Recombinant Helianthus annuus ATP synthase subunit c, chloroplastic (atpH)

How does the c-ring stoichiometry affect ATP synthesis efficiency?

The c-ring stoichiometry (number of c subunits forming the ring) directly determines the ion-to-ATP ratio, which is a critical bioenergetic parameter defining how many protons are required to produce one ATP molecule. Different organisms have evolved different c-ring stoichiometries, ranging from c₈ in animal mitochondria to c₁₅ in cyanobacteria like Spirulina platensis, with some bacteria like Burkholderia pseudomallei having as many as c₁₇ subunits .

In plant chloroplasts with a c₁₄-ring (such as found naturally in tobacco), 14 protons are required for one complete rotation of the c-ring, producing 3 molecules of ATP (ion-to-ATP ratio = 4.6). Given that linear photosynthetic electron transport translocates 12 protons across the thylakoid membrane per 2 molecules of NADPH, this results in an ATP/NADPH ratio of only 1.29, which is insufficient to meet the ATP demands of the Calvin-Benson cycle . Plants compensate for this deficiency through cyclic electron transport around photosystem I, which generates additional proton motive force without net NADPH production .

What conserved structural motifs are critical for c-ring assembly and function?

The c-subunit contains a series of conserved glycine repeats (the GxGxGxG motif) located in the N-terminal α-helix of the c-subunit hairpin. This motif establishes very tight α-helical packing within the c-ring structure, which is essential for its stability and proper function .

In angiosperms including sunflower, the sequence surrounding the glycine repeats is highly conserved: GLAVGLASIGPGVGQGT. Modifications to this sequence can alter the c-ring stoichiometry, as demonstrated in experimental studies where changing this sequence to match that of Spirulina platensis (aLAVGigSIGPGlGQGq) resulted in a c₁₅-ring instead of the native c₁₄-ring .

What are the most effective methods for isolating and purifying recombinant Helianthus annuus ATP synthase subunit c for structural studies?

For effective isolation and purification of recombinant Helianthus annuus ATP synthase subunit c, researchers should employ a comprehensive approach:

• Expression System Selection: While bacterial expression systems like E. coli can be used, eukaryotic expression systems may provide better post-translational modifications. For plant proteins, many researchers use specialized suppliers such as CUSABIO TECHNOLOGY LLC, which has expertise in recombinant plant protein production .

• Purification Protocol:

  • Employ affinity chromatography using His-tags or other fusion tags

  • Perform ion-exchange chromatography to separate based on charge differences

  • Use size-exclusion chromatography for final purification

• C-ring Isolation: For specific c-ring isolation, researchers can follow protocols similar to those used for tobacco ATP synthase, which involve:

  • Membrane solubilization with detergents

  • Density gradient centrifugation

  • Blue native PAGE for isolation of intact c-rings

• Stability Verification: Test the stability of isolated c-rings by treating samples with trichloroacetic acid (TCA), which dissociates the c-rings into monomeric c subunits. This approach allows confirmation that the isolated proteins are indeed c-rings rather than unrelated proteins .

How can I design chloroplast transformation experiments to modify the c-ring stoichiometry in Helianthus annuus?

Based on successful experiments with tobacco, the following approach can be adapted for Helianthus annuus:

• Vector Design Strategy:

  • Identify the plastid atpH gene within the atpI-H-F-A operon in H. annuus

  • Design a vector containing:

    • Homologous recombination regions flanking the atpH gene

    • Selectable marker cassette (e.g., aadA for spectinomycin resistance)

    • Modified atpH gene with targeted mutations in the glycine-repeat motif

• Target Sequence Modification:

  • To increase c-ring stoichiometry, modify codons in the glycine-repeat region to match sequences from organisms with larger c-rings

  • For a c₁₅-ring, use the Spirulina platensis sequence (aLAVGigSIGPGlGQGq) as a template

  • For control experiments, insert only the selection marker without modifying atpH

• Transformation Protocol:

  • Use biolistic transformation for chloroplast targeting

  • Select transformants on spectinomycin-containing media

  • Confirm homoplasmy through multiple rounds of selection

  • Verify transformation by PCR and sequencing

• Considerations:

  • Not all sequence modifications will produce viable plants, as observed in tobacco-SAG experiments where plants were albino and non-viable

  • Include multiple controls, including wild-type and vector-only transformants

What methodologies are effective for characterizing c-ring stoichiometry in transformed plants?

To accurately determine c-ring stoichiometry in transformed plants, employ these complementary approaches:

• Blue Native PAGE Analysis:

  • Isolate thylakoid membranes from transformed plants

  • Solubilize membranes with mild detergents

  • Separate protein complexes by blue native PAGE

  • Use the established correlation between electrophoretic mobility and c-ring stoichiometry

  • Compare mobility with reference samples of known stoichiometry (e.g., from Spirulina platensis for c₁₅)

• TCA Dissociation Test:

  • Treat isolated c-ring samples with trichloroacetic acid

  • Confirm dissociation into monomeric c subunits

  • Verify that the monomers match the expected molecular weight

• Mass Spectrometry:

  • Analyze intact c-rings by mass spectrometry

  • Calculate total mass to determine the number of subunits

• Electron Microscopy:

  • Use cryo-electron microscopy for direct visualization

  • Count individual c-subunits within the ring structure

Research has demonstrated that c-ring stability may differ between wild-type and modified versions, with the non-native c₁₅-ring showing less resistance to SDS solubilization and signs of complex degradation . This indicates the importance of assessing both stoichiometry and stability.

What methods are most effective for measuring changes in proton motive force (pmf) composition in plants with modified ATP synthase?

To accurately assess changes in pmf composition in plants with modified ATP synthase, researchers should employ these specialized techniques:

• Electrochromic Shift (ECS) Measurements:

  • Monitor the absorbance changes of photosynthetic pigments that respond to the electric field across the thylakoid membrane

  • Use ECS to track both the generation and relaxation of the electric field component of pmf

  • The fast relaxation kinetics of ECS after a light-to-dark transition directly reflects ATP synthase activity

  • Compare relaxation half-times between wild-type and modified plants

• ΔpH Assessment:

  • Use pH-dependent fluorescent probes to estimate lumen pH

  • Analyze NPQ parameters as indirect indicators of ΔpH

  • Measure violaxanthin de-epoxidation state as a biochemical indicator of lumen acidification

• Combined pmf Quantification:

  • Total pmf can be estimated from the total amplitude of the ECS signal

  • The relative contributions of ΔpH and Δψ can be determined through inhibitor treatments (e.g., nigericin to collapse ΔpH)

  • Alternative methods include monitoring P515 absorption changes

• Practical Considerations:

  • Ensure consistent leaf development stage and pre-measurement dark adaptation

  • Control environmental conditions during measurements

  • Use multiple technical and biological replicates

  • Perform measurements under various light intensities to assess light-dependent responses

These methods have revealed that plants with c₁₅-rings show faster relaxation of the thylakoid membrane pmf compared to wild-type plants, indicating compensatory mechanisms that maintain photosynthetic electron transport despite the altered stoichiometry .

How can engineering ATP synthase c-ring stoichiometry be leveraged to enhance crop productivity under varying environmental conditions?

Engineering ATP synthase c-ring stoichiometry represents a promising approach for enhancing crop productivity through several mechanisms:

• Drought Tolerance Optimization:

  • Reduced c-ring stoichiometry could improve water-use efficiency by enhancing ATP production per proton

  • This may be particularly advantageous under water-limited conditions where maintaining photosynthetic efficiency with reduced stomatal conductance is crucial

• Temperature Stress Adaptation:

  • Different c-ring stoichiometries may provide optimal ATP synthesis at different temperatures

  • Engineering temperature-specific variants could enhance crop resilience to climate change

• Light Utilization Efficiency:

  • Modified c-rings could alter the balance between photochemical and non-photochemical processes

  • Optimizing this balance for specific light environments (high light vs. shade conditions) may increase net carbon assimilation

• Experimental Approach:

  • Create a library of c-ring variants with different stoichiometries

  • Screen these variants under diverse environmental conditions

  • Combine physiological measurements with -omics approaches to understand whole-plant responses

• Considerations and Challenges:

  • Maintain the stability of engineered c-rings for consistent expression levels

  • Ensure compatibility with other components of the photosynthetic machinery

  • Address potential tradeoffs between optimizing for different environmental stressors

Research on tobacco has demonstrated that plants can adapt to significant changes in ATP synthase composition and activity through adjustments in the components of pmf, suggesting considerable flexibility in the photosynthetic apparatus that could be exploited for crop improvement .

What are the current methodological challenges in studying the protein-protein interactions between atpH and other ATP synthase subunits?

Investigating protein-protein interactions between ATP synthase subunit c (atpH) and other components of the complex presents several methodological challenges:

• Membrane Protein Solubilization:

  • ATP synthase is an integral membrane complex, making it difficult to solubilize while maintaining native interactions

  • Challenge: Finding detergents that preserve structural integrity without disrupting critical interactions

  • Solution: Screen multiple detergents and nanodisc systems for optimal solubilization conditions

• C-ring Stability Issues:

  • Modified c-rings often show decreased stability compared to wild-type versions

  • Challenge: Distinguishing between native interactions and artifacts from destabilized complexes

  • Solution: Use multiple complementary techniques and compare with wild-type controls

• Stoichiometry Determination:

  • Different ATP synthase components have distinct stoichiometries

  • Challenge: Accurately quantifying subunit ratios and interaction affinities

  • Solution: Combine structural methods (cryo-EM) with quantitative proteomics and biophysical techniques

• Chloroplast-Specific Interactions:

  • Plant ATP synthases have unique regulatory interactions not present in bacterial or mitochondrial counterparts

  • Challenge: Identifying plant-specific interaction partners and regulatory mechanisms

  • Solution: Comparative interactomics between different organisms and organellar ATP synthases

• Technical Approaches:

  • Cross-linking mass spectrometry to capture transient interactions

  • Surface plasmon resonance for measuring binding kinetics

  • FRET-based assays for monitoring interactions in reconstituted systems

  • Native mass spectrometry for intact complex analysis

Current research suggests that the stability of the ATP synthase complex is significantly affected by c-ring modifications, suggesting important interaction interfaces between the c-ring and other subunits that require further characterization .

How do chloroplast ATP synthase c-ring stoichiometries compare across different plant species, and what are the evolutionary implications?

The comparative analysis of ATP synthase c-ring stoichiometries across plant species reveals important evolutionary patterns:

• Known Stoichiometry Distribution:

  • Most angiosperms studied to date (including tobacco) possess c₁₄-rings in their chloroplast ATP synthase

  • Cyanobacteria show greater diversity, with species like Spirulina platensis having c₁₅-rings

  • This suggests evolutionary divergence in ATP synthesis efficiency after endosymbiosis

• Sequence Conservation and Divergence:

  • The glycine-repeat motif (GxGxGxG) is highly conserved across plant species

  • In angiosperms, the sequence surrounding these repeats (GLAVGLASIGPGVGQGT) is remarkably conserved

  • Specific residues within this region have diverged in cyanobacteria with different c-ring stoichiometries

• Methodological Approaches for Comparative Studies:

  • Genome mining to identify atpH sequences across plant species

  • Structural prediction to model potential c-ring stoichiometries

  • Experimental validation through isolation and characterization of c-rings

  • Phylogenetic analysis to correlate stoichiometry with evolutionary relationships

• Bioenergetic Adaptation Hypotheses:

  • C-ring stoichiometry may reflect adaptation to specific environmental niches

  • Larger c-rings (requiring more protons per ATP) may be advantageous in high-light environments

  • Smaller c-rings may provide a competitive advantage in low-light or energy-limited conditions

The experimental success in engineering tobacco to express a c₁₅-ring suggests that c-ring stoichiometry is determined primarily by protein sequence rather than cellular environment, opening possibilities for understanding the evolutionary pressures that shaped ATP synthase diversity .

What quality control measures should be implemented when working with recombinant Helianthus annuus ATP synthase subunit c?

Implementing rigorous quality control for recombinant Helianthus annuus ATP synthase subunit c requires a multi-faceted approach:

• Expression Verification:

  • Western blotting with specific antibodies against ATP synthase subunit c

  • Mass spectrometry confirmation of protein identity and sequence

  • SDS-PAGE analysis to verify protein size and purity

• Functional Assessment:

  • Blue native PAGE to verify assembly into proper c-ring structures

  • TCA dissociation test to confirm c-ring integrity and stability

  • Reconstitution assays to test proton translocation activity

• Structural Integrity:

  • Circular dichroism spectroscopy to assess secondary structure content

  • Thermal stability assays to determine melting temperature

  • Limited proteolysis to evaluate proper folding

• Contamination Testing:

  • Endotoxin testing for proteins expressed in bacterial systems

  • Nucleic acid contamination assessment

  • Host cell protein analysis for recombinant expression systems

• Storage Stability:

  • Monitor protein stability under various storage conditions

  • Test freeze-thaw stability if applicable

  • Establish shelf-life parameters based on activity retention

When sourcing commercially produced recombinant proteins, researchers should request detailed quality control documentation from suppliers such as CUSABIO TECHNOLOGY LLC , including certificates of analysis with specifications for purity, activity, and identity confirmation methods.

What are the most effective strategies for optimizing chloroplast transformation efficiency in studies of ATP synthase subunit c?

Optimizing chloroplast transformation for ATP synthase subunit c studies requires specialized approaches:

• Vector Design Optimization:

  • Include homologous recombination sequences specific to the target region in the plastid genome

  • Position the selection marker (e.g., aadA cassette) to minimize impact on downstream gene expression

  • For atpH modifications, consider the operon structure (atpI-H-F-A) and potential polar effects

• Transformation Method Refinement:

  • Biolistic delivery parameters: optimize gold particle size, helium pressure, and target distance

  • Tissue preparation: use young, actively growing tissue for transformation

  • Recovery media: include osmotic support and appropriate hormones

• Selection Strategy:

  • Apply multiple rounds of selection on spectinomycin-containing media to achieve homoplasmy

  • Use alternating antibiotic selection if applicable

  • Consider shoot regeneration from primary transformants rather than seedling selection

• Homoplasmy Verification:

  • PCR-based assays to detect wild-type versus transformed plastid genomes

  • Southern blotting to confirm complete replacement of wild-type plastid DNA

  • Seed tests to verify maternal inheritance and stable transformation

• Efficiency Metrics:

  • Track transformation efficiency (transformants per bombardment)

  • Monitor time to homoplasmy achievement

  • Assess phenotypic stability across generations

• Considerations Specific to ATP Synthase:

  • ATP synthase is essential for photosynthesis, so complete knockout mutations may be lethal or result in albino phenotypes, as observed in ΔatpH tobacco plants

  • When introducing modified sequences, consider designing multiple variants with varying degrees of modification