Recombinant Dioscorea elephantipes ATP synthase subunit c, chloroplastic (atpH)

Expression System Selection:

• E. coli is the preferred host for recombinant expression, with BL21(DE3) strains being particularly suitable .

• Expression as a fusion protein with maltose binding protein (MBP) significantly enhances solubility and facilitates purification .

• Employing T7-based expression vectors with controlled induction parameters optimizes yield.

Purification Strategy:

• Initial purification using affinity chromatography (e.g., amylose resin for MBP-fusion proteins)

• Cleavage of fusion tag using specific proteases (e.g., TEV protease)

• Secondary purification steps such as size exclusion chromatography

• Buffer optimization with appropriate detergents to maintain protein stability

Critical Considerations:

• Expression temperature should be lowered (16-25°C) to minimize inclusion body formation

• Induction with lower IPTG concentrations (0.1-0.5 mM) often improves soluble protein yield

• Protein folding verification using circular dichroism spectroscopy is essential to confirm native α-helical structure

• Storage in glycerol-containing buffers (typically 50%) at -20°C maintains stability for extended periods

This approach has yielded significant quantities of highly purified c-subunit with confirmed α-helical secondary structure .

• How is ATP synthase activity regulated in photosynthetic organisms?

ATP synthase in photosynthetic organisms employs a sophisticated light-dependent regulatory mechanism that differs significantly from other systems:

Redox-Regulated Mechanism:

In photosynthetic organisms, ATP synthase activity is modulated through a thioredoxin-mediated redox system that responds to light intensity . This regulatory pathway operates as follows:

• Under sufficient light conditions, linear electron flow reduces thioredoxin

• Reduced thioredoxin subsequently reduces a specific redox switch in the γ-subunit of ATP synthase

• In this reduced state, ATP synthase maintains full activity for ATP synthesis

• In low light or darkness, the redox switch becomes oxidized, inhibiting enzyme activity

This mechanism prevents wasteful ATP hydrolysis when light is unavailable for photosynthesis, effectively conserving cellular energy resources .

Structural Basis:

Recent cryo-EM studies have elucidated the structural basis of this regulation, revealing that the γ-subunit serves as the key structural element in redox regulation . Conformational changes between oxidized (inactive) and reduced (active) states determine enzyme activity.

The oxidized state can be experimentally converted to the active form using reducing agents such as dithiothreitol (DTT), providing a valuable experimental tool for investigating this regulatory mechanism .

Advanced Research Questions

• What factors influence c-ring stoichiometry in ATP synthases from different photosynthetic organisms?

The c-ring stoichiometry in ATP synthases varies across different photosynthetic organisms, with significant implications for bioenergetic efficiency:

Observed Stoichiometric Variations:

• Plant chloroplasts: c14 ring structure

• Cyanobacteria: c13-c15 ring structures

• Total range across all organisms: c8-c17

These variations directly influence the ion-to-ATP ratio, which ranges from 4.7 in plants to 4.3-5.0 in cyanobacteria, significantly higher than in most non-photosynthetic organisms .

Bioenergetic Implications:

The higher ion-to-ATP ratios observed in photosynthetic organisms reflect an evolutionary adaptation that enables ATP synthesis under conditions of low proton motive force. This adaptation is particularly valuable in photosynthetic systems where light fluctuations can cause rapid changes in pmf .

Under high-light conditions, photosynthetic organisms can establish substantial pH gradients across thylakoid membranes (up to 2.5 units), corresponding to approximately -180 mV of total pmf . The larger c-ring allows ATP synthesis to continue efficiently even when this gradient diminishes under lower light conditions.

Methodological Approaches for Studying c-ring Stoichiometry:

• High-resolution structural determination via cryo-electron microscopy

• X-ray crystallography of isolated c-rings

• Mass spectrometry-based approaches for subunit counting

• Biochemical cross-linking followed by electrophoretic analysis

• Biophysical techniques such as atomic force microscopy

Understanding the factors that influence c-ring stoichiometry provides insights into the bioenergetic adaptations of photosynthetic organisms to their specific environmental niches.

• How can researchers investigate the structure-function relationship of ATP synthase subunit c through site-directed mutagenesis?

Site-directed mutagenesis represents a powerful approach for elucidating structure-function relationships in ATP synthase subunit c:

Key Residues for Targeted Mutagenesis:

• Proton-binding residues in transmembrane helices (particularly conserved acidic residues)

• Residues involved in subunit-subunit interactions within the c-ring

• Amino acids at interfaces with other ATP synthase components

• Residues potentially involved in regulatory mechanisms

Experimental Design Framework:

A comprehensive mutagenesis strategy should include:

Methodological Workflow:

• Design mutations based on sequence conservation analysis and structural information

• Generate mutant constructs using PCR-based mutagenesis techniques

• Express and purify mutant proteins using established protocols

• Assess structural integrity via circular dichroism and thermal stability assays

• Evaluate functional impact through ATP synthesis/hydrolysis assays

• Determine effects on c-ring assembly and protein-protein interactions

This approach enables researchers to systematically map functional domains within the ATP synthase subunit c and understand how specific amino acid residues contribute to its catalytic mechanism, assembly, and regulation.

• What are the challenges and solutions in c-ring reconstitution from recombinant subunits?

Reconstitution of functional c-rings from recombinantly expressed subunits presents significant challenges that must be addressed through specialized methodologies:

Major Challenges:

• Assembly Barriers:

  • Ensuring correct stoichiometry in the reconstituted ring

  • Maintaining proper orientation of highly hydrophobic subunits

  • Achieving thermodynamically stable subunit interactions

  • Preventing non-specific aggregation during assembly

• Functional Verification:

  • Demonstrating proton translocation capability

  • Assessing rotational competence

  • Verifying interaction with other ATP synthase components

  • Comparing properties with native c-rings

Methodological Solutions:

The recombinant production of individual c1 subunits has been successfully demonstrated using fusion protein approaches in E. coli expression systems . The key steps toward successful reconstitution include:

• Optimized Detergent Selection:

  • Screening multiple detergents for optimal solubilization

  • Introducing lipids during purification to enhance stability

  • Using mild detergents that preserve native-like interactions

• Controlled Assembly Conditions:

  • Gradual detergent removal through dialysis

  • Manipulation of pH and ionic strength to promote correct oligomerization

  • Temperature-controlled assembly protocols

  • Step-wise assembly with monitored intermediate states

• Verification Approaches:

  • Size exclusion chromatography to confirm homogeneous assembly

  • Circular dichroism to verify α-helical secondary structure

  • Thermal stability assays to assess complex integrity

  • Functional reconstitution into liposomes for activity testing

If successful, recombinant reconstitution provides unique opportunities to explore c-ring assembly factors and investigate the molecular determinants of c-ring stoichiometry, which remain incompletely understood despite their significant bioenergetic implications .

• How do inhibitors target ATP synthase in photosynthetic organisms and what are their research applications?

ATP synthase inhibitors provide valuable research tools for investigating enzyme structure, function, and regulation in photosynthetic organisms:

Inhibitor Classes and Mechanisms:

Research Applications:

• Structural studies:

  • Co-crystallization with inhibitors reveals binding pockets

  • Identification of functional domains through differential inhibition

  • Elucidation of conformational states through inhibitor stabilization

• Mechanistic investigations:

  • Dissection of rotational steps using step-wise inhibition

  • Analysis of catalytic cooperativity through partial inhibition

  • Discrimination between ATP synthesis and hydrolysis mechanisms

• Physiological studies:

  • Manipulation of energy-dependent processes in chloroplasts

  • Investigation of regulatory responses to energetic stress

  • Analysis of ATP synthase's role in photoprotection

Experimental Approaches:

• In vitro enzyme assays with purified ATP synthase to determine inhibition kinetics

• Binding studies using isothermal titration calorimetry or surface plasmon resonance

• Structural analysis of enzyme-inhibitor complexes via cryo-EM or X-ray crystallography

• Physiological studies in isolated chloroplasts or intact photosynthetic cells

Understanding the interaction of inhibitors with ATP synthase in photosynthetic organisms not only advances basic knowledge but also provides potential applications in herbicide development and pharmacological targeting .

• What methodologies are most effective for studying redox regulation of chloroplastic ATP synthase?

The redox regulation of chloroplastic ATP synthase represents a unique adaptation of photosynthetic organisms that requires specialized methodological approaches:

Key Experimental Approaches:

• Biochemical Activity Assays Under Controlled Redox Conditions:

  • ATP synthesis measurements using luciferin/luciferase systems

  • ATPase activity assays with phosphate release detection

  • Comparison of activity in oxidized vs. DTT-reduced states

  • Thiol-trapping experiments to track reduction state

• Structural Analysis of Redox-Dependent Conformational Changes:

  • Cryo-electron microscopy of ATP synthase in oxidized vs. reduced states

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational dynamics

  • Fluorescence resonance energy transfer (FRET) to monitor distance changes between subunits

  • Cross-linking analysis under different redox conditions

• Redox Sensor Integration:

  • Site-directed incorporation of fluorescent probes at redox-sensitive sites

  • Real-time monitoring of conformational changes using spectroscopic techniques

  • Development of genetically encoded redox sensors fused to ATP synthase components

Technical Considerations:

• Maintenance of defined redox potential during experiments through buffer optimization

• Use of physiologically relevant thioredoxin systems rather than chemical reductants alone

• Correlation of structural changes with functional effects through combined approaches

• Light-dependent activation considerations in experimental design

Recent structural studies have revealed that the γ-subunit serves as the key element in redox regulation, with tentoxin being useful for stabilizing the reduced state during structural analysis . These approaches have provided valuable insights into how light-dependent redox changes modulate ATP synthase activity in photosynthetic organisms.

• What bioinformatic approaches are valuable for comparative analysis of ATP synthase subunit c across photosynthetic species?

Bioinformatic analysis provides critical insights into evolutionary relationships, functional conservation, and structural determinants of ATP synthase subunit c across photosynthetic species:

Essential Bioinformatic Methods:

• Sequence Alignment and Conservation Analysis:

  • Multiple sequence alignment of ATP synthase subunit c sequences from diverse photosynthetic organisms

  • Identification of highly conserved residues critical for function

  • Calculation of conservation scores to highlight functionally important regions

  • Correlation of sequence conservation with known structural elements

• Phylogenetic Analysis:

  • Construction of phylogenetic trees to examine evolutionary relationships

  • Analysis of sequence divergence patterns in relation to c-ring stoichiometry

  • Investigation of clade-specific adaptations in photosynthetic organisms

  • Correlation with environmental niches and photosynthetic strategies

• Structural Bioinformatics:

  • Homology modeling based on available crystal structures

  • Molecular dynamics simulations to assess conformational flexibility

  • Protein-protein interaction prediction for c-c subunit interfaces

  • Electrostatic potential mapping to identify proton pathways

Comparative Analysis Framework:

Analysis of the Dioscorea elephantipes ATP synthase subunit c (atpH) sequence (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) against other plant species reveals:

These bioinformatic approaches can identify candidate residues for site-directed mutagenesis, predict functional effects of sequence variations, and provide insights into the molecular basis of c-ring stoichiometry differences observed across photosynthetic species.