Recombinant Saccharum officinarum ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in chloroplasts?

The chloroplast ATP synthase subunit c is a critical component of the membrane-embedded Fo motor of the ATP synthase complex. It forms a ring structure embedded in the thylakoid membrane that rotates during ATP synthesis. This rotation is mechanically coupled to ATP production in the F1 head of the complex through a central stalk. The c-subunit ring plays a crucial role in proton translocation across the membrane along an electrochemical gradient established during photosynthesis .

In structural terms, the c-subunit has an alpha-helical secondary structure, with hydrophobic regions allowing it to be embedded within the thylakoid membrane. The number of c-subunits forming the oligomeric ring (cn) varies between organisms and affects the ratio of protons translocated to ATP synthesized . This stoichiometric variation directly impacts the bioenergetic efficiency of photosynthesis.

How does recombinant expression of chloroplastic atpH differ from native protein isolation?

Recombinant expression offers several significant advantages over native protein isolation:

• Yield control: Recombinant systems can provide significantly higher quantities of purified protein compared to isolation from plant tissues.

• Genetic manipulation: The gene sequence can be optimized for expression in the host system, enhancing protein yields.

• Fusion tag options: Expression as fusion proteins (e.g., MBP-fusion) can improve solubility of otherwise hydrophobic membrane proteins.

• Purification efficiency: Column purification methods can achieve higher purity levels than traditional isolation.

What expression systems are suitable for producing recombinant Saccharum officinarum atpH?

For hydrophobic membrane proteins like ATP synthase subunit c, the bacterial expression system with fusion partners has been successfully employed. The approach of expressing the hydrophobic c-subunit as a soluble MBP-fusion protein, followed by cleavage and reversed phase column purification, enables significant quantities of purified protein with correct alpha-helical secondary structure to be obtained .

What purification methods are most effective for recombinant atpH?

Based on existing research methodologies, an effective purification strategy for recombinant ATP synthase subunit c involves:

• Initial fusion protein approach: Express as a fusion protein with a solubility-enhancing partner (e.g., MBP) to overcome the hydrophobic nature of the c-subunit.

• Affinity chromatography: Purify the fusion protein using affinity chromatography (e.g., amylose resin for MBP-tagged proteins).

• Protease cleavage: Precisely cleave the fusion tag using a specific protease.

• Reversed phase chromatography: Purify the cleaved c-subunit using reversed phase column chromatography, which is particularly effective for hydrophobic membrane proteins.

• Verification steps: Confirm the secondary structure of the purified protein (e.g., circular dichroism to verify alpha-helical structure) .

This multi-step approach has proven successful for obtaining highly purified c-subunit with the correct structural properties.

How does the c-ring stoichiometry in Saccharum officinarum ATP synthase impact its bioenergetic efficiency?

The c-ring stoichiometry (the number of c-subunits per oligomeric ring) directly determines the proton-to-ATP ratio, a crucial parameter affecting photosynthetic efficiency. In chloroplast ATP synthases, this stoichiometry varies between species and influences the thermodynamic efficiency of energy conversion.

The relationship between c-ring stoichiometry and bioenergetic efficiency can be expressed as:

$\text{Efficiency} = \frac{\Delta G_{ATP}}{\text{n} \times \Delta\mu_{H^+}}$

Where:

• $\Delta G_{ATP}$ is the free energy of ATP synthesis (approximately 51 kJ/mol under physiological conditions in chloroplasts)

• $\text{n}$ is the number of c-subunits in the ring

• $\Delta\mu_{H^+}$ is the electrochemical potential across the membrane

In studying Saccharum officinarum, researchers need to determine the specific c-ring stoichiometry, as it may differ from other photosynthetic organisms. Understanding this parameter is critical for comparative studies of photosynthetic efficiency across different plant species and for engineering approaches aimed at optimizing energy conversion.

Current research suggests that the rotation of the c-ring in chloroplast ATP synthase occurs in three unequal steps (103°, 112°, and 145°), corresponding to 4, 4.4, and 5.6 c-subunits per step, respectively . This asymmetric rotation mechanism has significant implications for understanding energy conversion efficiency.

What methodological approaches can resolve structural interactions between recombinant atpH and other ATP synthase subunits?

Resolving the structural interactions between ATP synthase subunits requires sophisticated methodological approaches:

• Cryo-electron microscopy (cryo-EM): High-resolution cryo-EM has successfully determined the structure of complete chloroplast ATP synthase complexes, resolving side chains of all protein subunits, nucleotides, and proton pathways. This approach can reveal how subunit c interacts with other components .

• Cross-linking coupled with mass spectrometry: This approach can identify interaction interfaces between subunits by chemically linking closely associated amino acids and identifying these linkages through mass spectrometry.

• Molecular dynamics simulations: Based on structural data, simulations can predict dynamic interactions between subunits during the rotation cycle.

• Site-directed mutagenesis: Systematic mutation of specific residues at potential interaction interfaces, followed by functional assays, can identify critical interaction points.

• Reconstitution experiments: Combining purified recombinant subunits to reconstruct functional subcomplexes can elucidate assembly mechanisms and subunit interdependencies.

These approaches can be implemented hierarchically, starting with high-resolution structural determination, followed by targeted investigations of specific interactions identified in the structural models.

How can researchers address the hydrophobicity challenges when working with recombinant atpH?

The extreme hydrophobicity of ATP synthase subunit c presents significant challenges for recombinant expression, purification, and functional studies. Advanced strategies to address these challenges include:

• Optimized fusion protein design: Beyond standard MBP fusions, researchers can:

  • Test multiple fusion partners with different properties (size, charge, solubility)

  • Explore dual fusion tags (N- and C-terminal)

  • Optimize linker length and composition between the fusion tag and atpH

• Membrane-mimetic environments for purified protein:

  • Detergent selection is critical - test a panel of detergents (mild, non-ionic, zwitterionic)

  • Lipid nanodisc incorporation to maintain native-like membrane environment

  • Amphipol stabilization for structural studies

• Expression condition optimization:

  • Controlled slow expression (reduced temperature, low inducer concentration)

  • Specialized E. coli strains designed for membrane protein expression

  • Co-expression with chaperones to aid proper folding

• Advanced purification approaches:

  • Detergent exchange during purification steps

  • On-column refolding protocols

  • Size-exclusion chromatography in appropriate detergent micelles

Implementation of these strategies must be empirically optimized for the specific properties of Saccharum officinarum atpH, as membrane protein behavior can vary considerably even between closely related proteins .

What factors affect the assembly of recombinant atpH into functional c-rings?

The assembly of individual c-subunits into functional c-rings is a complex process influenced by multiple factors:

Experimental approaches to study c-ring assembly include:

• In vitro reconstitution of purified c-subunits under controlled conditions

• Atomic force microscopy to visualize assembly intermediates

• Chemical cross-linking to capture assembly states

• Native mass spectrometry to determine oligomeric state distributions

• Förster resonance energy transfer (FRET) between labeled subunits to monitor assembly kinetics

Understanding the assembly process is crucial for producing functional recombinant c-rings for structural and functional studies .

How can researchers verify the functional integrity of recombinant atpH?

Verifying the functional integrity of recombinant ATP synthase subunit c requires multiple complementary approaches:

• Structural verification:

  • Circular dichroism spectroscopy to confirm alpha-helical secondary structure

  • NMR spectroscopy for atomic-level structural characterization

  • Thermal stability assays to assess protein folding quality

• Assembly competence:

  • Oligomerization assays to verify ability to form c-rings

  • Electron microscopy to visualize assembled structures

  • Cross-linking studies to assess subunit-subunit interactions

• Functional reconstitution:

  • Incorporation into liposomes or nanodiscs with other ATP synthase components

  • Proton conductance measurements in reconstituted systems

  • ATP synthesis/hydrolysis assays in reconstituted complexes

• Comparative analysis:

  • Direct comparison with native protein isolated from Saccharum officinarum

  • Complementation assays in mutant systems lacking functional c-subunits

These verification steps are essential before using the recombinant protein for further structural or functional studies, as improperly folded or non-functional protein would lead to misleading results in downstream applications.

Genetic engineering offers powerful approaches to modify ATP synthase subunit c for various research objectives:

• Site-directed mutagenesis applications:

  • Altering proton-binding residues to study proton translocation mechanisms

  • Modifying subunit-subunit interaction sites to investigate assembly determinants

  • Introducing cysteine residues for site-specific labeling and cross-linking studies

  • Creating chimeric proteins with c-subunits from different species to identify functional domains

• Fluorescent protein fusions:

  • Strategic placement of fluorescent proteins or peptides for tracking without disrupting function

  • FRET pairs to monitor conformational changes or protein-protein interactions

  • pH-sensitive fluorescent tags to monitor local proton concentration changes

• Affinity tag engineering:

  • Developing minimally disruptive tags for purification

  • Inducible degron tags for controlled protein depletion studies

  • Split-protein complementation systems to study assembly in vivo

• Expression control elements:

  • Inducible promoters for temporal control of expression

  • Tissue-specific promoters for spatial control in plant systems

  • Synthetic ribosome binding sites for expression level optimization

Each modification strategy requires careful consideration of the structure-function relationship to ensure that the modified protein retains relevant native properties while incorporating the desired engineered features .

What technical challenges exist in comparing mitochondrial and chloroplastic ATP synthase from Saccharum officinarum?

Researchers face several technical challenges when comparing the mitochondrial and chloroplastic ATP synthases from Saccharum officinarum:

• Isolation complexity:

  • Different subcellular fractionation protocols required for each organelle

  • Cross-contamination between organelles during isolation

  • Maintaining integrity of membrane protein complexes during purification

• Structural and compositional differences:

  • Different subunit composition between mitochondrial and chloroplastic complexes

  • Structural variations affecting antibody recognition and purification strategies

  • Distinct lipid requirements for maintaining native structure and function

• Functional assay considerations:

  • Different optimal conditions for activity measurements

  • Variations in regulatory mechanisms requiring distinct assay designs

  • Challenges in normalizing activity between different complexes

• Genetic manipulation limitations:

  • Different genetic systems governing expression (nuclear vs. organellar genomes)

  • Challenges in creating comparable mutations in both systems

  • Difficulty in obtaining viable cells with ATP synthase deletions, as demonstrated in studies with related organisms

Addressing these challenges requires integrated approaches combining:

• Careful subcellular fractionation techniques

• Differential tagging strategies for specific isolation

• Complementary functional assays under standardized conditions

• Advanced structural biology methods to compare assembled complexes

Recent studies on mitochondrial genome structural variants in Saccharum species provide valuable insight for comparative analysis of organellar ATP synthases .

How might the study of recombinant atpH contribute to understanding cytoplasmic male sterility in Saccharum officinarum?

Cytoplasmic male sterility (CMS) in Saccharum officinarum presents a significant breeding challenge and research opportunity. The connection between ATP synthase function and CMS involves several potential mechanisms:

• Energy production impairment: Altered ATP synthase efficiency could limit energy availability during pollen development. Recombinant atpH studies can help quantify energy production differences between fertile and sterile lines.

• Mitochondrial-chloroplast interactions: While cytoplasmic male sterility is typically associated with mitochondrial function, interactions between chloroplastic and mitochondrial energy production systems are crucial, particularly in developing anthers. The study of recombinant atpH can help elucidate these organellar interactions .

• Comparative functional analysis: Expressing recombinant atpH from both sterile and fertile Saccharum officinarum lines can reveal functional differences that might contribute to CMS.

• Chimeric protein effects: Recent research has identified that mitochondrial chimeric gene ORF113 is highly expressed in male-sterile S. officinarum flowers and significantly inhibits ATP synthesis when expressed in yeast cells. Understanding how such chimeric proteins interact with ATP synthase components, including potentially the chloroplastic c-subunit, could provide key insights into CMS mechanisms .

This research direction has significant implications for sugarcane breeding programs, as pollen sterility in S. officinarum currently restricts its role to being a female parent in crosses with S. spontaneum, resulting in a narrow genetic basis for modern sugarcane cultivars .

What are the latest techniques for studying proton translocation through the c-ring in real-time?

Advanced techniques for real-time monitoring of proton translocation through ATP synthase c-rings represent the cutting edge of bioenergetics research:

• Single-molecule FRET imaging:

  • Strategic placement of fluorophore pairs on c-subunits and adjacent components

  • Real-time monitoring of conformational changes during proton translocation

  • Correlation of FRET signals with ATP synthesis rates

• pH-sensitive fluorescent probes:

  • Site-specific incorporation of pH-sensitive fluorescent amino acids

  • Integration of genetically encoded pH sensors near proton channels

  • Microfluidic platforms for rapid modulation of pH gradients during measurements

• Electrical recording techniques:

  • Solid-supported membrane electrophysiology for monitoring proton currents

  • Nanoscale electrochemical detection systems integrated with protein reconstitution platforms

  • Patch-clamp techniques adapted for organellar membranes

• Advanced spectroscopic methods:

  • Time-resolved infrared spectroscopy to detect protonation state changes

  • NMR spectroscopy with isotope-labeled specific residues involved in proton translocation

  • Raman spectroscopy for monitoring structural changes associated with proton movement

• Computational approaches:

  • Quantum mechanics/molecular mechanics simulations of proton transfer events

  • Markov state modeling of proton movement through the c-ring

  • Machine learning analysis of experimental data to identify proton translocation patterns

These techniques can be applied to recombinant Saccharum officinarum atpH systems to understand the specific properties of proton translocation in this species, with potential implications for optimizing photosynthetic efficiency .

How does the redox regulation of ATP synthase differ between Saccharum officinarum and other plant species?

Redox regulation represents a critical control mechanism for chloroplast ATP synthase activity, adapting energy production to changing environmental conditions. Species-specific differences in this regulation may reflect evolutionary adaptations to different habitats and photosynthetic requirements.

Key aspects of ATP synthase redox regulation include:

• γ-subunit redox switch: Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark. Species variations in this regulatory element can significantly impact energy conservation strategies .

• Thioredoxin interaction sites: The number, location, and redox potential of regulatory cysteines can vary between species, potentially leading to different thresholds for activation/inactivation.

• Kinetics of redox response: The rate at which ATP synthase activity responds to changing redox conditions may differ between species, reflecting adaptation to fluctuating light environments.

• Integration with other photosynthetic processes: Coordination between ATP synthase regulation and other redox-regulated photosynthetic processes may show species-specific optimization.

Experimental approaches to compare redox regulation between species include:

• Site-directed mutagenesis of potential regulatory cysteines

• Activity assays under controlled redox conditions

• Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

• Comparative structural analysis of the γ-subunit regulatory region

Saccharum officinarum, as a C4 plant, may exhibit distinctive redox regulation characteristics compared to C3 species, potentially contributing to its photosynthetic efficiency under high-light, high-temperature conditions .

What experimental approaches can determine if the atpH gene in Saccharum officinarum is essential under various growth conditions?

Determining the essentiality of the atpH gene under different growth conditions requires sophisticated experimental approaches that overcome the challenges of manipulating genes essential for energy production:

• Conditional knockout strategies:

  • Inducible gene silencing using RNA interference or CRISPR interference

  • Temperature-sensitive mutations that maintain function under permissive conditions

  • Chemical-inducible protein degradation systems

• Complementation approaches:

  • Introduction of heterologous atpH genes under control of inducible promoters

  • Variant atpH alleles with altered function under specific conditions

  • The gene transfer agent transduction combined with conjugation method, as demonstrated in related ATP synthase studies

• Growth condition parameters to test:

  • Varying light intensities and spectral qualities

  • Different carbon sources and concentrations

  • Aerobic versus anaerobic conditions

  • Temperature stress conditions

  • Drought and osmotic stress

• Phenotypic and molecular analyses:

  • Photosynthetic efficiency measurements

  • Growth rate and biomass accumulation

  • Metabolomic profiling to detect compensatory pathways

  • Transcriptomic analysis to identify adaptive responses

Previous research has demonstrated that ATP synthase genes can be essential under standard growth conditions, as attempts to obtain viable cells carrying deletions were unsuccessful despite extensive efforts . This emphasizes the need for sophisticated conditional approaches when studying potentially essential genes like atpH.

What are the optimal buffer conditions for maintaining stability of purified recombinant atpH?

Maintaining stability of purified recombinant ATP synthase subunit c requires carefully optimized buffer conditions that address its hydrophobic membrane protein nature:

Additional stability considerations include:

• Storage temperature (typically 4°C for short-term, -80°C for long-term with flash-freezing)

• Avoidance of freeze-thaw cycles

• Protection from light if photosensitive components are present

• Addition of protease inhibitors to prevent degradation

Empirical stability testing using techniques such as size-exclusion chromatography, light scattering, and activity assays over time is essential to optimize conditions for specific recombinant protein preparations .

How can researchers troubleshoot low expression yields of recombinant atpH?

Low expression yields of recombinant ATP synthase subunit c are a common challenge due to its hydrophobic nature. A systematic troubleshooting approach includes:

• Genetic construct optimization:

  • Codon optimization for expression host (critical for plant genes in bacterial systems)

  • Evaluation of different fusion partners beyond standard MBP (e.g., TrxA, SUMO, GST)

  • Optimization of ribosome binding site strength and spacing

  • Assessment of rare codon usage and tRNA supplementation needs

• Expression conditions optimization:

  • Induction parameter screening (inducer concentration, temperature, timing)

  • Growth media formulation (rich vs. minimal, supplementation with specific amino acids)

  • Cell density at induction (mid-log vs. late log phase)

  • Duration of expression (short high-intensity vs. longer lower-intensity)

• Host strain selection:

  • Testing specialized strains designed for membrane proteins

  • Evaluation of strains with modified proteases or chaperone systems

  • Consideration of eukaryotic expression systems for complex proteins

• Solubilization and detection methods:

  • Verification that protein is expressed but in inclusion bodies

  • Optimization of cell lysis conditions to prevent aggregation

  • Evaluation of different detergents for efficient extraction

  • Enhanced detection methods for low-abundance expression

A systematic approach using design of experiments (DOE) methodology to simultaneously optimize multiple parameters can efficiently identify conditions for improved yields . For example, successful recombinant expression of spinach chloroplast ATP synthase subunit c was achieved using codon optimization and an MBP fusion approach in E. coli BL21 cells .

What experimental controls are essential when studying the interaction of recombinant atpH with other ATP synthase subunits?

• Protein quality controls:

  • Verification of proper folding of all protein components

  • Assessment of oligomeric state and homogeneity

  • Confirmation of functional activity where possible

  • Thermal stability analysis before interaction studies

• Binding specificity controls:

  • Non-binding mutants of interaction partners

  • Competition with unlabeled proteins

  • Irrelevant proteins of similar structural characteristics

  • Detergent/lipid-only controls to exclude non-specific effects

• Technical method controls:

  • For pull-down assays: control beads without bait protein

  • For FRET: single-labeled controls and acceptor photobleaching

  • For co-immunoprecipitation: non-specific antibodies and pre-immune serum

  • For surface plasmon resonance: reference surfaces and buffer injections

• Assembly state controls:

  • Monomeric vs. oligomeric forms of components

  • Time-dependent assembly monitoring

  • Concentration-dependent effects on interactions

  • Effects of energy state (ATP/ADP ratio) on interactions

• Environmental condition controls:

  • pH dependence of interactions

  • Salt concentration effects

  • Temperature effects on binding kinetics

  • Lipid composition influence on protein-protein interactions

Implementation of these controls ensures that observed interactions are specific, biologically relevant, and not artifacts of the experimental system .

How can functional assays for recombinant atpH be designed to assess proton translocation efficiency?

Designing functional assays to assess proton translocation efficiency of recombinant ATP synthase subunit c requires sophisticated approaches that link structural integrity to functional capacity:

• Reconstitution systems:

  • Liposome reconstitution with purified recombinant c-subunits

  • Co-reconstitution with minimal components required for proton translocation

  • Creation of defined proton gradients across membranes

  • Integration with other ATP synthase components to form functional complexes

• Proton flux measurement approaches:

  • pH-sensitive fluorescent dyes entrapped in liposomes

  • Ion-selective microelectrodes to measure local pH changes

  • Patch-clamp electrophysiology of reconstituted membranes

  • Isotope exchange measurements using deuterium or tritium

• Coupling to ATP synthesis:

  • Linked enzyme assays to measure ATP production

  • Luciferase-based real-time ATP detection systems

  • Correlation of proton flux with ATP synthesis rates

  • Determination of H⁺/ATP ratios under varying conditions

• Comparative performance metrics:

  • Maximal proton translocation rate (Vmax)

  • Proton affinity (apparent Km for protons)

  • Efficiency of coupling between proton translocation and ATP synthesis

  • Thermodynamic efficiency calculations based on measured parameters

These assays should incorporate appropriate controls, including inactive mutants, uncoupled systems, and comparative analysis with native protein complexes. The development of standardized assays would facilitate comparison between different experimental systems and species-specific variations in ATP synthase function .