Recombinant Guizotia abyssinica ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c in Guizotia abyssinica and what is its role in chloroplasts?

ATP synthase subunit c (atpH) in Guizotia abyssinica is a hydrophobic membrane protein that forms part of the F₀ sector of chloroplastic ATP synthase. The protein plays a critical role in the rotational mechanism of ATP synthesis during photosynthesis. Specifically, it forms an oligomeric ring (c-ring) embedded in the thylakoid membrane that couples proton translocation to ATP synthesis. When protons move through the membrane along an electrochemical gradient, they drive the rotation of this c-ring, which is mechanically coupled to the γ-subunit in the F₁ sector, ultimately catalyzing the synthesis of ATP from ADP and inorganic phosphate .

How does the c-ring structure contribute to ATP synthesis mechanics?

The c-subunits assemble into a ring structure (c-ring) within the thylakoid membrane, with each individual subunit containing essential proton-binding sites. The number of c-subunits in this ring (stoichiometry) directly influences the proton-to-ATP ratio during energy conversion. As protons move through the membrane along the electrochemical gradient, they bind to and release from specific sites on the c-subunits, driving the rotation of the entire c-ring .

This rotational motion is mechanically coupled to the γ-subunit in the F₁ sector, which extends into the catalytic α₃β₃ complex. For each complete rotation of the c-ring, the enzyme synthesizes three ATP molecules. Therefore, the ratio of protons translocated to ATP synthesized equals the number of c-subunits (n) divided by three . This stoichiometric relationship is fundamentally important for understanding the bioenergetics of photosynthesis in different plant species.

What expression systems are most effective for producing recombinant Guizotia abyssinica atpH?

The most effective expression system documented for Guizotia abyssinica atpH involves E. coli using codon-optimized synthetic genes. Similar to methods developed for spinach ATP synthase subunit c, the recombinant approach typically employs BL21 derivative E. coli cells with the following optimization strategies:

• Fusion protein approach: Expression as a fusion protein with a solubilizing partner (e.g., maltose binding protein, MBP) to overcome the extreme hydrophobicity of the c-subunit .

• Codon optimization: Synthetic genes with E. coli-optimized codons significantly enhance expression levels .

• Vector selection: Comparative studies indicate that pMAL-c2x vectors with MBP fusion tags provide superior results compared to other expression vectors such as pET-32a(+) and pFLAG-MAC .

• Expression conditions: Induction at lower temperatures (16-25°C) and reduced IPTG concentrations often yields better results for membrane proteins like atpH.

The expression system can produce milligram quantities of highly purified protein, comparable to methods used for spinach c₁ subunit production.

What purification strategies yield the highest purity of recombinant atpH?

The most effective purification strategy for recombinant Guizotia abyssinica atpH involves a multi-step approach:

• Initial purification: Affinity chromatography utilizing the fusion tag (e.g., MBP or His-tag) to capture the fusion protein from cell lysates .

• Protease cleavage: Controlled proteolytic cleavage to separate the atpH protein from its fusion partner. Factor Xa or TEV protease is commonly used for this purpose .

• Secondary purification: Reversed-phase HPLC for final purification of the hydrophobic c-subunit. A C18 or C4 column with an acetonitrile/TFA gradient is typically employed .

• Buffer exchange: Final buffer exchange into a stabilizing buffer (often Tris/PBS with 6% trehalose for stability).

This purification approach regularly achieves >85% purity as confirmed by SDS-PAGE, with final yields in the milligram range from typical bacterial culture volumes. The purified protein can be stored as a lyophilized powder and reconstituted in sterile water (0.1–1.0 mg/mL) with glycerol for long-term storage.

What quality control methods confirm proper folding and functionality of the recombinant protein?

Quality control for recombinant Guizotia abyssinica ATP synthase subunit c involves multiple analytical techniques:

• Secondary structure analysis: Circular dichroism (CD) spectroscopy to confirm the proper α-helical secondary structure characteristic of the native protein. This is particularly important for membrane proteins like atpH, where proper folding is critical for function .

• Mass spectrometry: To verify the exact molecular weight and sequence integrity of the purified protein .

• Functional reconstitution: Assembly assays to test the ability of the recombinant c-subunits to form oligomeric rings or integrate into proteoliposomes .

• Binding assays: Evaluation of specific binding to known ATP synthase inhibitors or interaction partners can provide functional validation .

• Stability testing: Thermal shift assays to assess protein stability under various buffer and storage conditions.

A crucial control experiment involves comparing the properties of the recombinant protein with those of the native protein isolated from Guizotia abyssinica chloroplasts or from closely related species when direct comparison is not possible.

What determines c-ring stoichiometry in chloroplast ATP synthase and why is it important?

The c-ring stoichiometry (number of c-subunits per ring) is a fundamental parameter that directly influences the bioenergetics of ATP synthesis. Several factors determine this stoichiometry:

• Genetic factors: The primary sequence of the c-subunit can influence packing arrangements and interface stability between adjacent subunits .

• Membrane environment: Lipid composition and membrane thickness can affect the optimal packing of the c-ring .

• Evolutionary adaptation: The stoichiometry appears to be species-specific and may represent adaptation to different energetic requirements and environmental conditions .

The importance of c-ring stoichiometry is evident in several aspects:

Understanding and potentially modifying c-ring stoichiometry has significant implications for photosynthetic efficiency, particularly under stress conditions. Engineering larger c-rings (e.g., c₁₅ in tobacco) has been shown to enhance proton flux without compromising growth, suggesting adaptability in chloroplast ATP synthases.

How does the c-subunit interact with other components of the ATP synthase complex?

The c-subunit interacts with several components of the ATP synthase complex:

• Interaction with a-subunit: The c-ring interfaces with the a-subunit (atpI) of the F₀ sector, forming the proton channel. This interface is critical for proton translocation across the membrane .

• Interaction with γ-subunit: The c-ring rotation is mechanically coupled to the γ-subunit, which extends into the F₁ sector. This coupling is essential for converting the rotational energy of the c-ring into catalytic energy in the F₁ sector.

• Interactions with other F₀ components: The c-ring also interacts with other components of the F₀ sector, including the b-subunit, which forms part of the peripheral stalk connecting F₀ to F₁ .

• Lipid interactions: The c-ring interacts extensively with membrane lipids, which can affect its stability and rotational properties. These interactions may be particularly important in chloroplasts, where thylakoid membrane composition is specialized .

What are the known inhibitors targeting chloroplastic ATP synthase c-subunit?

Several inhibitors are known to target the chloroplastic ATP synthase, with specific interactions involving the c-subunit:

• Oligomycin: While primarily known as a mitochondrial ATP synthase inhibitor, oligomycin sensitivity is conferred by the OSCP (oligomycin sensitivity-conferring protein), which interacts with the c-ring .

• Tentoxin: This cyclic tetrapeptide inhibits chloroplast ATP synthase in sensitive plant species (though not in all plants). It acts as an uncompetitive inhibitor of ATPase activity, with a complex mode of action that can also stimulate activity at high concentrations .

• Amphiphilic peptides: Synthetic peptides including Syn-A2 and Syn-C have been shown to inhibit ATP synthase activity, potentially binding at the same site as natural inhibitors .

• Angiostatin: This protein has been found to inhibit ATP generation by binding to F₁ and completely inhibiting ATPase activity, although its effect on chloroplastic ATP synthase is less well-characterized than on mitochondrial forms .

These inhibitors provide valuable tools for research on ATP synthase function and may serve as templates for the development of more specific agents targeting ATP synthase in various applications .

How does Guizotia abyssinica ATP synthase c-subunit compare to homologs in other plant species?

Comparative analysis reveals both conservation and variation in ATP synthase c-subunit across plant species:

The exact evolutionary significance of variations in the c-subunit across species remains an active area of research, particularly with respect to how these variations might contribute to adaptation to different light environments or other ecological factors.

What methodological approaches are used to study c-ring assembly and stoichiometry?

Several complementary approaches are used to study c-ring assembly and determine its stoichiometry:

• Cryo-electron microscopy (cryo-EM): This technique has been pivotal in resolving the structure of complete ATP synthase complexes, including the c-ring. It allows visualization of the assembled complex in near-native conditions.

• Atomic force microscopy (AFM): AFM can be used to determine the diameter and subunit composition of isolated c-rings .

• Cross-linking studies: Chemical cross-linking coupled with mass spectrometry has been used to study the interactions between c-subunits and other components of the ATP synthase complex .

• Reconstitution experiments: In vitro reconstitution of c-rings from purified recombinant subunits allows investigation of the factors that influence ring assembly and stoichiometry .

• Genetic approaches: Modification of the c-subunit sequence through site-directed mutagenesis can provide insights into the determinants of ring assembly and stability.

These methodologies have collectively advanced our understanding of c-ring structure and function, though the exact stoichiometry and assembly mechanism in Guizotia abyssinica specifically remains to be characterized.

How can recombinant Guizotia abyssinica ATP synthase c-subunit be used to study photosynthetic efficiency?

Recombinant Guizotia abyssinica ATP synthase c-subunit offers several approaches to study photosynthetic efficiency:

• Engineered c-ring stoichiometry: Modifying the c-subunit to alter ring size could potentially enhance ATP synthesis efficiency under various stress conditions. Studies in tobacco have demonstrated that engineering larger c-rings (e.g., c₁₅) enhances proton flux without compromising growth.

• Structure-function analysis: Site-directed mutagenesis of key residues can provide insights into the molecular basis of proton translocation and coupling to ATP synthesis, potentially identifying targets for improving photosynthetic efficiency .

• Inhibitor studies: Recombinant c-subunit can be used to screen for and characterize compounds that modify ATP synthase function, potentially identifying agents that enhance photosynthetic efficiency .

• Reconstitution systems: Purified recombinant c-subunit can be used to develop in vitro reconstitution systems that allow controlled study of ATP synthesis under various conditions, providing insights into factors limiting efficiency in vivo .

• Comparative studies: Comparing the properties of c-subunits from different species adapted to various light environments can provide insights into natural strategies for optimizing photosynthetic efficiency .

These approaches collectively offer powerful tools for understanding and potentially enhancing photosynthetic efficiency, with implications for improving crop productivity under challenging environmental conditions.

How can structural insights from ATP synthase c-subunit inform drug development for human diseases?

Structural studies of ATP synthase c-subunit, including those from plants like Guizotia abyssinica, have significant implications for drug development:

• Conserved mechanisms: Despite evolutionary divergence, fundamental mechanisms of ATP synthase function are conserved across species. Insights from plant systems can inform understanding of human ATP synthase, which is implicated in various diseases .

• Therapeutic targets: ATP synthase has emerged as a potential molecular target for treating human diseases including cancer, obesity, and autoimmune disorders. For example, the drug Bz-423, developed for systemic lupus erythematosus, inhibits mitochondrial ATP synthase by binding to the OSCP subunit .

• Anti-cancer applications: Inhibition of ATP synthase has been suggested as an antiangiogenic therapeutic strategy to block tumor angiogenesis. Studies show that ATP synthase inhibitors can markedly inhibit migration and proliferation of endothelial cells .

• Selective targeting: Understanding the structural differences between ATP synthases from different organisms can enable the development of highly selective inhibitors that target pathogen ATP synthases while sparing human enzymes .

• Novel inhibitor scaffolds: The rich diversity of natural ATP synthase inhibitors, some of which target the c-subunit, provides valuable scaffolds for developing new therapeutics .

These applications highlight the translational potential of basic research on ATP synthase structure and function across different species, including plants like Guizotia abyssinica.

What is the significance of atpH in understanding chloroplast genome evolution and plant adaptation?

The atpH gene encoding ATP synthase subunit c provides significant insights into chloroplast genome evolution and plant adaptation:

• Conserved gene arrangement: In Guizotia abyssinica, as in other plants, atpH is part of the chloroplast genome (Ordered Locus Names: GuabCp013) . Its location and arrangement relative to other genes can provide insights into chloroplast genome evolution.

• Selection pressure: While some ATP synthase subunits (atpA, atpB, and atpI) show evidence of positive selection, suggesting adaptation to different environments, atpH appears to be under stronger purifying selection, reflecting its critical functional role .

• C-ring stoichiometry variation: The variation in c-ring size across species may represent adaptation to different environmental conditions, influencing the efficiency of ATP synthesis under varying light and temperature regimes .

• Interspecific hybridization: Studies of Guizotia species show that despite morphological similarities between species, there are genetic differences that can be traced through chloroplast genes, including those encoding ATP synthase subunits .

• Adaptation to ecological niches: The specific sequence characteristics of atpH and other ATP synthase components may reflect adaptation to the ecological niche of Guizotia abyssinica as an oilseed crop in East Africa .

Understanding these evolutionary patterns not only provides insights into plant adaptation but may also inform strategies for crop improvement, particularly in relation to photosynthetic efficiency and stress tolerance.

What emerging technologies might enhance our understanding of ATP synthase c-subunit dynamics in vivo?

Several emerging technologies show promise for advancing our understanding of ATP synthase c-subunit dynamics in vivo:

• Single-particle cryo-EM advances: Improvements in resolution and sample preparation techniques allow visualization of conformational changes during ATP synthesis, providing dynamic insights beyond static structures.

• Super-resolution microscopy: Techniques such as PALM, STORM, and STED microscopy enable visualization of ATP synthase distribution and dynamics in intact chloroplasts with unprecedented resolution .

• Time-resolved spectroscopy: Advanced spectroscopic methods can track conformational changes in the c-ring during proton translocation and ATP synthesis in real time .

• Genetic editing tools: CRISPR-Cas9 and related technologies enable precise modification of the atpH gene in chloroplasts, allowing investigation of structure-function relationships in vivo .

• Computational approaches: Molecular dynamics simulations and quantum mechanical calculations provide insights into the energetics and mechanics of c-ring rotation that are difficult to access experimentally .

• Artificial photosynthetic systems: Reconstitution of ATP synthase components, including the c-subunit, in artificial membrane systems allows controlled study of function under defined conditions .

These technologies collectively promise to provide a more comprehensive understanding of c-subunit function in the context of photosynthetic energy conversion, potentially informing strategies for enhancing photosynthetic efficiency and developing novel biomimetic energy conversion systems.