Recombinant Crucihimalaya wallichii ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c is a critical component of the chloroplastic ATP synthase complex, specifically forming the c-ring structure within the F0 motor portion embedded in the thylakoid membrane. This protein plays a central role in energy conversion during photosynthesis, as it forms a rotor ring whose rotation is mechanically coupled to ATP synthesis .

The c-subunit typically features two hydrophobic transmembrane alpha-helical domains connected by a short polar loop, with a conserved carboxylate residue in the second transmembrane helix that is essential for proton translocation . The c-ring consists of multiple c-subunits arranged in a circle, and its rotation is driven by the flow of protons across the thylakoid membrane along an electrochemical gradient .

Importantly, the number of c-subunits in the ring (c-ring stoichiometry) determines the proton-to-ATP ratio, a key factor in photosynthetic efficiency. This ratio varies among species and may represent an evolutionary adaptation to different environmental conditions .

What methods are recommended for cloning and expressing the atpH gene from Crucihimalaya wallichii?

For successful recombinant expression of C. wallichii atpH, researchers should consider the following methodological approach:

• Gene design and optimization: Based on the known sequence of atpH in Brassicaceae, design a synthetic gene with codons optimized for expression in E. coli or other host systems. Add appropriate restriction sites for subsequent cloning .

• Expression vector selection: Select vectors that accommodate the hydrophobic nature of the c-subunit. Fusion tags such as maltose-binding protein (MBP) significantly enhance solubility and facilitate purification .

• Host strain selection: Use specialized E. coli strains designed for membrane protein expression, such as C41(DE3) or C43(DE3), which better tolerate potentially toxic membrane proteins .

• Expression conditions: Optimize growth temperature (typically 18-25°C), inducer concentration (0.1-0.3 mM IPTG), and induction time (16-20 hours) to improve proper folding and reduce toxicity .

• Protein extraction and purification: Employ gentle detergent extraction followed by affinity chromatography using the fusion tag, with potential subsequent tag removal depending on experimental requirements .

How does RNA editing impact atpH expression and what are the implications for recombinant production?

RNA editing, particularly C-to-U conversions, is a critical post-transcriptional modification in plant chloroplasts that can significantly affect gene expression and protein function . While specific data for C. wallichii atpH is not directly available, research on related chloroplast genes provides valuable insights.

In Arabidopsis, disruption of the ATP synthase γ subunit (ATPC1) affects the editing of multiple chloroplast transcripts, indicating a complex relationship between ATP synthase components and RNA processing machinery . Importantly, several ATP synthase-related transcripts show altered editing levels when ATP synthase function is compromised, including atpH-3′UTR-13210, which showed increased editing in atpc1 mutants .

When designing recombinant expression systems for C. wallichii atpH, researchers should consider:

• Using cDNA sequences that reflect the edited transcript rather than genomic DNA

• Accounting for potential regulatory elements in untranslated regions that might be affected by editing

• Recognizing that recombinant systems lack the native RNA editing machinery, potentially affecting expression outcomes

These considerations are particularly important given that RNA editing can create start/stop codons, alter amino acid sequences, or influence translation efficiency .

What is known about the taxonomy and evolutionary context of Crucihimalaya wallichii?

Crucihimalaya wallichii (Hook.f. & Thomson) Al-Shehbaz, O'Kane & R.A.Price belongs to the Brassicaceae family (cruciferous plants), which includes model organisms like Arabidopsis thaliana . This plant has been known by several synonyms, including Arabidopsis wallichii and Sisymbrium wallichii, reflecting taxonomic revisions as understanding of evolutionary relationships improves .

Crucihimalaya wallichii is commonly known as Wallich Rock-Cress and is adapted to alpine environments in the Himalayan region . As a member of Brassicaceae, it belongs to a plant family characterized by specific floral structure (cruciform corolla, tetradynamous stamen) and fruit type (capsular) .

The Brassicaceae family has been subject to extensive phylogenetic studies, revealing three major evolutionary lineages (I, II, and III) . Modern chloroplast genome sequencing has helped resolve many taxonomic relationships within this family, though the specific placement of Crucihimalaya within these lineages requires further investigation .

Understanding the evolutionary context of C. wallichii provides valuable background for studies of its chloroplast genes like atpH, potentially revealing adaptations to its native alpine environment.

What experimental approaches can be used to determine the c-ring stoichiometry of Crucihimalaya wallichii ATP synthase?

Determining c-ring stoichiometry is crucial for understanding the bioenergetic properties of ATP synthase. For C. wallichii, researchers should consider these complementary approaches:

• Atomic Force Microscopy (AFM):

  • Purify and reconstitute the c-ring structure

  • Image using high-resolution AFM

  • Measure ring diameter and calculate subunit number based on known dimensions of individual c-subunits

• Mass Spectrometry of Intact Rings:

  • Isolate intact c-rings using mild detergent solubilization

  • Analyze by native mass spectrometry

  • Determine molecular weight to deduce the number of c-subunits

• Cryo-Electron Microscopy:

  • Purify the entire ATP synthase complex or isolated c-rings

  • Obtain high-resolution structures using cryo-EM

  • Count subunits directly from the structural data

• Crosslinking Studies:

  • Use chemical crosslinkers to stabilize the c-ring structure

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

  • Determine stoichiometry from crosslinking patterns

Comparative analysis of c-ring stoichiometry could reveal adaptations to the alpine environment where C. wallichii naturally grows, potentially showing differences from temperate species like Arabidopsis or spinach .

How might the amino acid sequence of Crucihimalaya wallichii ATP synthase subunit c reflect adaptation to alpine environments?

Alpine plants face unique environmental challenges including low temperatures, high light intensity, and rapid temperature fluctuations. The ATP synthase c-subunit may exhibit specific adaptations to maintain functionality under these conditions:

• Cold Stability Adaptations:

  • Increased proportion of hydrophobic residues to enhance membrane integration

  • Strategic placement of proline residues to maintain structural flexibility at low temperatures

  • Modifications to ion-binding sites to maintain proton conductance at lower temperatures

• Functional Adaptations:

  • Altered c-ring stoichiometry to optimize the H+/ATP ratio for alpine light conditions

  • Modified proton-binding residues to function efficiently in the pH ranges typical of alpine chloroplasts

  • Structural changes enhancing stability during freeze-thaw cycles

• Methods for Adaptation Analysis:

  • Comparative sequence analysis with lowland Brassicaceae species

  • Homology modeling to identify structurally significant differences

  • Site-directed mutagenesis to test the functional significance of unique residues

  • Thermal stability assays comparing recombinant proteins from alpine vs. temperate species

Chloroplast genome studies in Brassicaceae have identified genes under positive selection, and ATP synthase components could be subject to such selection pressures in extreme environments . Understanding these adaptations could provide insights for engineering crops with enhanced photosynthetic efficiency under stress conditions.

What challenges arise in reconstituting functional c-rings from recombinant Crucihimalaya wallichii ATP synthase subunit c?

Reconstituting functional c-rings presents several complex challenges that researchers must address:

• Protein Aggregation and Misfolding:

  • The highly hydrophobic nature of subunit c promotes aggregation

  • Solution: Utilize specialized detergents (DDM, OG) and lipid mixtures that mimic the native thylakoid membrane environment

  • Monitor folding using circular dichroism spectroscopy to verify alpha-helical content

• Assembly Factors and Chaperones:

  • Native assembly likely requires specific chaperones absent in recombinant systems

  • Solution: Consider co-expression with known assembly factors or develop step-wise reconstitution protocols

  • Test various lipid compositions that may facilitate self-assembly

• Verification of Functional Assembly:

  • Develop assays to confirm that reconstituted c-rings maintain native-like properties

  • Use proton translocation assays with pH-sensitive fluorescent dyes

  • Apply negative-stain electron microscopy or AFM to verify ring formation

Success in reconstitution would enable detailed functional studies of C. wallichii ATP synthase c-rings, potentially revealing unique properties related to its alpine adaptation .

How does the RNA editing profile of atpH in Crucihimalaya wallichii potentially differ from model plants and what are the functional implications?

RNA editing plays a crucial role in chloroplast gene expression and can vary significantly between species, potentially reflecting evolutionary adaptations to different environments . While specific data for C. wallichii atpH RNA editing is not directly available, insights can be drawn from research on related plants.

In Arabidopsis, disruption of the ATP synthase γ subunit (ATPC1) affects RNA editing at multiple sites, including changes in atpH-3′UTR-13210 editing levels . This suggests a complex regulatory network connecting ATP synthase function and RNA processing.

Key aspects to consider regarding RNA editing in C. wallichii atpH include:

• Potential Editing Sites:

  • Coding region sites that alter amino acid sequence

  • UTR sites that may affect translation efficiency

  • Sites that create or remove regulatory elements

• Functional Consequences:

  • Altered protein structure or stability adapted to alpine conditions

  • Modified translation efficiency under temperature stress

  • Changes in regulatory networks affecting energy metabolism

• Experimental Approaches to Characterize Editing:

  • RT-PCR and Sanger sequencing to identify C-to-U editing sites

  • High-throughput RNA sequencing to quantify editing efficiency

  • Comparative analysis with lowland relatives to identify environment-specific editing patterns

Understanding the RNA editing landscape of C. wallichii atpH could provide insights into post-transcriptional adaptation mechanisms and inform the design of recombinant expression systems that accurately reflect the native protein .

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

Purifying hydrophobic membrane proteins like ATP synthase subunit c requires specialized approaches. Based on successful protocols for similar proteins, we recommend the following strategy:

• Solubilization Optimization:

  • Test multiple detergents including DDM, OG, and LDAO at various concentrations

  • Include glycerol (10-20%) as a stabilizing agent

  • Maintain pH between 7.0-8.0 to preserve protein stability

• Fusion Tag Selection and Purification:

  • MBP tag significantly enhances solubility and enables affinity purification

  • His6 tag provides an alternative for IMAC purification

  • Consider dual tagging strategies for enhanced purity

• Chromatographic Approach:

  • Begin with affinity chromatography based on the fusion tag

  • Follow with size exclusion chromatography to remove aggregates

  • Consider ion exchange as a polishing step if necessary

• Tag Removal Considerations:

  • Evaluate whether the tag interferes with downstream applications

  • If removal is necessary, optimize protease digestion conditions to prevent aggregation

  • Perform a second affinity step to separate cleaved protein from the tag

Proper handling throughout the purification process is essential to maintain protein stability and prevent aggregation, including keeping samples cold and minimizing exposure to air .

What analytical methods can confirm the proper folding and oligomeric state of recombinant ATP synthase subunit c?

Confirming proper folding and assembly of recombinant ATP synthase subunit c requires multiple complementary analytical approaches:

• Secondary Structure Analysis:

  • Circular Dichroism (CD) Spectroscopy: Verify the alpha-helical content characteristic of properly folded c-subunit

  • FTIR Spectroscopy: Provide additional structural information, particularly useful for membrane proteins

  • Compare spectral features with those of well-characterized c-subunits from other species

• Oligomeric State Assessment:

  • Blue Native PAGE: Analyze native oligomeric state under mild conditions

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): Determine absolute molecular weight of protein-detergent complexes

  • Analytical Ultracentrifugation: Provide information on size distribution and homogeneity

• Structural Visualization:

  • Negative-stain Electron Microscopy: Directly visualize ring formation

  • Atomic Force Microscopy: Measure dimensions and topology of reconstituted c-rings

  • Crosslinking Mass Spectrometry: Identify specific subunit-subunit interactions

• Functional Assessment:

  • Proton Translocation Assays: Confirm functionality of reconstituted c-rings

  • Inhibitor Binding Studies: Verify preservation of specific binding sites (e.g., for DCCD)

  • Reconstitution with Partner Subunits: Test interaction with other ATP synthase components

These analytical approaches provide complementary information about different aspects of protein structure and function, together building a comprehensive picture of the recombinant protein's properties .

How can researchers effectively compare the functional properties of recombinant versus native Crucihimalaya wallichii ATP synthase c-subunit?

Comparing recombinant and native protein properties is essential to validate the biological relevance of recombinant systems. For C. wallichii ATP synthase c-subunit, consider these approaches:

• Structural Comparison:

  • Proteolytic Fingerprinting: Compare fragmentation patterns between native and recombinant proteins

  • Mass Spectrometry: Identify post-translational modifications present in native but not recombinant protein

  • CD Spectroscopy: Compare secondary structure profiles under various conditions

• Functional Comparison:

  • Reconstitution Studies: Assess the ability of both proteins to form functional c-rings

  • Proton Conductance: Measure and compare proton translocation efficiency

  • Thermal Stability: Determine if both proteins exhibit similar stability profiles across temperature ranges

• Interaction Analysis:

  • Pull-down Assays: Compare binding to other ATP synthase subunits

  • Lipid Interaction Studies: Assess binding preferences to various membrane lipids

  • Response to Inhibitors: Compare sensitivity to known ATP synthase inhibitors

• Complementation Studies:

  • Express recombinant C. wallichii atpH in model organisms with atpH mutations

  • Assess the ability to restore ATP synthase function

  • Compare with native gene complementation efficiency

Creating a comprehensive comparison requires isolating native protein from C. wallichii chloroplasts, which may be challenging due to limited biomass availability. Researchers may need to develop specialized extraction protocols or consider alternative approaches such as heterologous expression of tagged versions in related plant species .

How might understanding Crucihimalaya wallichii ATP synthase c-subunit adaptations inform strategies for engineering stress-tolerant crops?

ATP synthase optimization represents a promising but underexplored approach for enhancing crop photosynthetic efficiency under stress conditions. Insights from C. wallichii could guide several engineering strategies:

• Cold Tolerance Engineering:

  • Identify specific amino acid substitutions in C. wallichii c-subunit that confer cold stability

  • Introduce these substitutions into crop ATP synthase c-subunits using precise genome editing

  • Test effects on photosynthetic performance under cold stress conditions

• Optimizing Bioenergetic Efficiency:

  • Determine if C. wallichii c-ring stoichiometry differs from model plants

  • Engineer crop c-rings with altered stoichiometry to modify the H+/ATP ratio

  • Fine-tune energy conservation versus photosynthetic output for specific environments

• Stress Resilience Mechanisms:

  • Characterize how C. wallichii ATP synthase maintains function during rapid temperature fluctuations

  • Identify regulatory mechanisms governing ATP synthase assembly under stress

  • Engineer similar regulatory pathways in crop species

This research could ultimately contribute to developing crops with enhanced photosynthetic efficiency under suboptimal conditions, potentially expanding growing ranges and increasing agricultural resilience to climate change .

What novel experimental approaches could advance our understanding of ATP synthase c-subunit function in alpine plants?

Advancing our understanding of ATP synthase adaptations in alpine plants like C. wallichii requires innovative experimental approaches:

These approaches would not only advance our understanding of C. wallichii ATP synthase but could also establish broadly applicable methodologies for studying protein adaptations to extreme environments .