Recombinant Solanum lycopersicum ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c in tomato chloroplasts forms part of the membrane-embedded Fo portion of the ATP synthase complex. This small 81 amino acid protein typically consists of two hydrophobic alpha-helices connected by a polar loop region . Multiple copies of this subunit arrange in a ring structure within the thylakoid membrane, containing conserved carboxyl groups that participate in proton translocation. During photosynthesis, the light-driven electron transport chain establishes a proton gradient across the thylakoid membrane. As protons flow through the c-ring, they cause it to rotate, which mechanically couples to the central stalk of the F1 portion. This rotation drives conformational changes in the catalytic sites where ADP and inorganic phosphate combine to form ATP. The c-subunit's role in this rotary mechanism is crucial for converting the energy of the proton gradient into the chemical energy of ATP.

How does the tomato ATP synthase subunit c differ from homologs in other plant species?

While ATP synthase subunit c is highly conserved across plant species due to its fundamental role in energy conversion, the Solanum lycopersicum variant exhibits subtle sequence differences that may reflect species-specific adaptations. These differences primarily occur in non-catalytic regions, as the proton-binding site and transmembrane domains remain highly conserved due to functional constraints. Comparative sequence analysis typically reveals higher variability in the connecting loop regions between the transmembrane helices, which might influence interactions with other ATP synthase subunits. These species-specific variations potentially affect the efficiency of ATP synthesis under different physiological conditions or environmental stresses particular to tomato's native habitat. Understanding these differences provides insights into the evolutionary adaptation of energy metabolism across plant species and may inform strategies for engineering more efficient photosynthetic systems.

What factors affect the assembly of ATP synthase subunit c into the complete ATP synthase complex?

The assembly of ATP synthase subunit c into the functional complex involves several critical factors. First, proper membrane insertion requires specific chaperones and insertion machinery in the thylakoid membrane. The c-subunits must correctly oligomerize to form the c-ring, a process influenced by lipid composition, particularly the presence of phosphatidylglycerol and other negatively charged lipids. The stoichiometry of the c-ring (typically 14 subunits in chloroplasts) must be precisely maintained for proper function. Assembly occurs in a coordinated fashion with other ATP synthase components, with specific protein-protein interactions guiding the process. Environmental factors such as pH, ionic strength, and temperature significantly affect assembly efficiency and stability. Disruptions in this assembly process can result in reduced ATP synthesis capacity and compromised photosynthetic efficiency. Research into these assembly factors provides important insights for successful recombinant expression and functional reconstitution of ATP synthase components.

What are the optimal expression systems for producing recombinant Solanum lycopersicum ATP synthase subunit c?

The expression of recombinant ATP synthase subunit c presents unique challenges due to its hydrophobic nature and small size. Several expression systems have been evaluated, each with distinct advantages and limitations:

For ATP synthase subunit c, the most successful approach typically involves E. coli expression with a fusion partner (SUMO or thioredoxin) to improve solubility, coupled with reduced induction temperature (18-20°C). Key parameters to optimize include induction duration (16-20 hours often yielding better results), inducer concentration, and the composition of the growth medium (rich media like TB or 2YT generally outperforming LB). For functional studies, expression systems that allow proper membrane insertion might be preferable despite potentially lower yields.

What purification strategies maintain the structural integrity of recombinant ATP synthase subunit c?

Purifying recombinant ATP synthase subunit c requires strategies that accommodate its hydrophobic nature while preserving structural integrity. A successful multi-step purification approach includes:

• Membrane Isolation: After cell lysis, differential centrifugation isolates membrane fractions (typically 20,000g to remove debris, followed by 100,000g to pellet membranes).

• Solubilization: The membrane fraction requires careful solubilization with appropriate detergents. Comparative analysis shows:

• Affinity Chromatography: When expressed with affinity tags, this provides the initial purification. Buffer conditions should maintain detergent concentrations above critical micelle concentration.

• Size Exclusion Chromatography: This final polishing step separates monomeric protein from aggregates and other impurities.

Throughout purification, maintaining a slightly alkaline pH (7.5-8.0) and including glycerol (10-15%) in all buffers helps stabilize the protein structure. For structural studies, reconstitution into nanodiscs or liposomes using controlled detergent removal provides a more native-like environment.

How can researchers assess the quality and functionality of purified recombinant ATP synthase subunit c?

Assessing the quality and functionality of purified ATP synthase subunit c requires multiple complementary approaches:

• Purity and Integrity Assessment:

  • SDS-PAGE with silver staining to detect impurities

  • Western blotting with specific antibodies to confirm identity

  • Mass spectrometry to verify the exact mass and detect modifications

  • N-terminal sequencing to confirm proper processing

• Structural Integrity Evaluation:

  • Circular dichroism spectroscopy to analyze secondary structure content

  • Fluorescence spectroscopy to monitor tertiary structure

  • Dynamic light scattering to confirm monodispersity

  • Thermal shift assays to determine stability

• Functional Analysis:

  • Reconstitution into liposomes for proton translocation assays

  • Assembly with other ATP synthase components to form functional complexes

  • Proton-dependent conformational change measurements

  • Inhibitor binding studies (e.g., oligomycin sensitivity)

A high-quality preparation should demonstrate the expected alpha-helical content (>70%), thermal stability consistent with membrane proteins, monodisperse behavior in detergent solutions, and the ability to assemble into higher-order structures. For functional validation, the reconstituted protein should demonstrate proton translocation activity when incorporated into liposomes and generate a proton-motive force when assembled with other ATP synthase components.

How should researchers design experiments to study proton translocation through ATP synthase subunit c?

Designing experiments to study proton translocation through ATP synthase subunit c requires careful consideration of the membrane environment and measurement techniques. A comprehensive approach includes:

• Reconstitution Systems: The purified protein should be incorporated into liposomes with controlled lipid composition. A mixture of phosphatidylcholine and phosphatidylethanolamine (70:30) with 5-10% cardiolipin often provides a suitable environment.

• Proton Gradient Establishment: Several methods can be used:

  • pH jump method: Create an initial pH difference across the membrane

  • Valinomycin/K+ system: Establish a K+ diffusion potential that drives proton uptake

  • Light-driven systems: Incorporate bacteriorhodopsin to pump protons in response to light

• Measurement Techniques:

  • Fluorescent pH indicators (ACMA or pyranine) to monitor pH changes

  • Potentiometric dyes (Oxonol VI) to monitor membrane potential

  • Radiolabeled proton flux measurements using rapid filtration

• Experimental Controls:

  • Empty liposomes without protein

  • Liposomes with known uncouplers (CCCP) as positive controls

  • Proteoliposomes with specific inhibitors of ATP synthase

• Data Analysis: Calculate proton flux rates, establish kinetic parameters, and determine the stoichiometry of proton translocation.

This experimental design should follow basic principles outlined in proper experimental methodology, including appropriate controls, replication, and statistical analysis to ensure validity of the results .

How can site-directed mutagenesis be effectively used to study structure-function relationships in ATP synthase subunit c?

Site-directed mutagenesis offers powerful insights into structure-function relationships of ATP synthase subunit c. An effective experimental design should include:

• Target Selection Strategy:

  • Conserved residues identified through multiple sequence alignment

  • Residues implicated in proton binding and translocation (particularly the conserved carboxylate)

  • Residues at subunit-subunit interfaces within the c-ring

  • Residues potentially involved in lipid interactions

• Mutation Types to Consider:

  • Conservative substitutions (maintaining similar properties)

  • Non-conservative substitutions (altering charge, size, or hydrophobicity)

  • Alanine scanning of specific regions

  • Introduction of reporter groups (cysteine for labeling)

• Functional Assays for Mutants:

• Control Experiments:

  • Wild-type protein analyzed in parallel

  • Multiple mutation variants with increasing severity of change

  • Rescue mutations to restore function in defective mutants

What approaches can be used to investigate the role of ATP synthase subunit c in regulating energy metabolism in tomato chloroplasts?

Investigating the regulatory role of ATP synthase subunit c in tomato chloroplast energy metabolism requires integrating multiple experimental approaches:

• Manipulating Expression Levels:

  • Generate transgenic tomato plants with altered expression of the ATP synthase subunit c gene

  • Use inducible promoters to control expression timing and level

  • Apply CRISPR/Cas9 genome editing to introduce specific mutations

• Physiological Measurements:

  • Chlorophyll fluorescence analysis to assess photosynthetic efficiency

  • Oxygen evolution measurements to quantify photosynthetic rate

  • ATP/ADP ratio determination in isolated chloroplasts

  • Carbon fixation rates using labeled CO2

• Integration with Metabolic Networks:

  • Metabolomic analysis to identify changes in metabolite pools

  • Flux analysis using isotope labeling to track carbon movement

  • Transcriptomic and proteomic analyses to identify compensatory responses

• Environmental Response Studies:

  • Compare wild-type and modified plants under different light intensities

  • Analyze responses to fluctuating light conditions

  • Assess performance under various temperature regimes

This approach aligns with findings that manipulating ATP supply can significantly affect plant metabolism, as demonstrated in studies of the tomato plastidic ATP/ADP transporter gene (SlAATP), which when overexpressed led to increased starch accumulation through up-regulation of starch biosynthesis genes .

How can researchers investigate the interaction between ATP synthase subunit c and other components of the photosynthetic apparatus?

Studying interactions between ATP synthase subunit c and other photosynthetic components requires techniques that can capture dynamic protein-protein interactions in membrane environments:

• Proximity-based Interaction Methods:

  • Chemical cross-linking coupled with mass spectrometry (CX-MS)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Proximity labeling techniques like BioID or APEX2

  • FRET or BRET between fluorescently labeled components

• Co-purification Approaches:

  • Affinity purification with tagged ATP synthase subunit c

  • Blue native PAGE followed by second dimension SDS-PAGE

  • Sucrose gradient fractionation of thylakoid membranes

  • GraFix method for stabilizing fragile complexes

• In vivo Visualization:

  • Confocal microscopy with fluorescently tagged proteins

  • Super-resolution microscopy techniques (STORM, PALM)

  • Electron microscopy with immunogold labeling

  • Förster resonance energy transfer (FRET) microscopy

• Functional Interaction Studies:

  • Measure effects of ATP synthase inhibitors on electron transport

  • Assess how disrupting supercomplexes affects proton gradient formation

  • Examine energy distribution between photosystems under varying conditions

Research in this area could build on findings about cooperation between adenine nucleotide translocator and ATP synthase in other systems , exploring whether similar cooperative mechanisms exist between chloroplastic ATP synthase and other components of the photosynthetic machinery.

How can structural biology techniques be applied to study conformational states of ATP synthase subunit c?

Structural biology offers powerful tools to capture the conformational states of ATP synthase subunit c during its functional cycle:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle analysis of the complete ATP synthase complex

  • Subtomogram averaging of membrane-embedded complexes

  • Time-resolved cryo-EM with rapid freezing after activation

  • Advantages: Can capture multiple conformational states in a single sample

• X-ray Crystallography:

  • Crystallization of the c-ring in different biochemical states

  • Use of antibody fragments to stabilize specific conformations

  • Soaking crystals with substrates, inhibitors, or pH-altering compounds

  • Advantages: Potentially higher resolution for small subunits

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Solution NMR of detergent-solubilized or nanodisc-reconstituted subunit c

  • Solid-state NMR of membrane-embedded c-rings

  • Chemical shift analysis to track protonation states of key residues

  • Advantages: Provides atomic-level information on dynamics and protonation

• Spectroscopic Techniques:

  • FTIR difference spectroscopy to track protonation changes

  • EPR spectroscopy with site-directed spin labeling

  • Time-resolved fluorescence spectroscopy with environmentally sensitive probes

  • Advantages: Can monitor specific chemical events during the functional cycle

The most informative approach involves trapping the protein in defined functional states for structural analysis, which can be achieved by manipulating pH, using non-hydrolyzable ATP analogs, employing specific inhibitors, or creating mutants that stall the rotary mechanism at particular steps.

How can comparative studies between Solanum lycopersicum ATP synthase subunit c and its homologs inform our understanding of energy metabolism evolution?

Comparative studies of ATP synthase subunit c across species provide valuable insights into energy metabolism evolution:

• Sequence-Based Evolutionary Analysis:

  • Construct phylogenetic trees using ATP synthase subunit c sequences

  • Calculate selection pressures (dN/dS ratios) to identify conserved regions

  • Perform ancestral sequence reconstruction to infer evolutionary trajectories

  • Conduct coevolution analysis to identify coordinated changes with other subunits

• Structure-Function Comparative Analysis:

  • Compare 3D structures across evolutionary diverse species

  • Identify conserved structural elements versus lineage-specific adaptations

  • Map functional residues onto structures to visualize evolutionary constraints

  • Examine c-ring stoichiometry differences across taxa and their functional implications

• Functional Complementation Studies:

  • Express ATP synthase subunit c from different species in a common background

  • Measure functional parameters (ATP synthesis rates, proton translocation efficiency)

  • Create chimeric proteins combining domains from different species

  • Correlate functional differences with ecological or physiological traits

• Ecological and Environmental Correlations:

  • Compare ATP synthase properties with habitat parameters

  • Analyze adaptations in extremophile plants

  • Correlate c-ring features with metabolic strategies (C3, C4, CAM photosynthesis)

  • Examine convergent evolution in unrelated species facing similar challenges

These comparative approaches provide a broader evolutionary context for interpreting functional studies and potentially identify novel features that could be exploited for improving crop energy efficiency.

What are common challenges in expressing recombinant ATP synthase subunit c and how can they be overcome?

Expressing recombinant ATP synthase subunit c presents several challenges due to its hydrophobic nature:

Additional strategies include codon optimization for the expression host, removing transit peptides that might interfere with expression, and using sequential extraction protocols to reduce contaminants. When troubleshooting, analyze each step separately: check mRNA levels to confirm transcription, use Western blotting to detect low protein levels, and examine membrane fractions specifically for hydrophobic proteins.

How can researchers troubleshoot functional assays for ATP synthase activity?

Troubleshooting functional assays for ATP synthase activity requires systematic identification of potential issues:

• Protein Quality Issues:

  • Problem: Inactive protein due to denaturation or aggregation

  • Diagnosis: Analyze protein by size exclusion chromatography or native PAGE

  • Solution: Optimize purification conditions, avoid freeze-thaw cycles

• Reconstitution Problems:

  • Problem: Improper orientation or incorporation into liposomes

  • Diagnosis: Fluorescent labeling to quantify incorporation, electron microscopy

  • Solution: Adjust lipid composition, optimize detergent removal rate

• Assay Sensitivity Limitations:

  • Problem: Signal-to-noise ratio too low

  • Diagnosis: Test with positive controls (known active preparations)

  • Solution: Increase protein concentration, use more sensitive detection methods

• Coupling System Failures:

  • Problem: ATP synthesis detection system not functioning

  • Diagnosis: Validate coupling enzymes separately

  • Solution: Prepare fresh coupling reagents, try alternative detection methods

• Absence of Essential Components:

  • Problem: Missing other ATP synthase subunits required for function

  • Diagnosis: Compare with isolated complete ATP synthase complex

  • Solution: Co-reconstitute with other essential subunits

A systematic approach should follow a logical decision tree, verifying protein folding, membrane incorporation, proton gradient formation, component completeness, and detection system functionality in sequence. For each step, appropriate controls help isolate problem sources.

What considerations are important when designing experiments to investigate environmental effects on ATP synthase subunit c?

Designing experiments to investigate environmental effects on ATP synthase subunit c requires consideration of multiple variables:

• Temperature Effects:

  • Experimental Design: Conduct assays across physiologically relevant temperatures (10-40°C)

  • Controls: Include thermostable enzymes as internal standards

  • Analysis: Generate Arrhenius plots to determine activation energy

• pH Considerations:

  • Experimental Design: Test function across pH range 5.5-8.5

  • Controls: Use multiple buffer systems with overlapping ranges

  • Analysis: Generate pH-activity profiles, consider protonation states

• Ionic Strength and Ion Species:

  • Experimental Design: Vary salt concentration and composition

  • Controls: Include ion chelators to verify specific requirements

  • Analysis: Determine optimal concentrations, identify competitive effects

• Lipid Environment:

  • Experimental Design: Test activity in liposomes with varied compositions

  • Controls: Compare native lipid extracts with synthetic mixtures

  • Analysis: Correlate membrane properties with activity

• Light Conditions (for chloroplastic proteins):

  • Experimental Design: Test under different light intensities and qualities

  • Controls: Include photosynthetic uncouplers to distinguish effects

  • Analysis: Correlate activity with light-induced gradients

When designing these experiments, it's crucial to consider the interplay between different environmental factors using factorial designs. Time-resolved measurements can reveal adaptation responses missed in endpoint assays, providing insights into how chloroplastic ATP synthase adapts to fluctuating environments.