Recombinant Aethionema grandiflora ATP synthase subunit c, chloroplastic (atpH)

What is the atpH gene and what does it encode in Aethionema grandiflora?

The atpH gene in Aethionema grandiflora encodes the ATP synthase subunit c, which is a critical component of the chloroplastic ATP synthase complex. This protein is located in the F₀ sector of the ATP synthase and functions as part of the proton channel that facilitates ATP synthesis. The protein has several alternative names including ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, and lipid-binding protein . The atpH-encoded protein spans 81 amino acids with the sequence MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV and is identified in the UniProt database with accession number A4QJI6 .

Where is the atpH gene located in the chloroplast genome of Brassicaceae plants?

The atpH gene is located in the plastid genome of Brassicaceae species, including Aethionema grandiflora. In land plants, the genes encoding ATP synthase subunits are arranged into two plastid operons . While some ATP synthase subunits (γ, δ, and b') are encoded by the nuclear genome, atpH along with genes encoding other subunits (α, β, ε, a, b, c) are part of the plastid genetic system . This gene arrangement is evolutionarily significant and has been conserved even in some non-photosynthetic organisms that have otherwise reduced plastid genomes, highlighting the fundamental importance of ATP synthase components .

How is the structure of ATP synthase subunit c related to its function in energy metabolism?

ATP synthase subunit c functions as a critical component of the F₀ portion of the ATP synthase complex. Structurally, it forms a ring in the membrane that rotates as protons pass through, driving conformational changes in the F₁ portion that catalyze ATP synthesis. The amino acid sequence of Aethionema grandiflora ATP synthase subunit c contains hydrophobic regions that anchor it within the thylakoid membrane, allowing it to participate in proton translocation . These structural features enable the protein to convert the energy of the proton gradient established during photosynthesis into mechanical rotation, ultimately powering the synthesis of ATP. The specific amino acid residues, particularly those forming transmembrane helices, are crucial for proper proton channeling and are generally highly conserved across species, reflecting their essential role in the molecular mechanism of energy transduction .

What are the optimal storage conditions for recombinant Aethionema grandiflora ATP synthase subunit c protein?

For recombinant Aethionema grandiflora ATP synthase subunit c protein, the optimal storage conditions include keeping the protein at -20°C for general storage, or at -80°C for extended preservation . The protein is typically stored in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein's stability . Working aliquots may be stored at 4°C for up to one week to minimize freeze-thaw cycles, as repeated freezing and thawing is not recommended and can compromise protein integrity and functionality . Researchers should prepare small aliquots upon initial thawing to prevent degradation from multiple freeze-thaw cycles, particularly when planning long-term experimental schedules.

What expression systems are most effective for producing recombinant Aethionema grandiflora atpH protein?

While the search results don't provide specific information about expression systems for Aethionema grandiflora atpH protein, general approaches for chloroplast proteins can be inferred. Bacterial expression systems, particularly E. coli, are commonly used for producing recombinant chloroplast proteins due to their simplicity and high yield. For ATP synthase subunit c, which is a membrane protein, specialized expression systems that facilitate proper membrane protein folding may be necessary. This could include E. coli strains optimized for membrane protein expression or eukaryotic systems like yeast or insect cells. The expression region for the Aethionema grandiflora atpH protein spans amino acids 1-81, representing the full-length protein , which should be considered when designing expression constructs. Tag selection should be carefully evaluated as it can affect protein folding, with the specific tag type typically determined during the production process to ensure optimal protein functionality .

What purification methods yield the highest activity for recombinant ATP synthase subunit c proteins?

Purification of recombinant ATP synthase subunit c proteins requires specialized approaches due to their hydrophobic nature and membrane association. While specific methods for Aethionema grandiflora atpH are not detailed in the search results, effective protocols generally involve:

• Initial extraction using detergent solubilization to release the protein from membranes

• Affinity chromatography utilizing the recombinant protein's tag

• Size exclusion chromatography to separate properly folded protein from aggregates

• Ion exchange chromatography for further purification

For functional studies, it's critical to monitor protein activity throughout purification to ensure the methods preserve the native structure. Research on chloroplast ATP synthase has shown that when reconstituting the catalytic core from subunits α, β, and γ, combining chaperone systems like CPN60 and HSP70 with assembly factors has achieved up to 95% of native ATP synthase activity . This suggests that proper folding and assembly factors are crucial considerations when purifying individual ATP synthase subunits for functional studies.

How can recombinant atpH be used in studies of ATP synthase biogenesis and assembly?

Recombinant Aethionema grandiflora atpH protein can serve as a valuable tool for investigating ATP synthase biogenesis and assembly mechanisms. Researchers can use the purified protein in reconstitution assays to study the stepwise assembly of the ATP synthase complex, particularly the integration of subunit c into the F₀ sector. The recombinant protein allows for controlled experiments examining protein-protein interactions between atpH and other ATP synthase subunits, as well as with assembly factors like PAB (PROTEIN IN CHLOROPLAST ATPASE BIOGENESIS) that have been shown to assist in the assembly of the catalytic core .

In vitro reconstitution experiments combining recombinant atpH with other subunits can help elucidate the precise sequence of assembly events and identify critical interaction domains. Additionally, labeled recombinant atpH can be used in pull-down assays to identify novel assembly factors or chaperones that may specifically interact with subunit c during complex formation. Such studies are particularly relevant given the complex coordination required between nuclear and plastid-encoded subunits during ATP synthase biogenesis in plants, where "precise coordination between gene expression and assembly is vital for the cF₁F₀ biogenesis process to ensure appropriate complex stoichiometry and prevent the accumulation of pre-complexes" .

What role does Aethionema grandiflora atpH play in evolutionary studies of Brassicaceae?

Aethionema grandiflora, including its atpH gene, serves as an important reference point in evolutionary and phylogenetic studies of Brassicaceae. Specifically, A. grandiflora is frequently used as an outgroup in phylogenetic analyses of the Brassicaceae family, helping to root phylogenetic trees and establish evolutionary relationships . This positioning reflects Aethionema's status as one of the earliest diverging lineages within the family.

The conservation of chloroplast genes like atpH across diverse plant species provides valuable markers for tracking evolutionary changes. Researchers studying chloroplast genome evolution can use atpH sequence data from Aethionema grandiflora as a comparative reference to examine patterns of sequence conservation, selection pressure, and structural rearrangements across the Brassicaceae family. Such analyses contribute to our understanding of how photosynthetic machinery has evolved and adapted across different plant lineages.

In comprehensive phylogenomic studies, chloroplast genes including atpH are often used in combination to reconstruct evolutionary histories with high resolution. As noted in phylogenetic analyses, "All the phylogenetic trees were rooted with Aethionema cordifolium and Aethionema grandiflorum as the outgroup" , highlighting the importance of these species in establishing the evolutionary framework for Brassicaceae studies.

How can structural studies of atpH contribute to understanding chloroplast ATP synthase engineering?

Structural studies of Aethionema grandiflora atpH can significantly contribute to chloroplast ATP synthase engineering efforts. Detailed knowledge of subunit c structure, particularly its interaction surfaces with other ATP synthase components, provides essential information for rational design approaches. By understanding the structure-function relationships in ATP synthase subunit c, researchers can identify potential modification sites that might alter properties such as proton conductance, rotational efficiency, or regulatory interactions.

Recent advances in chloroplast ATP synthase engineering have recognized that "tailoring the activity of chloroplast ATP synthases and modeling approaches can be applied to modulate photosynthesis" . Structural insights derived from studies of atpH can inform these engineering efforts by identifying conserved domains that must be preserved versus regions amenable to modification without compromising function. Additionally, comparative structural analyses across species can reveal natural variations that have evolved to optimize ATP synthase performance under different environmental conditions, potentially providing blueprints for engineered improvements.

As noted in current research, "advances in genetic manipulation and protein design tools will significantly expand the scope for testing new strategies in engineering light-driven nanomotors" . Structural characterization of key components like atpH will be fundamental to these approaches, potentially enabling the development of ATP synthases with altered efficiency, regulatory properties, or environmental responses to enhance photosynthetic performance.

What methods are most effective for analyzing selection pressure on atpH across Brassicaceae species?

For analyzing selection pressure on atpH across Brassicaceae species, researchers should employ multiple complementary approaches. The most effective methodological framework includes:

• Sequence Alignment and Codon-Based Analyses: Multiple sequence alignment of atpH genes from diverse Brassicaceae species, followed by calculation of synonymous (dS) and non-synonymous (dN) substitution rates. The dN/dS ratio (ω) provides a primary indicator of selection pressure, with ω<1 suggesting purifying selection, ω=1 indicating neutral evolution, and ω>1 pointing to positive selection.

• Site-Specific Models: Implementing maximum likelihood methods such as PAML to identify specific codon positions under different types of selection. This approach can reveal functional domains under strong evolutionary constraints versus regions experiencing adaptive evolution.

• Branch-Site Models: These methods detect episodic selection on specific lineages, which is particularly relevant when examining atpH evolution across the Brassicaceae phylogeny where different selective pressures might apply to different clades.

• Sliding Window Analysis: For detecting localized regions of selection within the gene that might be missed by whole-gene analyses.

Research comparing codon usage frequency between related species, such as that performed between Cardamine resedifolia and Cardamine impatiens, has successfully identified genes under positive selection at the family-wide level . Similar methodologies could be applied to analyze selection patterns on atpH specifically, potentially revealing how evolutionary forces have shaped this critical component of the photosynthetic machinery across Brassicaceae.

How does atpH conservation compare between photosynthetic and non-photosynthetic organisms?

The conservation of atpH between photosynthetic and non-photosynthetic organisms represents a fascinating aspect of plastid genome evolution. Studies of non-photosynthetic algae have provided important insights into this phenomenon. In Prototheca wickerhamii, a heterotrophic unicellular alga closely related to photosynthetic Chlorella vulgaris, researchers found that "no genes for photosynthetic functions have been found, except for sequences encoding six subunits of the ATP synthase (atpA, atpB, atpE, atpF, atpH, and atpI)" .

This conservation pattern is particularly significant because P. wickerhamii has a drastically reduced plastid genome (54,100 bp) compared to its photosynthetic relative C. vulgaris (150,613 bp) . The retention of ATP synthase genes, including atpH, in an organism that has lost photosynthetic function suggests these genes serve essential functions beyond photosynthesis. This observation aligns with the hypothesis that "in the reduced plastid genome of Prototheca, genes coding for components of the plastid translational apparatus have been preferentially retained, and might be needed for the expression of the atp genes" .

Comparative analyses between Aethionema grandiflora atpH and homologs in non-photosynthetic organisms could reveal which protein domains are under the strongest selective constraints, potentially identifying regions essential for functions beyond photosynthetic ATP production, such as maintaining plastid homeostasis or participating in non-photosynthetic metabolic processes.

What are the challenges in studying atpH expression and regulation in Aethionema grandiflora?

Studying atpH expression and regulation in Aethionema grandiflora presents several methodological challenges that researchers must address:

• Coordination of Nuclear and Plastid Gene Expression: Since ATP synthase biogenesis requires "precise coordination between gene expression and assembly" of subunits encoded by both nuclear and plastid genomes , studying atpH regulation necessitates examining cross-talk between these genetic compartments. This requires integrated approaches examining both plastid gene expression and nuclear regulatory factors.

• Tissue-Specific and Environmental Responsiveness: ATP synthase components show differential expression under varying environmental conditions. For example, research has shown that "cF₁ dissociated from the thylakoid membrane after cold treatment" . Studying such responses in Aethionema grandiflora requires controlled growth conditions and tissue-specific analyses.

• Assembly-Dependent Protein Stability: Studies have shown that ATP synthase subunits exhibit "assembly-dependent protein stability and a fast degradation of unassembled subunits" . This means that measuring atpH transcript levels alone may not accurately reflect functional protein levels, necessitating complementary protein analyses.

• Technical Limitations for a Non-Model Organism: Aethionema grandiflora lacks many of the molecular tools available for model plants like Arabidopsis. Researchers must adapt or develop species-specific protocols for RNA extraction, protein isolation, and gene expression analysis.

Addressing these challenges requires multifaceted approaches combining transcript analysis, protein detection, and assembly studies across different tissues and environmental conditions to fully characterize atpH expression and regulation in this phylogenetically significant Brassicaceae species.

How can recombinant Aethionema grandiflora atpH be used in antibody production and immunological studies?

Recombinant Aethionema grandiflora ATP synthase subunit c provides an excellent antigen for developing specific antibodies for immunological studies. For optimal antibody production, researchers should consider:

• Immunization Strategy: The full-length recombinant protein (81 amino acids) can be used for immunization, but researchers may also consider using specific peptide regions predicted to be antigenic and accessible in the native protein. For membrane proteins like atpH, careful epitope selection is crucial.

• Cross-Reactivity Considerations: Due to the highly conserved nature of ATP synthase components across species, researchers must validate antibody specificity through comprehensive cross-reactivity testing against ATP synthase subunits from related Brassicaceae species.

• Applications in Protein Localization: The resulting antibodies can be employed in immunohistochemistry and immunogold electron microscopy to study the distribution of ATP synthase complexes within chloroplast membranes under different developmental or environmental conditions.

• Quantitative Analysis: For quantitative studies of ATP synthase abundance, antibodies against atpH can be used in enzyme-linked immunosorbent assays (ELISA) and Western blotting, ideally with recombinant protein standards to enable absolute quantification.

• Assembly Studies: Anti-atpH antibodies are particularly valuable for investigating ATP synthase assembly processes through co-immunoprecipitation experiments that can identify interaction partners during different stages of complex formation.

The available recombinant protein, stored in a Tris-based buffer with 50% glycerol , provides a stable antigen source for these applications. This approach can yield valuable tools for examining ATP synthase biology not only in Aethionema grandiflora but potentially across the Brassicaceae family.

What experimental approaches can determine if structural variations in atpH affect ATP synthase efficiency?

To determine how structural variations in atpH affect ATP synthase efficiency, researchers should implement a multi-layered experimental approach:

• Site-Directed Mutagenesis: Introducing specific amino acid substitutions in the recombinant Aethionema grandiflora atpH protein based on comparative sequence analysis or structural predictions. Key targets would include residues involved in proton translocation and those at interaction interfaces with other subunits.

• In Vitro Reconstitution Assays: Reconstituting ATP synthase complexes with wild-type or mutant atpH proteins to directly measure enzymatic activity. This approach has proven effective, as demonstrated by studies where "CPN60 as well as the heat shock protein HSP70 systems were combined in an in vitro reconstitution assay of the cF₁ catalytic core from the subunits α, β, and γ, [achieving] 95% of that observed with native Arabidopsis core cF₁F₀" .

• Biophysical Characterization: Using techniques such as circular dichroism spectroscopy to assess structural changes in mutant proteins, and isothermal titration calorimetry to measure binding affinities with interacting subunits.

• Liposome Reconstitution and Proton Pumping Assays: Incorporating purified wild-type or mutant atpH proteins into liposomes to measure proton translocation efficiency under controlled conditions.

• Heterologous Expression Systems: Testing the functional effects of atpH variants in model organisms with modified ATP synthase genes, potentially using complementation approaches in ATP synthase-deficient mutants.

These methodologies can reveal how specific structural features of the atpH protein contribute to ATP synthase assembly, stability, and catalytic efficiency, providing insights that could inform future engineering efforts aimed at optimizing photosynthetic energy conversion.

How can knowledge of atpH structure and function contribute to chloroplast engineering for improved photosynthesis?

Knowledge of atpH structure and function can make significant contributions to chloroplast engineering strategies aimed at improving photosynthetic efficiency through several mechanisms:

• Optimizing Energy Balance: Research has established that "tailoring the activity of chloroplast ATP synthases and modeling approaches can be applied to modulate photosynthesis" . By engineering atpH to fine-tune ATP synthase activity, researchers could potentially optimize the ATP/NADPH ratio produced during photosynthesis to better match the demands of carbon fixation, potentially reducing energy losses.

• Enhancing Environmental Adaptability: Understanding how atpH responds to environmental stresses could enable the engineering of variants with improved stability or activity under suboptimal conditions, such as temperature extremes or fluctuating light intensities.

• Manipulating Proton Gradient Regulation: The c-subunit ring of ATP synthase plays a crucial role in determining the proton-to-ATP ratio. Engineering atpH to alter this stoichiometry could potentially influence energy coupling efficiency and the thylakoid lumen pH, which affects both photosynthetic electron transport and photoprotection mechanisms.

• Synthetic Biology Approaches: As noted in current research, "advances in genetic manipulation and protein design tools will significantly expand the scope for testing new strategies in engineering light-driven nanomotors" . This could include developing synthetic atpH variants with novel properties or regulatory features.

• Balancing Source-Sink Relationships: Modulating ATP synthase activity through atpH engineering could affect the availability of ATP for carbon fixation and other chloroplast metabolic processes, potentially addressing limitations in photosynthetic capacity.

These approaches align with broader efforts to enhance crop productivity through photosynthetic improvements, representing "one of the most significant challenges in plant biotechnology... to maintain or even enhance crop productivity in the face of increasing external stress conditions" .