Recombinant Lolium perenne ATP synthase subunit c, chloroplastic (atpH)

What is the structural characterization of Lolium perenne ATP synthase subunit c?

Lolium perenne ATP synthase subunit c (atpH) is a membrane-embedded protein component of the CF₀ subcomplex of the chloroplast ATP synthase. The full-length protein consists of 81 amino acids with the sequence: MNPLIAAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . This protein functions as part of the proton channel that converts the proton motive force into rotational motion, which is then coupled to ATP synthesis by the CF₁ subcomplex. The hydrophobic nature of this protein reflects its role in the membrane-embedded portion of the ATP synthase complex .

How does ATP synthase subunit c contribute to photosynthetic energy conversion?

ATP synthase subunit c forms part of the CF₀ sector that facilitates proton translocation across the thylakoid membrane. This subunit forms a ring structure in the membrane that rotates as protons flow through the complex, driven by the proton motive force (pmf) generated during photosynthetic electron transport. The rotational energy is transferred to the CF₁ sector, causing conformational changes in the catalytic sites that drive ATP synthesis from ADP and inorganic phosphate. The activity of this complex is tightly regulated by light conditions and metabolic factors, serving as a critical control point between the light and dark reactions of photosynthesis .

What expression systems are most effective for producing recombinant Lolium perenne ATP synthase subunit c?

For expression of recombinant Lolium perenne ATP synthase subunit c, bacterial expression systems (particularly E. coli) are commonly employed using codon-optimized constructs. When designing expression vectors, researchers should consider:

• Inclusion of appropriate purification tags (His-tags are commonly used)

• Use of expression vectors with strong, inducible promoters

• Optimization of growth conditions (temperature, induction time)

• Incorporation of solubility-enhancing fusion partners

For membrane proteins like ATP synthase subunit c, specialized extraction and purification methods using detergents or lipid nanodiscs may be necessary to maintain proper folding and function. Expression optimization typically requires testing multiple constructs and conditions to achieve sufficient protein yield and purity .

What storage conditions maintain stability of recombinant ATP synthase subunit c?

Optimal storage conditions for recombinant Lolium perenne ATP synthase subunit c include:

• Short-term storage (up to one week): 4°C in appropriate buffer

• Medium-term storage: -20°C in buffer containing 50% glycerol

• Long-term storage: -80°C in aliquots to prevent repeated freeze-thaw cycles

The protein is typically maintained in a Tris-based buffer optimized for stability. It's critical to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of activity. Working aliquots should be prepared during initial storage to minimize freeze-thaw events .

How can researchers investigate redox-dependent regulation of Lolium perenne ATP synthase activity?

To investigate redox-dependent regulation of ATP synthase activity in Lolium perenne, researchers should consider the following methodological approach:

• Thiol modification analysis: Use site-directed mutagenesis to modify conserved cysteine residues in the γ subunit that may participate in disulfide/sulfhydryl redox regulation. Compare wild-type and mutant proteins using circular dichroism spectroscopy to assess structural changes under different redox conditions.

• Thioredoxin interaction studies: Perform in vitro assays with purified thioredoxin and ATP synthase components to assess direct protein-protein interactions and subsequent functional changes. This can be coupled with biochemical assays measuring ATP synthesis/hydrolysis rates.

• Electrochromic shift (ECS) measurements: Monitor the dark-interval relaxation kinetics of the electrochromic shift signal in vivo to determine proton motive force (pmf) magnitude and thylakoid conductivity (gH+) under varying light conditions and redox states .

• Comparative analysis: Compare redox regulation across different photosynthetic organisms (cyanobacteria, algae, and higher plants) to establish evolutionary conservation of regulatory mechanisms, similar to studies conducted with PGR5 protein .

What role does ATP synthase subunit c play in temperature stress tolerance in Lolium perenne?

ATP synthase subunit c contributes to temperature stress tolerance in Lolium perenne through multiple mechanisms:

To investigate these aspects, researchers should combine transcriptomic, proteomic, and functional analyses of ATP synthase before and during temperature stress treatments.

How can researchers distinguish between light-dependent and metabolism-dependent regulation of ATP synthase activity?

To differentiate between light-dependent and metabolism-dependent regulation of ATP synthase activity, researchers should employ the following experimental approaches:

• Site-directed mutagenesis: Generate mutants with modifications to specific residues in the γ subunit, particularly focusing on conserved acidic amino acids that have been shown to affect light-dependent but not metabolism-induced regulation .

• Controlled light experiments: Utilize experimental setups that allow precise manipulation of light intensity and quality (wavelength), including transitions between dark, low light (LL), and high light (HL) conditions, while monitoring ATP synthase activity through:

  • Electrochromic shift (ECS) measurements to determine thylakoid conductivity

  • Assessment of proton motive force (pmf) formation and dynamics

  • Analysis of photosynthetic control and non-photochemical quenching (NPQ)

• Metabolic perturbation assays: Introduce specific metabolic changes independent of light conditions by:

  • Application of metabolic inhibitors

  • Modification of carbon dioxide levels

  • Altering substrate availability for dark reactions

• Redox state monitoring: Track the redox state of critical regulatory thiols using fluorescent probes or redox proteomics approaches during both light and metabolic transitions to determine if they respond differently .

Recent research has demonstrated that mutations in γ subunit affect light-induced regulation but not metabolism-induced regulation, suggesting these regulatory mechanisms operate through distinct pathways .

What experimental designs are optimal for studying the interaction between ATP synthase and PGR5-dependent regulation?

To investigate interactions between ATP synthase and PGR5-dependent regulation, the following experimental design is recommended:

• Genetic approach:

  • Generate single and double mutants affecting both ATP synthase components and PGR5

  • Create transgenic lines with tagged versions of both proteins for interaction studies

  • Use CRISPR/Cas9 system to introduce specific mutations in the regulatory domains

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation experiments to test direct interaction between AtPGR5 and AtCF1γ

  • Use bimolecular fluorescence complementation (BiFC) to visualize interactions in vivo

  • Employ yeast two-hybrid or pull-down assays to confirm specific binding domains

• Functional measurements:

  • Monitor proton motive force (pmf) dynamics using electrochromic shift (ECS) measurements

  • Assess thylakoid conductivity (gH+) under varying light regimes

  • Evaluate photosynthetic control and non-photochemical quenching (NPQ) in wild-type and mutant lines

  • Measure electron transport rates and PSI/PSII activity ratios

• Comparative analysis across species:

  • Examine PGR5-ATP synthase interactions in multiple photosynthetic organisms including cyanobacteria (Synechocystis), algae (Chlamydomonas), and higher plants (Arabidopsis, Setaria viridis)

  • This approach can reveal evolutionary conservation of regulatory mechanisms

Research has demonstrated that PGR5 may function as an inhibitor of ATP synthase, as AtPGR5 has been shown to interact with AtCF1γ. This interaction appears to be part of a mechanism for downregulating ATP synthase under high irradiance conditions via a thiol redox state-dependent pathway .

What methodologies can detect conformational changes in ATP synthase subunit c during environmental stress responses?

To detect conformational changes in ATP synthase subunit c during environmental stress responses, researchers should employ the following methodologies:

• Spectroscopic techniques:

  • Circular dichroism (CD) spectroscopy to monitor protein folding transitions at different temperatures

  • Fluorescence spectroscopy using intrinsic tryptophan fluorescence or extrinsic probes to detect subtle structural shifts

  • FTIR spectroscopy to analyze secondary structure changes

• Structural biology approaches:

  • Cryo-electron microscopy of the intact ATP synthase complex under different conditions

  • X-ray crystallography of isolated subunit c or subcomplexes

  • NMR spectroscopy for dynamic structural analysis

• Molecular dynamics simulations:

  • In silico modeling of subunit c behavior under various stress conditions

  • Simulation of protein-lipid interactions in the thylakoid membrane environment

• Protein modification tracking:

  • Mass spectrometry to detect post-translational modifications induced by stress

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility

Studies with similar proteins, like the antifreeze protein from Lolium perenne, have demonstrated clear folding transition temperatures detected by circular dichroism spectroscopy. For example, LpAFP showed three distinct folding transitions, including one between 10-15°C, suggesting that environmental temperature changes can trigger conformational modifications that may affect protein function or stability .

How can researchers overcome difficulties in measuring ATP synthase activity in isolated chloroplasts?

When measuring ATP synthase activity in isolated chloroplasts, researchers often encounter several challenges. To overcome these difficulties, consider the following approaches:

• Chloroplast isolation optimization:

  • Use gentle isolation techniques to maintain membrane integrity

  • Include appropriate osmolytes and protease inhibitors in isolation buffers

  • Perform isolation in dim light or darkness to prevent photodamage

  • Conduct rapid isolation to minimize degradation of ATP synthase components

• Activity measurement techniques:

  • Employ dark-interval relaxation kinetics (DIRK) of the electrochromic shift (ECS) signal to non-invasively assess ATP synthase conductivity

  • Measure ATP synthesis rates using luciferase-based ATP detection systems

  • Monitor proton gradient formation and dissipation using pH-sensitive fluorescent dyes

• Control experiments:

  • Include appropriate inhibitors (oligomycin, tentoxin) as negative controls

  • Compare intact and ruptured chloroplasts to identify stromal component effects

  • Use thiol-modifying agents to assess redox regulation impacts

• Data interpretation considerations:

  • Recognize that differences may exist between in vitro and in vivo regulation

  • Account for the observation that elevated thylakoid conductivity upon high light exposure is not observed in isolated and ruptured chloroplast preparations, suggesting the involvement of stromal components that are lost during isolation

What are the recommended approaches for analyzing the differential expression of ATP synthase subunit c under varying environmental conditions?

To analyze differential expression of ATP synthase subunit c under varying environmental conditions, researchers should implement the following approaches:

• Transcriptomic analysis:

  • Quantitative RT-PCR targeting atpH and related genes

  • RNA-Seq to capture global expression changes including ATP synthase components

  • Time-course experiments to track expression dynamics during stress responses

• Protein-level analysis:

  • Western blotting with specific antibodies against ATP synthase subunit c

  • Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Global proteomics to identify co-regulated proteins

• Experimental design considerations:

  • Include appropriate time points (early and late responses)

  • Gradually increase stress intensity to capture acclimation responses

  • Compare different tissues and developmental stages

• Data analysis strategies:

  • Normalize expression against stable reference genes or proteins

  • Use statistical approaches that account for biological variability

  • Employ clustering analysis to identify co-expressed genes

Studies with Lolium perenne have shown that stress-responsive transcripts increase in abundance within 1-2 days of exposure to cold temperatures (4°C). Similar timeframe monitoring should be applied when examining ATP synthase subunit expression changes in response to environmental stressors .

What are the emerging perspectives on engineering ATP synthase for enhanced photosynthetic efficiency?

Emerging perspectives on engineering ATP synthase for enhanced photosynthetic efficiency include:

• Optimization of regulatory mechanisms:

  • Modifying the thiol redox regulation of the γ subunit to adjust ATP synthase activation thresholds

  • Engineering PGR5-dependent regulation to optimize ATP synthase activity under fluctuating light conditions

  • Fine-tuning the balance between ATP synthesis and pmf dissipation

• Structural modifications:

  • Altering c-ring stoichiometry to modify the H⁺/ATP ratio

  • Engineering subunit interfaces to enhance complex stability under stress conditions

  • Modifying regulatory domains to optimize activity under variable environmental conditions

• Integration with other photosynthetic components:

  • Coordinated engineering of ATP synthase with electron transport components

  • Balancing ATP/NADPH production ratios for optimal carbon fixation

  • Enhancing coordination between light and dark reactions

• Cross-species optimization:

  • Incorporating regulatory features from stress-tolerant organisms into crop plants

  • Creating chimeric ATP synthase complexes with beneficial properties from different species

  • Leveraging the understanding of evolutionary differences in ATP synthase regulation across photosynthetic organisms

These approaches could be particularly valuable for developing crops with improved productivity under fluctuating environmental conditions, including temperature stress and variable light intensity.

How might ATP synthase subunit c modifications contribute to development of climate-resilient crops?

ATP synthase subunit c modifications could significantly contribute to climate-resilient crop development through:

• Temperature stress tolerance enhancement:

  • Engineering ATP synthase subunit c to maintain optimal function across broader temperature ranges

  • Incorporating structural features from extremophile organisms

  • Modifying regulatory mechanisms to respond appropriately to temperature fluctuations

• Improved energy balance under stress:

  • Optimizing ATP production during environmental stress conditions

  • Enhancing coordination between energy production and utilization pathways

  • Maintaining appropriate ATP/ADP ratios during stress recovery phases

• Integration with other stress tolerance mechanisms:

  • Coordinating ATP synthase function with antifreeze protein production in cold-tolerant crops

  • Enhancing ATP availability for stress response pathways, including those mediated by melatonin

  • Supporting energetically demanding acclimation processes

• Practical research approaches:

  • Comparative analysis of ATP synthase components from stress-tolerant wild relatives

  • CRISPR/Cas9 editing of regulatory domains

  • Field testing of transgenic lines under fluctuating environmental conditions

Research with Lolium perenne has demonstrated that stress response mechanisms (including antifreeze proteins) have practical applications for developing genetically-modified crops with enhanced freeze tolerance. Similar principles could be applied to ATP synthase engineering for broader climate resilience .

What quality control parameters are essential when working with recombinant ATP synthase subunit c preparations?

When working with recombinant ATP synthase subunit c preparations, researchers should verify the following quality control parameters:

• Purity assessment:

  • SDS-PAGE analysis with appropriate visualization methods

  • Mass spectrometry to confirm protein identity and detect contaminants

  • Size exclusion chromatography to assess aggregation state

• Structural integrity:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Fluorescence spectroscopy to assess tertiary structure

  • Dynamic light scattering to evaluate size distribution and homogeneity

• Functional validation:

  • Reconstitution studies in liposomes to assess proton channel formation

  • Assembly assays with other ATP synthase components

  • Proton conductance measurements in appropriate membrane systems

• Storage stability monitoring:

  • Tracking protein integrity over time under recommended storage conditions

  • Evaluating the impact of freeze-thaw cycles on structure and function

  • Assessing buffer compatibility and optimization

What are the recommended protocols for incorporating recombinant ATP synthase subunit c into functional membrane systems?

For incorporating recombinant ATP synthase subunit c into functional membrane systems, researchers should follow these recommended protocols:

• Protein preparation:

  • Use freshly purified protein or properly stored aliquots

  • Verify protein purity and structural integrity before incorporation

  • Remove any detergents or solubilizing agents that might interfere with membrane incorporation

• Membrane system selection:

  • Liposomes: For controlled compositional studies

  • Nanodiscs: For single-complex studies and structural analysis

  • Proteoliposomes: For functional assays requiring gradient formation

  • Thylakoid membrane preparations: For integration into native-like environments

• Incorporation methods:

  • Detergent-mediated reconstitution followed by detergent removal (dialysis, biobeads)

  • Direct incorporation during liposome formation

  • Cell-free expression systems with simultaneous membrane incorporation

• Functional validation:

  • Orientation analysis to ensure correct topology

  • Proton conductance measurements using pH-sensitive dyes

  • Assembly verification with other ATP synthase components

  • ATP synthesis/hydrolysis assays in reconstituted systems

• Analytical techniques:

  • Freeze-fracture electron microscopy to visualize membrane-embedded complexes

  • Fluorescence recovery after photobleaching (FRAP) to assess lateral mobility

  • Atomic force microscopy to examine surface topology and organization