Recombinant Fagopyrum esculentum subsp. ancestrale ATP synthase subunit c, chloroplastic (atpH)

What is the genomic context of atpH in Fagopyrum esculentum subsp. ancestrale?

The atpH gene, encoding ATP synthase subunit c, is found in the chloroplast genome of Fagopyrum esculentum subspecies ancestrale. It belongs to the photosynthesis-related gene category, specifically within the ATP synthase gene group that includes atpA, atpB, atpE, atpF, atpH, and atpI . The chloroplast genome of Fagopyrum species is well-characterized, with atpH being conserved across the genus. In F. esculentum subsp. ancestrale, like other Fagopyrum species, the chloroplast genome contains a complete set of genes required for photosynthetic function, including those encoding components of ATP synthase complexes that are crucial for energy conversion during photosynthesis .

How does the codon usage in atpH compare to other genes in Fagopyrum esculentum subsp. ancestrale?

Codon usage analysis of F. esculentum subsp. ancestrale shows specific patterns that affect protein expression. For instance, the RSCU (Relative Synonymous Codon Usage) values indicate preferences for certain codons over others encoding the same amino acid . For alanine, GCU is preferred (RSCU=1.64) over GCC (RSCU=0.78), GCA (RSCU=1.11), and GCG (RSCU=0.47). Similarly, for isoleucine, AUU is preferred (RSCU=1.48) over AUA (RSCU=0.96) and AUC (RSCU=0.56) . Understanding these codon preferences is essential when designing recombinant expression systems for the atpH gene to ensure optimal protein production.

What is the functional role of ATP synthase subunit c in chloroplasts?

The ATP synthase subunit c forms the c-ring in the F₀ component of the ATP synthase complex, which is embedded in the thylakoid membrane of chloroplasts. This subunit is crucial for proton translocation across the membrane, which drives ATP synthesis from ADP and inorganic phosphate . In chloroplasts, the proton gradient is established during photosynthesis, and the c-subunit ring rotates as protons pass through the membrane, converting this mechanical energy into chemical energy in the form of ATP . This protein is therefore essential for energy metabolism in photosynthetic organisms, linking light reactions to the production of ATP needed for carbon fixation and other metabolic processes.

What are the optimal conditions for heterologous expression of recombinant F. esculentum subsp. ancestrale atpH?

When expressing the recombinant F. esculentum subsp. ancestrale atpH gene, several factors must be considered to optimize yield and functionality. Based on experience with similar membrane proteins:

• Expression system selection: E. coli systems with modified codon usage are often suitable, as demonstrated in the expression of the Bacillus PS3 ATP synthase .

• Vector design: Incorporate a His-tag for purification, preferably at the C-terminus to avoid interfering with membrane insertion.

• Induction conditions: Use lower temperatures (16-20°C) during induction to minimize inclusion body formation.

• Membrane protein extraction: Solubilize using gentle detergents such as glycol-diosgenin (GDN) at 1% (w/v) .

A typical purification protocol would include:

• Membrane isolation through centrifugation

• Solubilization with GDN or similar detergents

• Affinity chromatography using HisTrap columns

• Size-exclusion chromatography for final purification

This approach allows for the isolation of functionally active recombinant protein suitable for subsequent structural and functional studies.

How can I verify the structural integrity of purified recombinant atpH protein?

Verification of structural integrity requires multiple complementary approaches:

• SDS-PAGE analysis: Confirms molecular weight and purity

• Western blot: Verifies protein identity using specific antibodies

• Circular dichroism spectroscopy: Assesses secondary structure content

• Fluorescence spectroscopy: Examines tertiary structure and stability

• Cryo-EM: Provides high-resolution structural information, similar to the approach used for bacterial ATP synthase

For membrane proteins like atpH, additional detergent-specific validation is necessary:

• Dynamic light scattering to confirm monodispersity

• Thermal stability assays in various detergent environments

• Limited proteolysis to check for proper folding

These methods collectively provide confidence in the structural integrity of the purified protein before proceeding to functional assays or crystallization attempts.

What methods are most effective for measuring the functional activity of recombinant atpH in vitro?

To assess the functional activity of recombinant atpH, researchers should employ several complementary approaches:

• ATP hydrolysis assay: Measures inorganic phosphate release using colorimetric methods like the malachite green assay

• ATP synthesis assay: Quantifies ATP production using luciferase-based luminescence

• Proton translocation measurements: Assesses H⁺ movement using pH-sensitive fluorescent dyes

• Reconstitution into liposomes: Creates a system to measure both synthesis and hydrolysis activities under controlled conditions

When establishing these assays, it's critical to:

• Include appropriate controls (heat-inactivated protein, known inhibitors)

• Create an artificial proton gradient to drive ATP synthesis

• Optimize detergent concentration or reconstitution conditions

• Account for the impact of lipid composition on activity

These functional assays should be performed across various pH values, temperatures, and ion concentrations to characterize the enzyme's kinetic parameters and optimal operating conditions.

How does the structure of F. esculentum subsp. ancestrale atpH compare to other plant species?

The structure of ATP synthase subunit c from F. esculentum subsp. ancestrale shares high conservation with other plant species, particularly in the transmembrane regions that form the c-ring. Comparative analysis of the amino acid sequences and predicted structures reveals:

• The two transmembrane helices connected by a short hydrophilic loop are highly conserved

• The essential carboxylate residue (typically Asp or Glu) involved in proton binding is preserved

• Species-specific differences mainly occur in the loop regions

While no direct structural data is available for F. esculentum subsp. ancestrale atpH, insights can be gained from comparable bacterial structures. The bacterial ATP synthase structure determined by cryo-EM provides a framework for understanding the arrangement of the c-ring subunits . The c-ring typically consists of multiple copies of the c subunit (8-15 depending on species), arranged in a circle to form the proton-conducting pathway.

What are the key residues in atpH responsible for proton translocation, and how can they be experimentally verified?

The key residues responsible for proton translocation in ATP synthase subunit c include:

• A conserved carboxylate residue (Asp or Glu) in the C-terminal transmembrane helix

• Several surrounding hydrophobic residues that create the proton-binding pocket

• Polar residues that participate in hydrogen bonding networks

These can be experimentally verified through:

• Site-directed mutagenesis: Systematically altering the conserved residues and measuring the impact on proton translocation and ATP synthesis

• Chemical modification: Using specific reagents that target carboxyl groups

• Isotope labeling: Incorporating deuterium or other isotopes to track proton movement

• Molecular dynamics simulations: Modeling proton movement through the c-ring

The experimental approach would be similar to those used in bacterial ATP synthase studies, where the architecture of the membrane region revealed the path of transmembrane proton translocation . This provides a model for understanding the roles of specific residues in the enzyme's function.

How do secondary metabolites in Fagopyrum species affect ATP synthase function?

Secondary metabolites in Fagopyrum species, particularly polyphenols, may interact with and modulate ATP synthase function in several ways:

• Direct interaction: Polyphenols can bind to protein complexes, potentially affecting their structural integrity or conformational changes

• Membrane effects: Alterations in membrane fluidity due to polyphenol incorporation can impact the rotational mobility of the ATP synthase complex

• Antioxidant properties: Polyphenols may protect the ATP synthase from oxidative damage during high light conditions

In F. esculentum, phenolic substances have significant interactions with proteins, especially after hydrothermal treatment . These interactions could potentially affect ATP synthase stability and function. Research has shown that phenolic compounds in buckwheat have inhibitory effects on protein digestion , which suggests they may form stable complexes with proteins including ATP synthase.

Future studies could explore:

• The binding affinity of specific buckwheat polyphenols to isolated ATP synthase

• Functional consequences of such interactions on ATP synthesis rates

• Protective effects against stress-induced damage to the ATP synthase complex

What evolutionary insights can be gained from comparing atpH sequences across Fagopyrum species?

Comparative analysis of atpH sequences across Fagopyrum species provides valuable evolutionary insights:

• Conservation patterns: The high degree of conservation in functional domains indicates strong selective pressure

• Adaptive evolution: Subtle sequence variations may reflect adaptation to different environmental conditions

• Phylogenetic relationships: atpH sequence analysis can complement whole chloroplast genome data for resolving taxonomic relationships

Data from chloroplast genome studies of eight Fagopyrum species shows that genes encoding ATP synthase subunits, including atpH, are highly conserved across the genus . This conservation extends to codon usage patterns, with similar RSCU values observed across species for most amino acids. For example, comparison of F. tataricum, F. cymosum, F. esculentum, F. esculentum subsp. ancestrale, F. longistylum, F. leptopodum, F. urophyllum, and F. luojishanense shows nearly identical patterns for many codons .

These evolutionary analyses provide context for understanding the functional constraints on ATP synthase structure and can help identify regions that may be involved in species-specific adaptation to different photosynthetic conditions.

How does the expression of atpH vary under different environmental conditions in F. esculentum subsp. ancestrale?

The expression of atpH in F. esculentum subsp. ancestrale is likely regulated in response to various environmental factors:

• Light intensity: Being part of the photosynthetic apparatus, atpH expression typically increases under higher light conditions

• Temperature stress: Both heat and cold stress may trigger adjustments in ATP synthase expression

• Drought conditions: Water limitation affects photosynthetic efficiency and energy requirements

• Nutrient availability: Particularly nitrogen status can influence photosynthetic gene expression

Methodological approaches to study these variations include:

• RT-qPCR to quantify mRNA levels under controlled stress conditions

• Proteomic analysis to measure protein abundance

• Chlorophyll fluorescence measurements to correlate with photosynthetic efficiency

• Western blotting with specific antibodies to track protein levels

Understanding these expression patterns is crucial for interpreting how F. esculentum subsp. ancestrale adapts its energy metabolism to environmental changes, which could inform breeding strategies for improved stress tolerance.

What is the relationship between ATP synthase activity and nutritional quality in Fagopyrum species?

The relationship between ATP synthase activity and nutritional quality in Fagopyrum species is complex and multifaceted:

In buckwheat, nutritional quality is particularly linked to protein content, starch properties (especially amylose concentration), and polyphenols like rutin . The digestibility of buckwheat proteins is affected by polyphenols, with phenolic substances showing significant inhibitory effects on protein digestion . This interaction between metabolites and proteins highlights the interconnected nature of energy metabolism and nutritional parameters.

Breeding programs focusing on nutritional quality should consider:

What are the most promising applications of recombinant F. esculentum subsp. ancestrale atpH in basic and applied research?

Recombinant F. esculentum subsp. ancestrale atpH offers several valuable applications:

• Basic research: Serves as a model for understanding chloroplast ATP synthase structure and function in ancient crop varieties

• Comparative studies: Enables structural comparisons between wild and domesticated Fagopyrum species to identify adaptations during domestication

• Protein engineering: Provides a template for designing ATP synthases with modified properties for biotechnological applications

• Agricultural applications: Insights could inform breeding strategies for improving energy efficiency in crops

Future studies should focus on:

These directions promise to expand our fundamental understanding of energy metabolism in plants while potentially contributing to improved crop varieties with enhanced energy efficiency and stress resilience.