Recombinant Cucumis sativus ATP synthase subunit c, chloroplastic (atpH)

What is the structural organization of ATP synthase subunit c in chloroplasts?

ATP synthase subunit c (atpH) in chloroplasts functions as part of a multimeric ring structure embedded in the thylakoid membrane. This c-ring is a critical component of the ATP synthase complex that produces adenosine triphosphate (ATP) required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of this c-subunit ring, which is driven by proton translocation across the thylakoid membrane along an electrochemical gradient .

In high-resolution structures, the c-ring consists of multiple identical c-subunits arranged in a circular formation. Each c-subunit contains alpha-helical secondary structures that span the membrane. The number of c-subunits in the ring varies among different organisms, with known stoichiometries ranging from c₁₀ to c₁₅ . Specifically, the c-ring from spinach chloroplasts has been determined to contain 14 subunits (c₁₄) .

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

The number of c-subunits per ring (n) is organism-dependent and directly affects the coupling ratio (ions transported : ATP generated). This ratio ranges from 3.3 to 5.0 among organisms with known c-ring stoichiometries . The coupling ratio is determined by the formula:

$\text{Coupling Ratio} = \frac{n}{3}$

Where n is the number of c-subunits and 3 represents the constant number of ATP molecules generated per c-ring rotation. For example, a c₁₄ ring (as found in spinach chloroplasts) would have a coupling ratio of approximately 4.7 .

This stoichiometric relationship is crucial because it determines the bioenergetic efficiency of the ATP synthase. A higher number of c-subunits requires more protons to complete a full rotation but produces the same three ATP molecules, making the process less energetically efficient but potentially more adaptable to certain environmental conditions .

What expression systems are most effective for recombinant production of chloroplastic ATP synthase subunit c?

For recombinant production of chloroplastic ATP synthase subunit c, Escherichia coli expression systems have proven effective. Based on methods developed for spinach chloroplast ATP synthase subunit c, researchers can construct a similar approach for Cucumis sativus:

• Gene design: Start with the known amino acid sequence of cucumber atpH and create a synthetic gene with codons optimized for E. coli expression.

• Expression vector: Use a vector containing a maltose-binding protein (MBP) fusion tag, which significantly improves solubility and expression levels of hydrophobic membrane proteins like subunit c.

• Expression conditions: Optimal induction typically occurs at lower temperatures (around 18-25°C) to allow proper folding of the membrane protein .

This expression system has been shown to yield significant quantities of properly folded subunit c with correct alpha-helical secondary structure when applied to spinach chloroplast ATP synthase .

What are the methodological approaches for studying intersubunit contacts in cucumber ATP synthase c-rings?

Studying intersubunit contacts in cucumber ATP synthase c-rings requires sophisticated structural analysis techniques:

• High-resolution crystallography: In meso crystallization has been successful for obtaining detailed structures of c-rings at 2.3 Å resolution, revealing the network of hydrogen bonds and intersubunit contacts .

• Comparative analysis: Researchers should compare intersubunit contacts between c-rings from different sources (plants, mitochondria, and bacteria) to understand evolutionary conservation and structural determinants of c-ring assembly.

• Mutational analysis: Targeted mutations of key residues involved in intersubunit contacts, followed by functional and structural analysis, can reveal the molecular mechanisms determining c-ring stoichiometry .

The high-resolution structure of spinach chloroplast c₁₄-ring reveals detailed molecular mechanisms of how intersubunit contacts occur, providing a template for similar studies in cucumber. These approaches help determine how specific amino acid residues coordinate to form stable c-rings with defined stoichiometries .

How can researchers investigate the unusual electron densities observed inside ATP synthase c-rings?

Recent high-resolution structural studies of chloroplast ATP synthase c-rings have revealed circular-like electron densities in the hydrophobic part of the internal pore, positioned approximately 5.4 Å apart along the central axis and parallel to the membrane plane . To investigate these features in cucumber ATP synthase c-rings, researchers should consider:

• Differential UV-Vis spectroscopy: This technique can detect potential isoprenoid quinones (such as plastoquinone in chloroplasts) that might be present within the c-ring structure .

• Mass spectrometry analysis: Extract and analyze potential cofactors from purified c-rings to identify their chemical composition.

• Functional assays: Assess the impact of these potential cofactors on c-ring stability and proton conductance by comparing native c-rings with those depleted of the cofactors.

This research direction is particularly important as these densities appear to be universal across ATP synthase c-rings from archaea and bacteria to eukaryotes, suggesting they may represent universal cofactors (possibly isoprenoid quinones) that stabilize the c-ring and prevent ion leakage .

What purification strategies yield functional recombinant ATP synthase subunit c for reconstitution studies?

Purifying functional recombinant ATP synthase subunit c for reconstitution studies requires a carefully designed protocol:

• Fusion protein approach: Express the cucumber atpH gene as a fusion protein with maltose-binding protein (MBP) to improve solubility.

• Affinity chromatography: Purify the fusion protein using amylose resin affinity chromatography.

• Protease cleavage: Remove the MBP tag using a specific protease (such as TEV or Factor Xa) with carefully optimized conditions to prevent aggregation.

• Secondary purification: Further purify the cleaved subunit c using size exclusion chromatography in the presence of appropriate detergents.

• Verification: Confirm correct alpha-helical secondary structure using circular dichroism spectroscopy .

This approach has been successfully used to obtain highly purified subunit c from spinach chloroplasts with the correct secondary structure, enabling further investigation into c-ring assembly and stoichiometry determination .

How should researchers design experiments to study factors affecting c-ring stoichiometry in cucumber?

Designing experiments to study factors affecting c-ring stoichiometry in cucumber ATP synthase should include:

• Comparative genomics: Analyze the atpH sequence across different plant species with known c-ring stoichiometries to identify potential sequence determinants.

• Site-directed mutagenesis: Create targeted mutations in the cucumber atpH gene at positions suspected to influence c-ring stoichiometry, based on comparative analysis.

• In vitro reconstitution: Develop systems to reconstitute purified recombinant c₁ subunits into multimeric rings under controlled conditions, testing the influence of:

  • pH and ionic strength

  • Lipid composition

  • Temperature

  • Presence of other ATP synthase subunits

• Structural analysis techniques: Use atomic force microscopy, electron microscopy, and mass spectrometry to determine the stoichiometry of reconstituted c-rings .

This comprehensive approach would help identify undefined factors affecting c-ring assembly and stoichiometry, advancing our understanding of the molecular mechanisms that determine the proton-to-ATP ratio in chloroplast ATP synthases .

What methodological challenges exist in expressing membrane proteins like ATP synthase subunit c from cucumber?

Recombinant expression of membrane proteins like cucumber ATP synthase subunit c presents several methodological challenges:

• Hydrophobicity: The highly hydrophobic nature of subunit c often leads to aggregation, misfolding, or toxicity to the host cells.

• Codon bias: Differences in codon usage between cucumber and E. coli can significantly impact expression efficiency.

• Post-translational modifications: Any plant-specific modifications may be absent in bacterial expression systems.

• Proper folding: Ensuring correct alpha-helical secondary structure formation in a heterologous system.

• Scale-up difficulties: Maintaining proper folding and avoiding aggregation during high-yield production.

Researchers can address these challenges through:

• Codon optimization using specialized software (e.g., Gene Designer)

• Fusion with solubility-enhancing tags like MBP

• Expression at reduced temperatures to slow protein synthesis and improve folding

• Testing multiple E. coli strains optimized for membrane protein expression

• Using specialized media formulations with osmolytes that stabilize membrane proteins

How does the ATP synthase subunit c from Cucumis sativus compare with other plant species?

While specific data on Cucumis sativus ATP synthase subunit c is limited, comparative analysis with other plant species reveals important insights:

Table 1: Comparison of ATP Synthase c-ring Stoichiometries Across Species

Based on evolutionary conservation among plants, we can hypothesize that cucumber chloroplastic ATP synthase likely contains a c-ring stoichiometry similar to that of spinach (c₁₄), although experimental verification is necessary .

The amino acid sequence of the c-subunit determines specific intersubunit contacts, which ultimately influence the c-ring stoichiometry. Comparative sequence analysis across plant species would help identify conserved residues critical for c-ring assembly and function .

How do environmental stresses affect ATP synthase expression and assembly in cucumber?

While specific data on cucumber ATP synthase response to stress is limited, insights can be drawn from proteomic studies of cucumber under iron deficiency stress:

Proteomic studies in cucumber have identified numerous proteins that change in abundance under stress conditions, including proteins involved in energy metabolism . Under iron deficiency, cucumber shows significant remodeling of metabolic pathways, including changes in:

• Carbohydrate-related metabolism

  • Alcohol dehydrogenases

  • Malate dehydrogenase

  • Fructose-bisphosphate aldolase

• Redox-related proteins

  • Heat shock proteins

  • Protein disulfide isomerase

• Iron-containing proteins

  • Aconitate hydratase

  • Peroxidase

For focused studies on ATP synthase response to stress, researchers should design experiments that:

• Analyze transcript levels of atpH under different stress conditions

• Perform proteomic analysis focused on chloroplast membrane proteins

• Investigate changes in ATP synthase assembly and activity in response to environmental stresses

What methods can be used to study the assembly of recombinant subunit c into functional c-rings?

To study the assembly of recombinant cucumber ATP synthase subunit c into functional c-rings, researchers should employ a multi-faceted approach:

• In vitro reconstitution: Develop protocols to reconstitute purified monomeric c₁ subunits into multimeric rings in artificial membrane systems such as:

  • Proteoliposomes

  • Nanodiscs

  • Lipid bilayers

• Analytical techniques for monitoring assembly:

  • Blue native polyacrylamide gel electrophoresis (BN-PAGE)

  • Size exclusion chromatography

  • Analytical ultracentrifugation

  • Dynamic light scattering

• Structural verification methods:

  • Negative-stain electron microscopy

  • Atomic force microscopy

  • Mass spectrometry to determine oligomeric state

• Functional assays:

  • Proton translocation measurements in reconstituted systems

  • ATP synthesis/hydrolysis coupling when combined with F₁ components

These methods would enable researchers to investigate the factors affecting c-ring assembly and the relationship between monomeric c₁ subunits and multimeric rings, which remains poorly understood .

How can researchers investigate the potential isoprenoid quinone cofactors in ATP synthase c-rings?

To investigate the potential isoprenoid quinone cofactors in cucumber ATP synthase c-rings, researchers should implement:

• Extraction and identification:

  • Organic solvent extraction from purified c-rings

  • HPLC separation of extracted compounds

  • Mass spectrometry for identification of plastoquinone or other isoprenoid quinones

• Spectroscopic analysis:

  • Differential UV-Vis spectroscopy to detect characteristic absorption patterns of quinones

  • Comparison with reference standards of plastoquinone and other isoprenoid quinones

• Functional studies:

  • Reconstitution of c-rings with and without the putative cofactors

  • Assessment of c-ring stability and proton conductance

  • Evaluation of the effect on ATP synthase assembly and function

• Structural validation:

  • Crystallographic studies of c-rings with and without cofactors

  • Molecular dynamics simulations to understand cofactor-protein interactions

This research is particularly significant as high-resolution structural studies have revealed unusual electron densities inside c-rings across diverse species, suggesting these potential cofactors may be universal components that stabilize the c-ring structure and prevent ion leakage through the central pore .