Recombinant Vitis vinifera ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c forms a multimeric ring (c₍ₙ₎) embedded in the thylakoid membrane of chloroplasts. This ring structure plays a critical role in energy conversion during photosynthesis. The rotation of the c-subunit ring is driven by the translocation of protons across the thylakoid membrane along an electrochemical gradient. This mechanical rotation is coupled to the γ-stalk in the F₁ region, driving the catalysis of ATP synthesis at the three α-β subunit interfaces . Each complete rotation produces 3 ATP molecules for every n protons that pass from the lumen to the stroma, where n represents the number of c-subunits in the ring . The stoichiometry of this ring varies between organisms and directly affects the bioenergetic efficiency of the ATP synthase.

Why is recombinant expression of chloroplastic ATP synthase subunit c valuable for research?

Recombinant expression offers several advantages for studying the structure and function of chloroplastic ATP synthase subunit c:

• It enables the production of significant quantities of purified protein that would be difficult to isolate from native sources .

• It allows researchers to introduce site-directed mutations for structure-function studies.

• It facilitates reconstitution experiments of the multimeric c-ring to investigate factors affecting stoichiometric variation .

• It enables the application of molecular biology techniques that cannot be applied to native c-rings.

• It provides material for biophysical and structural characterization without the complexities of purifying from plant tissue.

This approach is particularly valuable for investigating the factors that influence the stoichiometric variation of the c-ring, which remains poorly understood despite its significance for bioenergetic efficiency.

What expression systems are suitable for producing recombinant ATP synthase subunit c?

Based on successful expression systems for similar proteins, several options are available:

• E. coli expression system: Most commonly used for initial attempts due to its simplicity and high yield. The subunit c protein can be expressed as a fusion protein with a solubility tag like maltose binding protein (MBP) to overcome hydrophobicity issues .

• Pichia pastoris: Offers advantages for expression of eukaryotic proteins with proper folding. This system has been successfully used for expressing Vitis vinifera proteins, as demonstrated with VPEs .

• Insect cell expression systems: May provide better post-translational modifications for plant proteins.

For chloroplastic ATP synthase subunit c specifically, an E. coli expression system with a codon-optimized gene insert and MBP fusion strategy has proven effective for the spinach homolog . The protein is first expressed as a soluble MBP-fusion protein, then cleaved from MBP and purified using reversed-phase chromatography.

How does the stoichiometry of the c-ring in Vitis vinifera ATP synthase compare to other species, and what are the implications?

The stoichiometry of the c-ring (the number of c-subunits per ring) varies among different organisms and has direct implications for the bioenergetic efficiency of ATP synthesis. The ratio of protons translocated to ATP synthesized is determined by this stoichiometry . While specific data for Vitis vinifera c-ring stoichiometry is not directly provided in the search results, research approaches would include:

• Comparing sequence homology with species of known c-ring stoichiometry

• Structural analysis through cryo-electron microscopy or X-ray crystallography of purified c-rings

• Functional studies measuring H⁺/ATP ratios

• Computational modeling based on sequence determinants known to influence ring size

Understanding this stoichiometry is critical because it affects the plant's bioenergetic efficiency and potentially its adaptation to different environmental conditions. A lower stoichiometry results in higher ATP yield per proton, which may be advantageous under certain metabolic conditions.

What factors contribute to successful reconstitution of the c-ring from recombinant monomers?

Reconstitution of functional c-rings from recombinant monomers represents a significant research challenge. Key factors to consider include:

• Lipid environment: The specific composition of lipids appears critical for proper assembly and function. Different lipid mixtures resembling the thylakoid membrane should be tested.

• pH and ionic conditions: These must be carefully optimized to promote proper folding and assembly while preventing aggregation of the hydrophobic subunits.

• Detergent selection: Critical for solubilizing the monomeric subunits while allowing proper interaction for ring formation.

• Temperature and time: Assembly is likely temperature-dependent and may require extended incubation periods.

• Additional factors: Native assembly may require other proteins or cofactors not yet identified.

Experimental approaches should include systematic testing of these variables, followed by analytical techniques such as size exclusion chromatography, native PAGE, and electron microscopy to confirm proper assembly. Functional assessment would require reconstitution in liposomes and measurement of proton translocation or rotation.

How does recombinant ATP synthase subunit c compare functionally to the native protein?

This question addresses a critical concern in recombinant protein research. Approaches to assess functional equivalence include:

• Secondary structure comparison: Circular dichroism (CD) spectroscopy to confirm the recombinant protein has the correct alpha-helical secondary structure, as confirmed for spinach c₁ .

• Assembly capability: Ability to form oligomeric rings similar to native protein.

• Proton binding and translocation: Functional assays in reconstituted systems.

• Interaction with other ATP synthase subunits: Particularly the a-subunit which forms the proton channel.

• Post-translational modifications: Analysis of any modifications present in the native protein that might be absent in the recombinant version.

Differences observed should be carefully analyzed to determine whether they represent fundamental functional differences or artifacts of the recombinant expression system.

What is the optimal strategy for cloning and expressing Vitis vinifera ATP synthase subunit c?

Based on successful approaches with similar proteins, a comprehensive strategy would include:

• Gene identification and optimization:

  • Identify the ATP synthase subunit c gene sequence from Vitis vinifera genome databases

  • Optimize codon usage for the chosen expression host (typically E. coli)

  • Include appropriate regulatory elements and fusion tags

• Expression construct design:

  • Fusion with solubility-enhancing partners (MBP has proven effective)

  • Include a cleavable linker between the fusion partner and target protein

  • Add affinity tags for purification (His-tag or other suitable options)

• Expression conditions:

  • Test multiple E. coli strains (BL21 derivatives work well for hydrophobic proteins)

  • Optimize induction conditions (IPTG concentration, temperature, duration)

  • Consider lower temperatures (16-25°C) for improved folding

• Protein extraction and purification:

  • Use methods that effectively solubilize membrane proteins

  • Employ affinity chromatography for initial purification

  • Consider reversed-phase chromatography for final purification after tag cleavage

For expression systems alternative to E. coli, such as Pichia pastoris, similar principles apply, with modifications to the vector design and induction protocols (methanol induction for Pichia) .

What purification methods are most effective for recombinant ATP synthase subunit c?

Effective purification of this hydrophobic membrane protein requires a strategic approach:

• Initial capture:

  • Affinity chromatography utilizing fusion tags (MBP-affinity or His-tag)

  • Gentle washing conditions to prevent protein aggregation

• Tag cleavage:

  • Enzymatic cleavage (TEV protease or Factor Xa, depending on construct)

  • Optimization of cleavage conditions to ensure complete processing

• Final purification:

  • Reversed-phase HPLC has proven effective for the spinach homolog

  • Size exclusion chromatography can remove aggregates

  • Ion exchange chromatography may provide additional purification

• Quality control:

  • SDS-PAGE with immunoblotting to confirm identity

  • Mass spectrometry for accurate molecular weight determination

  • Circular dichroism to verify proper secondary structure

A typical purification protocol might involve initial MBP-affinity purification, followed by tag cleavage and reversed-phase chromatography. This approach has yielded highly purified c₁ subunit with the correct alpha-helical secondary structure for the spinach homolog .

What analytical methods can verify the structural integrity of purified recombinant ATP synthase subunit c?

Multiple complementary techniques should be employed:

• Circular Dichroism (CD) Spectroscopy:

  • Confirms alpha-helical secondary structure characteristic of subunit c

  • Enables comparison with native protein or published spectra

  • Can monitor thermal stability and structural changes under varying conditions

• Mass Spectrometry:

  • Confirms exact molecular weight and sequence integrity

  • Can identify post-translational modifications

  • Useful for detecting proteolytic degradation

• Protein NMR Spectroscopy:

  • Provides detailed structural information in membrane-mimetic environments

  • Can detect proper folding and tertiary structure

• Electron Microscopy:

  • For visualization of reconstituted c-rings

  • Negative staining for initial assessment

  • Cryo-EM for higher resolution structural analysis

• Functional Assays:

  • Proton binding studies

  • Reconstitution with other ATP synthase components

  • Assessment of oligomerization capacity

These methods collectively provide a comprehensive assessment of structural integrity before proceeding to functional studies.

How can researchers determine the stoichiometry of reconstituted c-rings?

Determining c-ring stoichiometry requires multiple complementary approaches:

• Mass Determination:

  • Native mass spectrometry can provide the molecular weight of intact c-rings

  • Comparison with the known mass of monomers allows calculation of subunit number

• Structural Analysis:

  • Cryo-electron microscopy with symmetry analysis

  • X-ray crystallography (if crystals can be obtained)

  • Atomic force microscopy to count subunits in membrane-embedded rings

• Crosslinking Studies:

  • Chemical crosslinking followed by SDS-PAGE analysis

  • Mass spectrometry of crosslinked products

• Functional Studies:

  • H⁺/ATP ratio measurements in reconstituted systems

  • These ratios directly correlate with c-ring stoichiometry

A table comparing known c-ring stoichiometries across species can provide context:

How should researchers interpret differences in expression levels between different constructs?

When evaluating expression levels of different ATP synthase subunit c constructs, consider:

• Codon Optimization Impact:

  • Compare expression levels between codon-optimized and non-optimized constructs

  • Analyze codon adaptation index (CAI) correlation with expression levels

• Fusion Partner Effects:

  • Quantify how different fusion partners (MBP, GST, SUMO, etc.) affect expression

  • Assess solubility vs. total expression to distinguish effects on folding vs. translation

• Regulatory Element Influence:

  • Compare promoter strengths and ribosome binding site efficiencies

  • Measure transcript levels via RT-qPCR to determine if differences occur at transcriptional or translational level

• Host Strain Variations:

  • Systematically compare expression across different E. coli strains

  • Consider strains with different capabilities (e.g., rare codon supplementation, chaperone co-expression)

• Statistical Analysis:

  • Use appropriate statistical tests (ANOVA with post-hoc analysis) to determine significance

  • Perform multiple independent experiments to ensure reproducibility

Expression level analysis should separate effects on total expression, soluble expression, and functional protein yield to comprehensively understand construct performance.

What research approaches can elucidate the evolutionary significance of variation in ATP synthase subunit c?

Investigating evolutionary aspects requires integrative approaches:

• Phylogenetic Analysis:

  • Construct phylogenetic trees of ATP synthase subunit c across species

  • Map known c-ring stoichiometries onto phylogenetic relationships

  • Identify conserved and variable regions that may influence ring size

• Sequence-Structure-Function Relationships:

  • Compare sequences between species with different ring sizes

  • Identify key residues that may determine oligomerization properties

  • Use site-directed mutagenesis to test hypotheses about these determinants

• Interorganellar Gene Transfer Analysis:

  • Investigate evidence of gene conversion or recombination between chloroplast and mitochondrial ATP synthase genes

  • Assess how these events might influence c-ring properties

• Correlation with Environmental Adaptation:

  • Compare c-ring properties across plants from different habitats

  • Test for correlations between environmental factors and ATP synthase efficiency

  • Consider how Vitis vinifera's evolutionary history might have shaped its ATP synthase properties

This research direction could provide insights into how bioenergetic efficiency has been shaped by evolutionary pressures and potentially inform bioenergetic engineering efforts.