, using coupled spectrophotometric assays (e.g., NADH oxidation or malate dehydrogenase coupling). Compare with wild-type ATP synthase complexes .
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Co-immunoprecipitation (Co-IP): Identify interactions between atpF2 and other subunits (e.g., subunit a, γ, or ε) or regulators like AtpQ . This can reveal structural dependencies or regulatory mechanisms.
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In vivo complementation: In knockout mutants of Cyanothece, assess growth defects and ATP synthase activity restoration upon reintroducing atpF2.
Data Table 1: Experimental Approaches for atpF2 Functional Analysis
| Method | Purpose | Key Controls/Parameters |
|---|---|---|
| Recombinant expression | Validate protein production | Western blot, ELISA, tag analysis |
| ATPase activity assay | Quantify ATP hydrolysis rates | Substrate (ATP) concentration, pH |
| Co-IP | Map subunit interactions | Antibody specificity, crosslinkers |
| In vivo complementation | Assess physiological relevance | Growth media, light/dark cycles |
How do subunit-specific regulatory mechanisms differ between cyanobacterial ATP synthases and other organisms?
Cyanobacteria employ unique regulatory strategies for ATP synthase, distinct from chloroplast or mitochondrial systems:
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AtpQ (Norf1): A small inhibitor protein encoded by atpT that binds ATP synthase to block ATP hydrolysis under low-energy conditions (e.g., darkness) . Unlike chloroplast γ-subunit redox regulation, AtpQ operates via direct steric hindrance.
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Subunit-specific inhibition: ADP and ε-subunit-mediated inhibition are conserved, but cyanobacteria lack redox-sensitive γ-subunit cysteine residues found in plants .
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Metabolic coupling: ATP synthase activity in Cyanothece strains is linked to nitrogenase-driven H₂ production, with PS II-derived ATP supporting nitrogenase function under nitrogen-limiting conditions .
Key Contrasts in Regulation
| Feature | Cyanobacteria | Chloroplasts |
|---|---|---|
| Hydrolysis inhibition | AtpQ binding, ADP/ε-subunits | Redox-sensitive γ-subunit |
| ATP/ADP sensing | γ-subunit-mediated | γ-subunit-mediated |
| Metabolic integration | PS II ↔ nitrogenase coupling | Cyclic vs. linear electron flow |
What challenges arise when interpreting multi-omic data for ATP synthase subunits in nitrogen-fixing cyanobacteria?
Integrating transcriptomic, proteomic, and metabolomic data for ATP synthase subunits in Cyanothece requires addressing:
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Temporal resolution: Nitrogenase activity and ATP synthase expression exhibit diurnal rhythms, necessitating time-resolved sampling (e.g., 12-h light-dark cycles) .
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Functional redundancy: Subunits like a2 (atpB2) and b2 (atpF2) may have overlapping roles, complicating interpretation of knockout phenotypes .
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Cross-pathway regulation: Glycogen breakdown (via glycogen phosphorylase) and PS II activity modulate ATP availability, indirectly affecting ATP synthase function .
Example Data Integration Workflow
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Transcriptomics: Quantify atpF2 mRNA levels during light/dark transitions.
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Proteomics: Measure atpF2 protein abundance via LC-MS/MS.
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Metabolomics: Track ATP/ADP ratios and glycogen levels.
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Bioinformatics: Use correlation networks to link atpF2 expression with nitrogenase (nif genes) or Calvin cycle enzymes .
How can researchers validate the authenticity of recombinant atpF2 protein?
Authentication requires multimodal validation:
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Primary structure:
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Mass spectrometry: Confirm peptide sequences via tryptic digestion and LC-MS.
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Edman degradation: Determine N-terminal sequence.
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Secondary/tertiary structure:
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Circular dichroism (CD): Assess α-helical/β-sheet content.
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Thermal shift assays: Measure denaturation temperatures.
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Functional validation:
Validation Workflow
| Step | Technique | Expected Outcome |
|---|---|---|
| Sequence confirmation | LC-MS, Edman sequencing | 100% match to predicted sequence |
| Structural analysis | CD, thermal shift assay | Consistent with membrane proteins |
| Functional testing | ATPase assays, Co-IP | Activity restoration, binding partners |
What unresolved questions persist in studying ATP synthase subunit dynamics in cyanobacteria?
Key gaps include:
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Structural basis of regulation: High-resolution cryo-EM structures of AtpQ-bound ATP synthase are lacking.
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Interplay with respiratory electron transport: How do TCA cycle-derived proton gradients influence ATP synthase activity in the dark?
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Evolutionary trade-offs: Why do cyanobacteria maintain distinct regulatory mechanisms compared to chloroplasts?
Hypothesis-Driven Research Opportunities
| Question | Proposed Approach |
|---|---|
| AtpQ binding site on ATP synthase | Cryo-EM of AtpQ-ATP synthase complexes |
| Respiratory ATP synthase activity | Real-time ATP/ADP monitoring in dark-grown cells |
| Evolution of AtpQ vs. γ-subunit redox | Phylogenetic analysis of ATP synthase regulators |
How does ATP synthase subunit expression correlate with nitrogenase activity in Cyanothece?
In Cyanothece sp. ATCC 51142, nitrogenase expression is tightly coupled to ATP synthase activity through:
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Light-dependent ATP synthesis: PS II-driven linear electron flow generates ATP for nitrogenase .
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Glycogen catabolism: In presence of glycerol, glycogen breakdown via glycogen phosphorylase provides reducing equivalents and ATP in the light, decoupling nitrogenase from dark-phase respiration .
Expression Correlation Data
| Condition | Nitrogenase Activity | ATP Synthase Subunit Expression |
|---|---|---|
| Nitrogen-limiting | High (dark period) | ↑ PS II subunits, ↓ PS I |
| Glycerol supplementation | High (light period) | ↑ Glycolytic enzymes, ↓ Calvin cycle |
What methodologies enable comparative analysis of ATP synthase subunits across cyanobacterial strains?
Comparative studies benefit from:
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Quantitative proteomics: LC-MS/MS-based label-free quantification of subunits in Cyanothece ATCC 51142 vs. PCC 7822 .
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Phylogenetic analysis: Align atpF2 sequences from diverse cyanobacteria to identify conserved motifs or lineage-specific adaptations.
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Biochemical profiling: Measure ATP synthase activity, proton translocation efficiency, and inhibitor sensitivity (e.g., AtpQ) across strains.
Strain-Specific Differences
| Strain | Nitrogenase Regulation | ATP Synthase Dependency |
|---|---|---|
| Cyanothece ATCC 51142 | Glycogen-dependent | PS II-coupled |
| Cyanothece PCC 7822 | Calvin cycle-supported | Mixed energy sources |
How can researchers resolve contradictions in ATP synthase subunit function inferred from omics vs. biochemical data?
Contradictions often arise from:
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Post-translational modifications: Proteomic data may miss phosphorylation or acetylation affecting subunit activity.
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Subcellular localization: Confocal microscopy or fractionation can clarify thylakoid vs. cytoplasmic ATP synthase pools.
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Experimental conditions: In vitro assays may not replicate in vivo proton gradient dynamics.
