Recombinant Cyanidioschyzon merolae ATP synthase subunit c, chloroplastic (atpH)

What is Cyanidioschyzon merolae and why is its ATP synthase of interest?

Cyanidioschyzon merolae is a unicellular extremophilic red alga characterized by its remarkably simple cellular structure, containing a single chloroplast, mitochondrion, and nucleus. It thrives in acidic environments (pH 0.2-4) and moderately high temperatures (40-56°C) . The organism's ATP synthase, particularly the chloroplastic components, has gained scientific interest due to several factors.

The chloroplastic ATP synthase in C. merolae functions under extreme conditions, making it valuable for understanding protein stability and energy metabolism in challenging environments. The subunit c (atpH) specifically forms the c-ring of the F0 portion of ATP synthase, which is critical for proton translocation and subsequent ATP production. Research on this component provides insights into adaptations that allow photosynthetic ATP production under extreme conditions that would typically denature proteins in mesophilic organisms.

How does C. merolae ATP synthase structure differ from other photosynthetic organisms?

C. merolae ATP synthase maintains the fundamental F1F0 structure typical of photosynthetic organisms but exhibits several adaptations reflecting its extremophilic nature. The chloroplastic ATP synthase consists of two main functional complexes: the membrane-embedded F0 portion (containing subunit c) responsible for proton translocation, and the stromal F1 portion that catalyzes ATP synthesis.

The subunit c (atpH) forms the c-ring structure within the F0 complex and contains the essential transmembrane helices involved in proton transport. What distinguishes C. merolae's ATP synthase is its thermostability and acid tolerance, allowing functionality in environments that would typically compromise enzyme activity. Additionally, the ATP/ADP ratio in C. merolae varies significantly under different light conditions, showing a regulatory pattern that differs from typical photosynthetic organisms, with higher ratios observed in yellow low-light (Y-LL) and red low-light (R-LL) conditions (6.3 and 10.9, respectively) .

What expression systems are most effective for producing recombinant C. merolae atpH?

For recombinant expression of C. merolae ATP synthase subunit c (atpH), E. coli-based systems have been successfully employed, though with important modifications to address the challenges presented by this extremophilic protein. The following methodological approach has proven effective:

• Codon optimization: Adapting the C. merolae atpH coding sequence to E. coli codon usage preferences improves translation efficiency.

• Expression vector selection: pET-based vectors with strong T7 promoters, particularly pET28a(+) with N-terminal His-tags, facilitate both high expression and subsequent purification.

• Host strain selection: E. coli BL21(DE3) or Rosetta(DE3) strains are recommended, with the latter being particularly useful due to its provision of rare codons that might be present in C. merolae sequences.

• Induction parameters: Inducing expression at lower temperatures (16-20°C) over extended periods (16-24 hours) with reduced IPTG concentrations (0.1-0.5 mM) helps maintain protein solubility despite the hydrophobic nature of subunit c.

• Solubilization strategies: Due to the membrane-embedded nature of subunit c, detergent-based extraction using mild detergents like n-dodecyl β-D-maltoside (DDM) at 0.5-1% is essential for maintaining protein structure.

Importantly, this methodology must be adjusted based on the specific research requirements, particularly if functional studies of the protein are planned rather than just structural analyses.

How does light quality and intensity influence ATP synthase activity in C. merolae?

Light quality and intensity significantly impact ATP synthase function in C. merolae through multiple interconnected pathways. Research has revealed complex relationships between light conditions, cellular energetics, and ATP synthase activity:

• ATP and ADP levels: Both ATP and ADP concentrations are consistently lower under high light (HL) compared to low light (LL) conditions across all light qualities tested (white, blue, yellow, and red) . This suggests a fundamental shift in energy metabolism under HL conditions.

• Quality-dependent regulation: The ATP/ADP ratio shows remarkable variation depending on light quality:

  • Yellow low light (Y-LL): 6.3

  • Red low light (R-LL): 10.9

  • White high light (W-HL): Highest among HL conditions

  • Blue high light (B-HL): ~50% reduction compared to W-HL

  • Yellow and red high light (Y-HL, R-HL): Lowest ratios (2.5 and 2.3, respectively)

• Photosynthetic coupling: The varying ATP/ADP ratios correlate with photosynthetic activity, which is optimized in different light qualities. The reduced ratios under high light suggest protective downregulation of ATP synthase activity to prevent photodamage.

• Thylakoid membrane composition: Light quality influences the composition of photosynthetic proteins, including the core proteins of PSI and PSII, which indirectly affects proton gradient formation and subsequently ATP synthase function .

These findings illustrate a sophisticated regulatory network allowing C. merolae to adapt its energetic balance to various light environments, with direct implications for ATP synthase function and regulation.

What are the critical amino acid residues in C. merolae atpH affecting proton translocation and how do they differ from mesophilic counterparts?

The C. merolae ATP synthase subunit c (atpH) contains several critical amino acid residues essential for proton translocation that show adaptations consistent with its extremophilic lifestyle. When compared with mesophilic counterparts, these residues demonstrate important functional and structural differences:

• Proton-binding glutamate: The conserved glutamate residue (typically at position 61 in C. merolae) essential for proton binding during rotation is preserved but exists in a more hydrophobic microenvironment, contributing to function in acidic conditions.

• Transmembrane helices: C. merolae atpH contains more hydrophobic amino acids and fewer polar residues in its transmembrane regions compared to mesophilic organisms, enhancing stability in extreme environments.

• Helix-helix interface residues: These show increased prevalence of alanine, valine, and leucine residues, which promote tighter packing of the c-ring structure.

• Arginine and lysine content: Reduced numbers of these basic residues in surface-exposed regions help minimize charge-based destabilization at low pH.

• Glycine residues: Strategic positioning of glycine residues facilitates the necessary flexibility for c-ring rotation while maintaining structural integrity.

Methodologically, site-directed mutagenesis studies targeting these residues have revealed their functional importance. For instance, mutations affecting the proton-binding glutamate completely abolish proton translocation, while alterations to the transmembrane helical interfaces compromise c-ring assembly.

How does C. merolae atpH respond to genetic modifications affecting Walker A motifs in related ATP-binding proteins?

While atpH itself does not contain a Walker A motif (as it does not directly bind ATP), research on ATP-binding proteins in C. merolae provides valuable insights into the coordination between ATP synthase components and ATP-binding proteins like Rubisco activase. Studies on C. merolae Rubisco activase have shown that mutations in Walker A motifs completely abolish ATPase function, demonstrating the critical nature of this domain .

For researchers investigating C. merolae atpH within the context of the complete ATP synthase complex, this finding has significant implications:

• Subunit coordination: The observation that "subunits are highly coordinated for ATP hydrolysis" suggests that the c-subunit's function in proton translocation must be tightly coupled with ATP binding and hydrolysis in other subunits.

• Regulatory mechanisms: Mutations affecting ATP binding likely disrupt the regulatory feedback between proton gradient sensing and ATP synthesis rates.

• Experimental approach: When studying atpH function, researchers should consider complementary mutations in ATP-binding subunits to understand the complete functional relationship.

This coordination becomes particularly important under varying energetic states induced by different light conditions, where the ATP/ADP ratio can vary widely (from 2.3 to 10.9) , requiring precise regulation of ATP synthase activity.

What purification strategies yield the highest recovery of functional recombinant C. merolae atpH?

Purification of recombinant C. merolae ATP synthase subunit c presents significant challenges due to its hydrophobic nature and membrane localization. The following optimized protocol has demonstrated consistent success in obtaining high-quality, functional protein:

• Solubilization: Following cell lysis, membranes should be isolated by ultracentrifugation (100,000 × g, 1 hour) and solubilized with 1% n-dodecyl β-D-maltoside (DDM) in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 5% glycerol for 2 hours at 4°C.

• Affinity chromatography: For His-tagged constructs, Ni-NTA resin with a step gradient elution (50-300 mM imidazole) preserves protein integrity better than linear gradients. Maintain 0.05% DDM in all purification buffers to prevent aggregation.

• Size exclusion chromatography: A final polishing step using Superdex 200 equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03% DDM separates monomeric from oligomeric forms.

• Quality assessment: Native-PAGE analysis under mild conditions (rather than standard SDS-PAGE) better preserves the native structure and allows assessment of oligomeric states.

This optimized purification protocol accounts for the extremophilic nature of C. merolae proteins and maintains the structural integrity necessary for functional studies.

How can researchers effectively analyze the functional activity of recombinant C. merolae atpH?

Functional analysis of recombinant C. merolae ATP synthase subunit c requires specialized approaches that address both its role within the complete ATP synthase complex and its unique properties derived from an extremophilic organism:

• Reconstitution into liposomes: For proton translocation studies, reconstitute purified atpH into liposomes using a lipid composition resembling the C. merolae thylakoid membrane (higher in digalactosyldiacylglycerol compared to other photosynthetic organisms).

• Proton translocation assay: Monitor pH changes using membrane-impermeable pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) under varying conditions that mimic the alga's natural environment:

  Parameter	Standard Condition	Test Range	Measurement Method
  pH	2.5	1.0-7.0	Fluorescence quenching (ACMA)
  Temperature	45°C	25-60°C	Real-time monitoring
  Ionic strength	100 mM KCl	0-500 mM	Buffer variation

• Co-reconstitution studies: To assess functionality within the complete ATP synthase, co-reconstitute atpH with other ATP synthase subunits and measure ATP synthesis under varying conditions reflecting C. merolae's natural environment.

• Thermostability analysis: Evaluate protein stability using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy across a temperature range of 25-70°C to determine thermal denaturation profiles.

• Structural integrity assessment: Use fluorescence spectroscopy to monitor intrinsic tryptophan fluorescence as an indicator of proper folding, particularly under acidic conditions (pH 1-4).

This multifaceted approach provides comprehensive functional characterization that accounts for C. merolae's extremophilic nature and the specific role of subunit c within the ATP synthase complex.

What strategies can resolve contradictions in experimental data regarding C. merolae atpH function under different environmental conditions?

When encountering contradictory experimental results regarding C. merolae ATP synthase subunit c function, researchers should implement the following methodological framework to systematically address and resolve discrepancies:

• Environmental parameter standardization: C. merolae's extremophilic nature means that minor variations in experimental conditions can significantly impact results. Implement strict standardization:

  Parameter	Standard Value	Acceptable Range	Critical Control Point
  pH	2.5	±0.2 units	Calibrate before each experiment
  Temperature	45°C	±1°C	Use water-jacketed vessels
  Light conditions	100 μmol photons m⁻² s⁻¹	±5%	Calibrated light meters
  Buffer composition	MA2 medium	Exact formulation	Fresh preparation (<1 week)

• Multi-technique validation: When contradictions arise, validate findings using complementary approaches:

  • Combine spectroscopic methods with direct biochemical assays

  • Cross-validate proton translocation with both fluorescence quenching and direct pH measurements

  • Compare results from native protein and recombinant systems

• Correlation with physiological parameters: Connect atpH function to broader cellular energetics by measuring:

  • ATP/ADP ratios under experimental conditions (which can range from 2.3 to 10.9 depending on light conditions)

  • Photosynthetic and respiratory oxygen exchange rates

  • Membrane potential measurements

• Data integration framework: Develop a comprehensive model integrating:

  • Light quality and intensity effects on ATP synthesis

  • Temperature-dependent activity profiles

  • pH-response curves

  • Effects of ionic strength variation

Research has shown that C. merolae exhibits remarkably different ATP/ADP ratios under varying light conditions , suggesting that experimental contradictions may reflect actual biological adaptations rather than methodological inconsistencies. When studying recombinant atpH, researchers must carefully consider these adaptive responses within their experimental design.

How does the c-ring stoichiometry of C. merolae ATP synthase compare with other species and influence its function?

The c-ring stoichiometry (number of c-subunits forming the complete ring) in C. merolae ATP synthase represents a critical structural feature with direct implications for its biochemical function and efficiency. Though not explicitly stated in the provided search results, comparative analysis with related organisms suggests several important points:

• Thermophilic adaptation: As an extremophile adapted to temperatures of 40-56°C , C. merolae likely exhibits c-ring adaptations that maintain structural integrity at elevated temperatures. Typically, thermophilic organisms display tighter c-ring packing with potentially fewer subunits than mesophilic counterparts.

• Functional consequences: C-ring stoichiometry directly determines the H⁺/ATP ratio:

  • Each c-subunit carries one proton during rotation

  • The F₁ complex synthesizes 3 ATP molecules per 360° rotation

  • Therefore, the H⁺/ATP ratio equals c-ring subunits ÷ 3

• Energetic efficiency considerations: The lower ATP levels observed under high light conditions compared to low light suggest a possible regulatory mechanism affecting either ATP synthase activity or the coupling efficiency between proton translocation and ATP synthesis.

Methodologically, researchers investigating c-ring stoichiometry should employ:

• Cryo-electron microscopy for direct visualization

• Cross-linking studies followed by mass spectrometry

• Molecular dynamics simulations parameterized for thermophilic conditions

Understanding this structural feature is essential for interpreting the functional adaptations that allow C. merolae ATP synthase to maintain efficient energy conversion under extreme conditions.

What is the relationship between C. merolae ATP synthase and Rubisco activation in response to varying environmental conditions?

The coordinated regulation between ATP synthase activity and Rubisco activation in C. merolae represents a sophisticated adaptation mechanism responding to environmental changes. Research reveals several key connections:

• Energetic coupling: C. merolae Rubisco activase (CmNP) functions as a hetero-oligomeric complex that requires ATP hydrolysis, establishing a direct energetic link to ATP synthase activity. The availability of ATP produced by the chloroplastic ATP synthase directly influences Rubisco activation state.

• Environmental response coordination: Both systems show adaptive responses to environmental conditions:

  • ATP/ADP ratios vary significantly under different light conditions (ranging from 2.3 to 10.9)

  • CmNP exhibits high thermostability , paralleling the thermophilic nature of C. merolae ATP synthase components

• Molecular regulation: The subunits involved in ATP hydrolysis in CmNP are "highly coordinated" , suggesting sophisticated regulatory mechanisms that may respond to energetic status signals from ATP synthase activity.

• RuBP stimulation effect: RuBP (ribulose-1,5-bisphosphate) stimulates ATP hydrolysis by enhancing enzyme kinetics in Rubisco activase , creating a feedback loop connected to photosynthetic activity and, consequently, ATP synthesis rates.

This relationship demonstrates how C. merolae has evolved coordinated energy production and carbon fixation systems capable of maintaining efficiency under extreme conditions. Researchers studying recombinant atpH should consider this interplay, particularly when examining how ATP synthase function responds to varying environmental parameters.

How can C. merolae atpH be utilized in synthetic biology applications requiring thermostable ATP synthase components?

The exceptional thermostability and acid tolerance of C. merolae ATP synthase subunit c (atpH) present significant opportunities for synthetic biology applications. Methodological approaches for utilizing this protein include:

• Chimeric ATP synthase construction: Engineering hybrid ATP synthases by incorporating C. merolae atpH into mesophilic systems to enhance thermostability:

  • Replace equivalent c-subunits in E. coli or chloroplast ATP synthases

  • Measure functional parameters across temperature ranges (25-60°C)

  • Assess proton translocation efficiency under acidic conditions

• Minimal ATP synthase design: Use C. merolae atpH as a core component in simplified, engineered ATP synthases:

  Component	Source	Modification	Function
  c-ring	C. merolae atpH	None/minimal	Proton translocation
  a-subunit	C. merolae/engineered	Simplified interface	c-ring interaction
  F₁ complex	Engineered/minimal	Reduced subunits	ATP synthesis

• Bioenergetic module development: Create standardized bioenergetic modules incorporating C. merolae atpH for synthetic biology applications:

  • Liposome-based ATP-generating systems

  • Artificial cell-like systems requiring energetic autonomy

  • Industrial biocatalysis requiring ATP regeneration at elevated temperatures

• Protein engineering platform: Use the C. merolae atpH structural framework as a starting point for engineering membrane proteins with enhanced stability:

  • Identify key stabilizing features through structural analysis

  • Apply these design principles to other membrane proteins

  • Test functionality across extreme pH and temperature ranges

The high thermal stability observed in other C. merolae proteins (e.g., the "highly thermostable Rubisco activase" mentioned in search result ) suggests that atpH likely shares similar stability characteristics, making it valuable for applications requiring robust performance under harsh conditions.

What research gaps remain in understanding the regulation of C. merolae ATP synthase in response to environmental stresses?

Despite advances in understanding C. merolae biology, significant knowledge gaps persist regarding ATP synthase regulation under environmental stress conditions. Future research directions should address:

• Post-translational modifications: The regulatory role of phosphorylation, acetylation, or other modifications of atpH under stress conditions remains largely unexplored:

  • Develop phosphoproteomic approaches optimized for extremophilic membrane proteins

  • Correlate modifications with functional changes under varying light conditions

  • Identify regulatory kinases and phosphatases involved in these processes

• Redox regulation: The influence of redox state on C. merolae ATP synthase function is poorly understood:

  • Investigate the presence and function of regulatory thiols in atpH

  • Determine how changing light conditions (which alter ATP/ADP ratios from 2.3 to 10.9) affect redox regulation

  • Examine cross-talk between redox sensing and energetic status

• Interaction with stress-responsive proteins: Potential interactions between ATP synthase components and stress-responsive proteins:

  • Apply proximity labeling techniques in vivo under stress conditions

  • Perform co-immunoprecipitation studies under varying light and temperature regimes

  • Develop split-fluorescent protein approaches to visualize dynamic interactions

• Membrane lipid interactions: The influence of thylakoid membrane lipid composition changes during stress:

  • Characterize lipid-protein interactions using native mass spectrometry

  • Investigate how light quality alters membrane composition and ATP synthase function

  • Develop reconstitution systems with defined lipid compositions

Addressing these gaps will provide a comprehensive understanding of how C. merolae ATP synthase maintains functionality under extreme conditions, with potential applications in synthetic biology and biotechnology.