Recombinant Nuphar advena ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, and what is its role in chloroplastic ATP synthesis?

ATP synthase subunit c is a critical component of the FoF1-ATP synthase complex, particularly within the Fo portion that facilitates proton translocation across membranes. In chloroplasts, this subunit forms an oligomeric ring structure (typically containing 8-15 subunits depending on the species) that rotates during proton movement, driving conformational changes in the F1 portion that catalyze ATP synthesis. The chloroplastic ATP synthase subunit c is encoded by the atpH gene and plays a fundamental role in converting the proton gradient established during photosynthesis into the mechanical energy needed for ATP production .

What is the molecular weight and basic structure of ATP synthase subunit c from Nuphar advena?

Based on comparative data with related species, the ATP synthase subunit c from Nuphar advena is expected to have a molecular weight of approximately 8 kDa, similar to that reported for Arabidopsis thaliana . The protein typically consists of two transmembrane alpha-helices connected by a small hydrophilic loop. The critical proton-binding site is usually a conserved glutamic acid residue (similar to E56 in thermophilic bacteria studies) that undergoes protonation and deprotonation during the catalytic cycle .

What methods are used to study ATP synthase subunit c structure and function?

Common methodologies include:

• Protein purification and reconstitution into proteoliposomes for functional studies

• Site-directed mutagenesis to identify critical residues

• Western blotting with specific antibodies (typical dilution 1:10,000)

• Proton transfer-coupled molecular dynamics simulations

• Membrane preparation techniques specific to chloroplastic proteins, including methods described by Lezhneva et al. and Chua & Bennoun for Arabidopsis and Chlamydomonas respectively

• Acid/base transition procedures to measure ATP synthesis in vitro

How do mutations in conserved residues of ATP synthase subunit c affect proton translocation and ATP synthesis activity?

Mutations in conserved residues, particularly those involved in proton binding like glutamic acid 56 (E56), significantly impact ATP synthase function. Research on thermophilic Bacillus PS3 has shown that a single E56D mutation reduces ATP synthesis activity but does not completely inhibit it. Interestingly, when double E56D mutations are introduced, the reduction in activity correlates with the distance between mutation sites – activity decreases further as the distance between the two mutations increases .

This spatial relationship indicates complex cooperative interactions among c-subunits during the catalytic cycle. Molecular simulations suggest that prolonged proton uptake duration in mutated c-subunits can be shared between adjacent subunits, creating a mechanistic basis for this cooperation. The degree of time-sharing decreases as the distance between mutation sites increases, explaining the observed activity patterns in biochemical assays .

What is the role of c-subunit cooperation in FoF1-ATP synthase rotation, and how does this impact experimental design?

The c-subunits in the c-ring demonstrate significant functional coupling during rotation. Research evidence indicates that at least three c-subunits at the a/c interface cooperate during c-ring rotation in the Fo portion of ATP synthase . This cooperation is reflected in the observation that multiple deprotonated carboxyl residues face the a-subunit simultaneously during the catalytic cycle.

This cooperative mechanism has important implications for experimental design:

• Studies using isolated single c-subunits may not capture the complex interactions present in the intact ring

• Mutation studies should consider not just the effect of individual mutations but also their relative positions

• Simulations and models should incorporate multiple c-subunits to accurately represent the functional unit

• Kinetic analyses need to account for cooperative effects rather than treating each c-subunit as an independent entity

How do pH gradients and membrane potential specifically affect ATP synthesis mediated by ATP synthase subunit c?

ATP synthesis is driven by the proton motive force (pmf), which comprises both the membrane potential (Δψ) and the proton gradient (ΔpH). In vitro studies with reconstituted ATP synthase demonstrate complex relationships between these parameters:

• A ΔpH of ≥1 is required for robust ATP synthesis when combined with a Δψ of ~140 mV

• ATP synthesis rates increase with increasing pmf up to a point, but plateau at higher N-side pH values (above 8)

• The absolute P-side pH value significantly impacts ATP synthesis rates, even when the total pmf remains constant

• With a constant ΔpH of 1.5 and Δψ of ~140 mV (total pmf ~230 mV), ATP synthesis at P-side pH 7/N-side pH 8.5 is only 10-20% of the activity at P-side pH 5.5/N-side pH 7

This indicates that while thermodynamic considerations are important, the absolute pH values on either side of the membrane significantly impact the molecular mechanisms of proton transport through the c-ring. This is likely due to asymmetric ion access half-channels to the c-ring binding sites from the P- and N-sides of the membrane .

What are the current challenges in expressing and purifying functional recombinant ATP synthase subunit c for structural studies?

Expression and purification of functional ATP synthase subunit c presents several challenges:

• The hydrophobic nature of the protein requires specialized solubilization and purification protocols

• Maintaining the native oligomeric state during purification is difficult but essential for functional studies

• Post-translational modifications that may be important for function can be lost in recombinant expression systems

• Reconstitution into liposomes requires careful control of lipid composition and protein orientation

• The small size (~8 kDa) makes standard protein purification tags potentially disruptive to structure and function

Researchers have addressed these challenges through approaches such as genetic fusion of multiple c-subunits into a single polypeptide to facilitate controlled mutation studies and using specialized membrane preparation techniques optimized for photosynthetic organisms .

How can researchers effectively use antibodies against ATP synthase subunit c in their studies?

Researchers can optimize antibody-based studies of ATP synthase subunit c through the following approaches:

• Antibody selection: Use polyclonal antibodies raised against KLH-conjugated peptides derived from ATP synthase subunit c sequences. For cross-species studies, antibodies raised against conserved regions of Arabidopsis thaliana (UniProt: P56760) may recognize homologous proteins in related species .

• Western blotting optimization:

  • Recommended dilution: 1:10,000

  • Expected molecular weight: ~8 kDa (based on Arabidopsis thaliana)

  • Sample preparation: Follow specialized membrane preparation protocols such as those described by Lezhneva et al. (2008) for Arabidopsis or Chua & Bennoun (1975) for Chlamydomonas

• Antibody storage and handling:

  • Store lyophilized or reconstituted antibodies at -20°C

  • Make aliquots after reconstitution to avoid repeated freeze-thaw cycles

  • Briefly spin tubes before opening to avoid loss of material

• Controls: Include appropriate positive controls (e.g., purified recombinant protein) and negative controls (e.g., samples from knockout organisms if available) to validate specificity.

What are the optimal protocols for reconstituting ATP synthase into proteoliposomes for functional studies?

Functional reconstitution of ATP synthase into proteoliposomes involves several critical steps:

How can mutation studies be designed to investigate cooperation among c-subunits in the ATP synthase complex?

Effective mutation studies to investigate c-subunit cooperation should:

• Use genetically fused single-chain c-rings:

  • This approach, demonstrated with thermophilic Bacillus PS3, allows precise control over the position and number of mutations within the c-ring

  • Multiple c-subunits are expressed as a single polypeptide, ensuring stoichiometric incorporation of wild-type and mutated subunits

• Design strategic mutation patterns:

  • Introduce mutations at conserved, functionally important residues (e.g., E56D in Bacillus PS3)

  • Create constructs with single mutations, double mutations at adjacent positions, and double mutations at distant positions within the c-ring

  • The relative positioning of mutations provides insights into cooperative mechanisms

• Employ complementary functional assays:

  • Measure ATP synthesis activity under standardized conditions

  • Assess proton pumping capability with pH-sensitive probes

  • Compare activities across different mutation patterns while keeping the total number of mutations constant

• Combine with molecular simulations:

  • Use proton transfer-coupled molecular dynamics simulations to interpret experimental results

  • Analyze trajectories for parameters such as proton uptake duration time

  • Identify mechanisms of time-sharing or other cooperative phenomena between adjacent or distant c-subunits

How do environmental factors influence ATP synthase subunit c function in different organisms?

Environmental factors significantly impact ATP synthase function through multiple mechanisms:

• pH effects:

  • The absolute pH value on both sides of the membrane critically affects ATP synthesis rates

  • Even with identical proton motive force, ATP synthesis at P-side pH 7/N-side pH 8.5 is only 10-20% compared to activity at P-side pH 5.5/N-side pH 7

  • This suggests specific pH requirements for optimal proton binding and release by the c-ring subunits

• Temperature considerations:

  • Thermophilic organisms (like Bacillus PS3) have temperature-adapted ATP synthases

  • Temperature affects both protein dynamics and proton equilibration rates

  • Experimental conditions must be optimized for the source organism of the ATP synthase being studied

• Lipid environment:

  • The composition of the lipid bilayer affects c-ring rotation and proton access channels

  • Reconstitution studies should consider the native lipid environment of the source organism

  • Lipid-protein interactions may contribute to species-specific functional adaptations

• Ionic strength:

  • Ion concentrations affect both membrane potential and protein conformational dynamics

  • Physiological ion gradients should be considered when designing in vitro experiments

What are the current limitations in computational modeling of ATP synthase subunit c function?

Computational modeling of ATP synthase subunit c faces several challenges:

How might the study of ATP synthase subunit c inform the development of bioinspired energy conversion systems?

ATP synthase subunit c research offers several insights for bioinspired technologies:

• Rotary molecular motor design:

  • The c-ring functions as a highly efficient rotary motor with nearly 100% energy conversion efficiency

  • Understanding the principles of proton-coupled rotation could inform synthetic molecular motor design

  • The cooperative mechanisms among c-subunits might inspire new approaches to coordinated molecular motion

• pH-gradient energy harvesting:

  • Natural ATP synthase effectively converts small pH gradients into useful chemical energy

  • This principle could be applied to harvest energy from industrial waste streams or natural pH gradients

  • The sensitivity to absolute pH values, not just gradients, should inform design parameters

• Self-assembly mechanisms:

  • The c-ring forms through the assembly of multiple identical subunits into a stable, functional complex

  • This self-assembly principle could inspire materials with programmable structure formation

  • Understanding the factors that determine c-ring stoichiometry could inform design rules for synthetic assemblies

• Biomimetic materials:

  • The structure of the c-ring balances rigidity (for maintaining structure) with flexibility (for function)

  • This balance could inspire new materials with tailored mechanical properties

  • The proton-binding sites could inform the design of ion-selective materials or membranes