Recombinant Oenothera glazioviana ATP synthase subunit c, chloroplastic (atpH)

What is Recombinant Oenothera glazioviana ATP synthase subunit c, chloroplastic (atpH)?

Recombinant Oenothera glazioviana ATP synthase subunit c, chloroplastic (atpH) is a partial-length recombinant protein derived from the Large-flowered evening primrose (Oenothera glazioviana, also known as Oenothera erythrosepala). This protein is a component of the chloroplastic ATP synthase complex, specifically part of the F0 sector. 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. It is commonly expressed in E. coli expression systems for research purposes and has a Uniprot accession number of B0Z548 .

Research methodologies involving this protein typically include structural studies of chloroplastic ATP synthase components, energy transduction mechanisms, and comparative analyses of ATP synthase subunits across plant species.

How should recombinant atpH be stored to maintain optimal activity?

The storage conditions for recombinant atpH significantly impact its stability and biological activity. For liquid formulations, the recommended storage is at -20°C/-80°C, which provides a shelf life of approximately 6 months. Lyophilized preparations demonstrate greater stability, with a shelf life of 12 months when stored at -20°C/-80°C .

To prevent protein degradation from repeated freeze-thaw cycles, working aliquots should be maintained at 4°C for no longer than one week. For reconstitution of lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage, the addition of glycerol to a final concentration of 5-50% (with 50% being standard) is recommended . This glycerol addition prevents ice crystal formation during freezing, which can damage protein structure.

What is the relationship between Oenothera glazioviana and its ATP synthase components?

Oenothera glazioviana (Large-flowered evening primrose) is a biennial plant in the Onagraceae family, growing up to 150 cm tall with medicinal properties attributed primarily to seed oils . While the plant itself has been studied for various medicinal applications, its chloroplastic ATP synthase components represent a distinct area of scientific interest.

The ATP synthase complex in chloroplasts is crucial for energy transduction during photosynthesis. The subunit c (atpH) is part of the membrane-embedded F0 portion of the complex, which forms a proton channel through the thylakoid membrane. In Oenothera species, ATP synthase components have been studied in the context of bioenergetics and proteomics, including under stress conditions such as copper exposure . The study of these components provides insights into energy metabolism adaptation in plants under environmental stress.

What expression optimization strategies are most effective for maximizing recombinant atpH yield?

Optimization of recombinant protein expression, including atpH, requires a systematic approach to culture media composition and growth conditions. Statistical optimization methods have demonstrated significant improvements in recombinant protein yields.

Recent research utilizing artificial neural network linked genetic algorithm (ANN-GA) modeling has shown superior predictive accuracy compared to traditional approaches, with documented increases in recombinant protein yields of up to 93.2% compared to baseline media formulations . For atpH production specifically, the following optimization table provides a framework for methodical improvement:

Applying response surface methodology (RSM) in conjunction with artificial neural networks provides a robust framework for identifying optimal conditions that account for complex interactions between variables . The most significant variables typically include glucose, yeast extract, and NH₄Cl concentrations, which function as essential carbon and nitrogen sources for E. coli metabolism and recombinant protein synthesis .

How can researchers validate the structural integrity of purified recombinant atpH?

Validation of recombinant atpH structural integrity requires a multi-method approach combining biophysical and functional analyses. The following methodological workflow is recommended:

• Purity Assessment: SDS-PAGE analysis should confirm >85% purity, as is standard for commercial preparations . Silver staining may provide higher sensitivity for detecting contaminants.

• Size Exclusion Chromatography (SEC-HPLC): This technique evaluates protein homogeneity and can detect aggregation or degradation. SEC-HPLC has demonstrated effectiveness in confirming the homogeneity of recombinant proteins after purification .

• Circular Dichroism (CD) Spectroscopy: CD analysis provides information about secondary structure content and can confirm proper folding by comparison with native atpH or published structural data.

• Mass Spectrometry: Techniques such as MALDI-TOF or ESI-MS verify the exact molecular weight and can identify post-translational modifications or truncations.

• Functional Assays: Reconstitution of atpH into liposomes or nanodiscs followed by proton transport measurements can confirm functional integrity.

Proteins that maintain both structural and functional integrity will show consistent results across these analytical platforms, providing a comprehensive validation of the recombinant protein's quality.

What approaches are recommended for studying protein-protein interactions involving atpH?

Investigating protein-protein interactions of atpH within the ATP synthase complex requires specialized techniques that preserve native-like conditions. The following methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to atpH or potential interaction partners to pull down protein complexes, followed by Western blot or mass spectrometry analysis.

• Bioluminescence Resonance Energy Transfer (BRET): Fusion of atpH and candidate interacting proteins with luciferase and fluorescent proteins to detect interactions in living cells.

• Surface Plasmon Resonance (SPR): Quantitative measurement of binding kinetics and affinity constants between purified atpH and interaction partners.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Identification of regions involved in protein-protein interactions by measuring changes in hydrogen-deuterium exchange rates upon complex formation.

• Cryo-Electron Microscopy: Structural visualization of atpH within the entire ATP synthase complex, providing insights into interaction interfaces at near-atomic resolution.

When designing these experiments, researchers should consider that the partial-length nature of some commercial recombinant atpH preparations may impact interaction studies . Using full-length constructs or carefully selecting fragments that contain known interaction domains is critical for meaningful results.

How does copper stress affect ATP synthase expression in Oenothera glazioviana?

Proteomic studies on Oenothera glazioviana roots under copper stress have revealed significant changes in the expression of energy metabolism proteins, including ATP synthase components. While specific data on atpH (subunit c) expression changes is limited, related ATP synthase subunits show altered expression patterns under stress conditions .

ATP synthase subunit alpha from the related species Oenothera villaricae demonstrates a 1.58-fold upregulation under copper stress conditions (p = 0.00195) . This suggests that plants may respond to metal stress by modulating energy production pathways, potentially as an adaptive mechanism to maintain ATP levels under adverse conditions.

The table below summarizes the documented changes in ATP synthase components under copper stress:

These findings indicate that copper stress induces specific changes in the ATP synthase complex, potentially affecting energy metabolism pathways. Researchers investigating recombinant atpH might consider studying its expression and function under similar stress conditions to better understand its role in plant adaptation mechanisms.

What reconstitution protocols are recommended for optimal recombinant atpH activity?

Proper reconstitution of recombinant atpH is crucial for maintaining its structural integrity and functional activity. The following protocol is recommended based on established methods for membrane proteins:

• Initial Preparation: Briefly centrifuge the vial containing lyophilized protein to ensure all material is at the bottom .

• Hydration: Reconstitute the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL. For membrane proteins like atpH, gradual addition of water with gentle agitation prevents aggregation .

• Stabilization: Add glycerol to a final concentration of 5-50% (with 50% being standard) for long-term storage stability. The glycerol acts as a cryoprotectant and helps maintain protein structure during freeze-thaw cycles .

• Membrane Reconstitution (for functional studies): For functional studies, further reconstitution into artificial lipid bilayers may be necessary:

  • Prepare liposomes using chloroplast lipid extracts or synthetic lipids (DOPC/DOPE/DOPG at a 7:2:1 ratio)

  • Solubilize liposomes with detergent (0.5-1% n-dodecyl-β-D-maltoside)

  • Add purified recombinant atpH at a protein:lipid ratio of 1:100-1:50

  • Remove detergent using Bio-Beads or dialysis

  • Validate reconstitution success using freeze-fracture electron microscopy or dynamic light scattering

• Quality Control: Perform SDS-PAGE analysis post-reconstitution to verify protein integrity and orientation assays to confirm proper insertion into membranes.

For researchers working with recombinant atpH in structural studies, maintaining a homogeneous preparation is critical, and techniques such as SEC-HPLC can be employed to verify protein quality after reconstitution .

How can researchers design experiments to compare the properties of native versus recombinant atpH?

• Source Material Preparation:

  • Native atpH: Extract from chloroplasts of Oenothera glazioviana using gentle detergent solubilization

  • Recombinant atpH: Express in E. coli and purify under conditions that minimize denaturation

  • Normalize protein concentrations using Bradford or BCA assays

• Structural Comparison:

  • Circular dichroism to compare secondary structure content

  • Limited proteolysis to assess domain folding and accessibility

  • Thermal stability assays (DSF or nanoDSC) to compare unfolding temperatures

  • Native PAGE to analyze oligomeric states

• Functional Comparison:

  • Reconstitution into liposomes for proton translocation assays

  • ATP synthesis/hydrolysis coupling efficiency measurements

  • Interaction studies with other ATP synthase subunits using microscale thermophoresis or SPR

• Data Analysis Framework:

  • Use statistical methods such as two-way ANOVA to assess both protein source (native vs. recombinant) and experimental conditions simultaneously

  • Employ non-parametric tests when appropriate, especially for small sample sizes

  • Calculate effect sizes (Cohen's d) to quantify the magnitude of differences

• Controls and Validation:

  • Include positive controls (well-characterized ATP synthase components)

  • Perform negative controls (denatured proteins, non-functional mutants)

  • Validate with orthogonal techniques for each major finding

This systematic approach ensures that observed differences between native and recombinant atpH can be attributed to their intrinsic properties rather than experimental artifacts.

How can recombinant atpH be utilized in synthetic biology applications?

Recombinant atpH has significant potential in synthetic biology applications, particularly in designing artificial bioenergetic systems. The following methodological approaches demonstrate its utility:

• Minimal ATP Synthase Construction: Recombinant atpH can be combined with other essential ATP synthase subunits to create minimal, functional ATP synthase complexes. These simplified systems allow researchers to study the fundamental principles of rotary catalysis and energy conversion.

• Biosensor Development: The proton-binding properties of atpH make it a candidate for developing pH-sensitive biosensors. By linking conformational changes in atpH to reporter systems (fluorescent proteins or FRET pairs), researchers can monitor proton gradients in real-time.

• Biohybrid Energy Systems: Recombinant atpH can be incorporated into artificial membranes alongside light-harvesting proteins to create biohybrid systems that convert light energy to ATP. This approach mimics natural photosynthesis in a controlled, engineered context.

• Protein Engineering Platforms: Using recombinant atpH as a template, researchers can apply directed evolution or rational design approaches to create modified c-subunits with altered properties such as ion selectivity, stability under extreme conditions, or coupling efficiency.

• Nanomotor Development: The c-ring formed by atpH subunits represents a biological rotary motor. Engineered versions could serve as components in nanoscale devices where controlled rotational motion is desired.

For these applications, the optimization approaches described for recombinant protein production are particularly relevant, as they can increase yield by over 90% compared to standard protocols . Such improvements in production efficiency make these ambitious synthetic biology applications more feasible.

What is the potential role of atpH in understanding the evolutionary adaptations of Oenothera glazioviana?

The study of atpH from Oenothera glazioviana provides valuable insights into evolutionary adaptations in energy metabolism systems. Methodological approaches to investigate these evolutionary aspects include:

• Comparative Sequence Analysis: Alignment of atpH sequences from Oenothera glazioviana with those from related species reveals conservation patterns indicating functional constraints and adaptive changes. This can be accomplished using tools like MUSCLE or CLUSTAL followed by selection pressure analysis (dN/dS ratios).

• Structural Homology Modeling: Building structural models of atpH based on crystallographic data from related species helps identify unique structural features that may represent adaptations to specific environmental conditions.

• Functional Adaptation Studies: Expression of recombinant atpH under conditions mimicking environmental stresses faced by Oenothera glazioviana (such as copper stress, as studied in proteomic analyses ) can reveal adaptive responses at the molecular level.

• Heterologous Expression Testing: Expressing Oenothera glazioviana atpH in model organisms adapted to different environments can reveal functional plasticity and constraints of this protein across environmental gradients.

• Ancestral Sequence Reconstruction: Inferring and synthesizing ancestral versions of atpH allows experimental testing of evolutionary hypotheses about functional changes over time.

Oenothera glazioviana has been studied for its medicinal properties and adaptability , but understanding the molecular basis of these adaptations requires detailed examination of key proteins involved in energy metabolism, including ATP synthase components. The proteomic changes observed under copper stress suggest that ATP synthase components play a role in stress adaptation mechanisms, potentially contributing to the plant's ability to thrive in diverse environments.