Recombinant Carica papaya ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c forms a multimeric ring (c₍ₙ₎) embedded in the thylakoid membrane of chloroplasts. This ring functions as part of the F₀ region and rotates when protons are translocated across the membrane along an electrochemical gradient. The rotation is mechanically coupled to the synthesis of ATP in the F₁ region of the enzyme. Each complete rotation of the c-ring produces 3 ATP molecules, with the number of protons required dependent on the number of c-subunits in the ring .

Methodologically, structural characterization typically involves:

• Circular dichroism to confirm alpha-helical secondary structure

• Electron microscopy or X-ray crystallography for tertiary and quaternary structure determination

• Molecular dynamics simulations to model membrane interactions

How does codon optimization affect the expression of recombinant ATP synthase subunit c?

Codon optimization significantly enhances expression levels of recombinant membrane proteins like ATP synthase subunit c. For chloroplastic proteins expressed in bacterial systems, codon optimization addresses the natural codon usage bias between plant chloroplasts and bacterial host cells.

Methodological approach:

• Analyze the native gene sequence and identify rare codons

• Design a synthetic gene with codons optimized for the expression host (e.g., E. coli)

• Add appropriate terminal restriction sites for cloning flexibility

• Use gene synthesis services or software like Gene Designer to create the optimized sequence

Gene optimization benefits include enhanced protein solubility, reduced formation of inclusion bodies, and increased yield. For hydrophobic membrane proteins like ATP synthase subunit c, codon optimization is particularly crucial for achieving functional expression.

What are effective fusion protein strategies for expressing recombinant ATP synthase subunit c?

The highly hydrophobic nature of ATP synthase subunit c presents significant challenges for recombinant expression. Fusion protein approaches have proven most effective:

The MBP fusion approach has been successfully implemented for spinach chloroplast ATP synthase subunit c and would likely be applicable to Carica papaya . The methodology involves:

• Cloning the atpH gene into a vector like pMAL-c2x at appropriate restriction sites

• Expressing the fusion protein in E. coli BL21 derivatives

• Purifying using affinity chromatography

• Cleaving the fusion tag with a specific protease

• Performing secondary purification (e.g., reverse-phase chromatography)

How can the stoichiometric variation in c-subunit rings across different species be investigated using recombinant approaches?

The stoichiometry of c-subunit rings varies across organisms (c₁₀ to c₁₅), affecting the coupling ratio of protons transported to ATP generated (3.3 to 5.0). Investigating this variation in Carica papaya compared to other species requires:

Methodological approach:

• Express and purify recombinant c₁ subunits from multiple species (including Carica papaya)

• Reconstitute multimeric rings in vitro under controlled conditions

• Analyze ring stoichiometry using techniques like:

  • Atomic force microscopy (AFM)

  • Cryo-electron microscopy

  • Native mass spectrometry

• Correlate stoichiometry with genetic and environmental factors

This approach enables comparative analysis of factors influencing stoichiometric differences, which may relate to evolutionary adaptations to specific environmental conditions or metabolic requirements.

What factors influence the assembly and stability of recombinant ATP synthase c-rings?

The assembly of monomeric c₁ subunits into functional c₍ₙ₎ rings depends on multiple factors:

Methodological investigation approach:

• Vary lipid composition during reconstitution experiments

• Test the impact of pH, temperature, and ionic strength on assembly

• Introduce site-directed mutations to identify key residues involved in subunit-subunit interactions

• Use molecular dynamics simulations to predict stability determinants

• Apply cross-linking techniques to capture assembly intermediates

Research suggests that the lipid environment, transmembrane hydrophobic matching, and specific amino acid interactions at the subunit interfaces all play critical roles in determining the final stoichiometry and stability of the c-ring .

How do post-translational modifications affect the function of chloroplastic ATP synthase subunit c?

While information on post-translational modifications (PTMs) specific to Carica papaya ATP synthase subunit c is limited, investigating potential PTMs would involve:

Methodological approach:

• Express the recombinant protein in eukaryotic systems capable of plant-like PTMs

• Compare with bacterial expression systems lacking specific PTMs

• Analyze using:

  • Mass spectrometry to identify and map modifications

  • Site-directed mutagenesis to remove potential modification sites

  • Functional assays to assess impact on activity

• Compare PTM patterns across different plant species including Carica papaya

Potential PTMs may include acetylation, phosphorylation, or lipid modifications that could affect membrane insertion, oligomerization, or proton translocation efficiency.

What expression system optimization strategies are most effective for maximizing yields of functional ATP synthase subunit c?

Optimizing recombinant expression of ATP synthase subunit c requires careful consideration of multiple factors:

For Carica papaya ATP synthase subunit c, a systematic approach testing these variables would be necessary, potentially adopting strategies that proved successful for similar proteins like the spinach chloroplast ATP synthase subunit c .

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

Purifying highly hydrophobic membrane proteins like ATP synthase subunit c requires specialized approaches:

Multi-step purification protocol:

• Initial capture using affinity chromatography (if expressed as a fusion protein)

• Protease cleavage to remove fusion tags

• Reversed-phase HPLC for final purification

• Detergent exchange or reconstitution into liposomes or nanodiscs for functional studies

Key considerations include:

• Selection of appropriate detergents for solubilization (DDM, LDAO, or FC-12)

• Buffer optimization to maintain stability during purification

• Validation of structural integrity after each purification step using circular dichroism

• Yield assessment and activity testing to ensure functionality

How can researchers verify the proper folding and functionality of recombinant ATP synthase subunit c?

Confirming proper folding and functionality requires multiple analytical approaches:

Methodological workflow:

• Secondary structure analysis:

  • Circular dichroism to confirm alpha-helical content

  • FTIR spectroscopy for secondary structure elements

• Tertiary structure assessment:

  • Intrinsic fluorescence spectroscopy

  • Limited proteolysis patterns

• Functional characterization:

  • Reconstitution into liposomes

  • Proton translocation assays

  • ATP synthesis coupling efficiency measurements

• Comparative analysis with native protein where available

For Carica papaya ATP synthase subunit c, establishing these verification methods would be essential for ensuring that recombinant protein research yields physiologically relevant results.

How can researchers determine the oligomeric state and stoichiometry of recombinant ATP synthase c-rings?

Determining c-ring stoichiometry requires sophisticated analytical approaches:

Methodological strategy:

• Native mass spectrometry:

  • Carefully solubilize intact c-rings

  • Analyze under conditions that preserve non-covalent interactions

• Cross-linking mass spectrometry:

  • Apply chemical cross-linkers to stabilize interfaces

  • Digest and analyze to identify interaction patterns

• Cryo-electron microscopy:

  • Visualize ring structures directly

  • Apply symmetry analysis to determine subunit number

• Atomic force microscopy:

  • Image membrane-embedded rings

  • Measure circumference and calculate subunit numbers

Data interpretation requires consideration of detergent effects, potential artifacts from recombinant expression, and comparative analysis with known c-ring structures from other organisms .

What bioinformatic approaches are useful for analyzing ATP synthase subunit c sequences across plant species?

Comparative genomic analysis provides valuable insights into evolutionary patterns:

Bioinformatic methodology:

• Multiple sequence alignment of atpH genes from diverse plant species including Carica papaya

• Conservation analysis of key functional residues

• Evolutionary rate analysis to identify regions under selective pressure

• Structural modeling based on known templates

• Correlation analysis between sequence features and known c-ring stoichiometries

How do lipid environments affect the structure and function of recombinant ATP synthase c-rings?

The lipid environment critically influences membrane protein function:

Research methodology:

• Reconstitute purified c-subunits in various defined lipid compositions

• Analyze structural parameters using:

  • Solid-state NMR spectroscopy

  • Fluorescence spectroscopy with environment-sensitive probes

  • Hydrogen-deuterium exchange mass spectrometry

• Measure functional parameters:

  • Proton translocation rates

  • Stability under different conditions

  • Assembly efficiency and stoichiometry

Results interpretation should consider native lipid compositions in Carica papaya chloroplasts compared to other species, as these differences may reflect adaptations to specific environmental conditions.

How can researchers address inclusion body formation during recombinant expression of ATP synthase subunit c?

Inclusion body formation is a common challenge with highly hydrophobic proteins:

Troubleshooting methodology:

• Prevention strategies:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Co-express molecular chaperones

  • Use fusion partners like MBP

• Recovery strategies:

  • Solubilize inclusion bodies with appropriate detergents

  • Use denaturing conditions followed by step-wise refolding

  • Apply on-column refolding during purification

Optimization must balance yield with proper folding, as improperly folded protein will not assemble into functional c-rings or may exhibit altered stoichiometry.

What approaches can overcome challenges in activation and processing of recombinant ATP synthase proteins?

Activation of ATP synthase components often requires specific processing:

Methodological approach:

• Test various activation conditions:

  • Reducing agents (DTT, cysteine-HCl)

  • pH conditions (typically acidic for protease activation)

  • Temperature optimization

• Monitor processing using:

  • Western blotting to confirm size changes

  • Activity assays to verify functional enhancement

• Optimize activation parameters:

  • Concentration of activating agents

  • Incubation time

  • Buffer composition

For example, with recombinant papain (another Carica papaya protein), activation with 5 mM cysteine for 30 minutes at room temperature was sufficient for complete processing and significantly enhanced activity from 1.6 to 20.7 U/mg .