Recombinant Adiantum capillus-veneris ATP synthase subunit c, chloroplastic (atpH)

How does the Adiantum capillus-veneris atpH gene differ from other fern species in terms of genomic organization?

The atpH gene in Adiantum capillus-veneris is located within the chloroplast genome, which has been completely sequenced. The circular chloroplast genome of A. capillus-veneris is 150,568 bp with a large single-copy region (LSC) of 82,282 bp, a small single-copy region (SSC) of 21,392 bp, and inverted repeats (IR) of 23,447 bp each .

The atpH gene is part of the ATP synthase complex genes found in the chloroplast genome. In comparison to other fern species, the genomic organization of Adiantum chloroplast shows some distinctive features. It belongs to the Pteridaceae family within a large clade of recently derived leptosporangiate families, representing a significant evolutionary position for comparative genomic studies .

What are the recommended storage and reconstitution protocols for recombinant atpH protein to maintain its stability?

For optimal stability and activity of recombinant Adiantum capillus-veneris ATP synthase subunit c, chloroplastic (atpH) protein, follow these research-validated protocols:

Storage:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquot reconstituted protein to prevent repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 50% and store at -20°C/-80°C

Reconstitution:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For optimal stability, add 5-50% glycerol (final concentration)

What expression systems have proven most effective for producing functional recombinant atpH protein?

The most effective expression system documented for producing functional recombinant Adiantum capillus-veneris ATP synthase subunit c is Escherichia coli. The recombinant full-length protein (amino acids 1-81) can be successfully expressed with an N-terminal His-tag in E. coli expression systems . This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE.

When designing your expression protocol, consider the following methodological aspects:

• Codon optimization for E. coli may improve expression yields

• N-terminal His-tag facilitates purification while maintaining protein functionality

• Expression in E. coli allows for scalable production while maintaining proper folding of this relatively small (81 amino acids) membrane protein

• Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0 maintains stability

What purification challenges are commonly encountered when isolating atpH protein, and how can they be addressed?

Several challenges commonly arise during the purification of recombinant atpH protein:

Challenge 1: Membrane protein solubility

• Solution: Use mild detergents during cell lysis and purification steps

• Methodology: Incorporate 0.5-1% non-ionic detergents (e.g., DDM or CHAPS) in lysis buffers to solubilize membrane-associated proteins while preserving native structure

Challenge 2: Protein aggregation

• Solution: Optimize buffer conditions and incorporate stabilizing agents

• Methodology: Include 6% trehalose in storage buffers as demonstrated effective for maintaining atpH stability

Challenge 3: Maintaining purity above 90%

• Solution: Multi-step purification approach

• Methodology: Initial IMAC (Immobilized Metal Affinity Chromatography) utilizing the His-tag, followed by size exclusion chromatography to achieve >90% purity as verified by SDS-PAGE

How can researchers effectively evaluate the functional activity of recombinant atpH protein in ATP synthesis experiments?

To evaluate the functional activity of recombinant atpH protein in ATP synthesis experiments, researchers should consider the following methodological approach:

Reconstitution Assay:

• Incorporate purified recombinant atpH protein into liposomes with other ATP synthase subunits

• Measure proton translocation activity using pH-sensitive fluorescent dyes

• Assess ATP synthesis by monitoring phosphate release via luciferase-based assays

Comparative Analysis:

• Compare activity with well-characterized ATP synthase c-subunits from model organisms

• Evaluate function across a pH gradient to determine optimal conditions

• Assess oligomerization capability, as the c-subunit forms a ring structure critical for ATP synthase function

Controls and Validation:

• Use site-directed mutagenesis to create non-functional variants as negative controls

• Verify proper incorporation into membrane systems using fluorescence microscopy with labeled protein

• Perform kinetic analyses to determine Km and Vmax values for the reconstituted complex

What potential therapeutic applications have been identified for Adiantum capillus-veneris extracts that may relate to ATP synthase function?

Research has identified several therapeutic applications for Adiantum capillus-veneris extracts, some of which may be connected to ATP synthase function:

Wound Healing Properties:
The aqueous extract of A. capillus-veneris has demonstrated significant angiogenic effects through both capillary-like tubular formations and proliferation of endothelial cells in vitro. Additionally, the aqueous and butanol fractions showed significant protective effects against oxygen free radical damage to fibroblasts at concentrations of 50 and 500 μg/ml .

Hepatoprotective Effects:
A. capillus-veneris L. extract can protect hepatic tissue and restore its functions in CBZ-treated rats by mitigating oxidative stress via upregulating antioxidant agents and neutralizing nitrogen and oxygen free radicals. It also relieves hepatic inflammation by decreasing NO, NF-κB, and pro-inflammatory cytokines (TNF-a, IL-6) in the liver .

While these effects haven't been directly linked to ATP synthase function, the plant's antioxidant properties may protect mitochondrial and chloroplast membranes where ATP synthase operates, potentially preserving energy metabolism in damaged tissues.

How does the structure of atpH from Adiantum capillus-veneris compare to its homologs across evolutionary lineages, and what implications does this have for chloroplast evolution?

The atpH gene in Adiantum capillus-veneris encodes an 81-amino acid protein that forms part of the ATP synthase complex in chloroplasts. Evolutionary analysis reveals several significant structural and functional insights:

Structural Conservation:

• The core functional domains of atpH are highly conserved across plant lineages

• The transmembrane regions that form the c-ring structure show particular conservation

• Key residues involved in proton translocation maintain evolutionary stability

Evolutionary Implications:
Adiantum capillus-veneris represents an important evolutionary position as a leptosporangiate fern within the Pteridaceae family. The complete chloroplast genome sequence provides critical insights into chloroplast evolution . The atpH gene and its protein product serve as molecular markers for understanding the evolution of the photosynthetic apparatus across plant lineages.

The chloroplast genome organization in A. capillus-veneris, including the positioning of the atpH gene, helps elucidate the evolutionary trajectory from bryophytes to seed plants, with ferns representing an important intermediate evolutionary stage .

What role might post-translational modifications play in regulating atpH function, and how can these be experimentally determined?

Post-translational modifications (PTMs) likely play crucial roles in regulating atpH function, affecting assembly, stability, and activity of the ATP synthase complex. The following methodological approaches can be employed to investigate these modifications:

Potential PTMs in atpH:

• Phosphorylation: May regulate assembly of the c-ring structure

• Acetylation: Potentially modulates protein-protein interactions within the complex

• Lipid modifications: May facilitate membrane insertion and stability

Experimental Determination Methods:

• Mass Spectrometry Approaches:

  • LC-MS/MS analysis of purified recombinant protein to identify modification sites

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to quantify modification changes under different conditions

  • Top-down proteomics to preserve intact protein structure and modification patterns

• Site-Directed Mutagenesis:

  • Create mutants at potential modification sites

  • Compare functional properties with wild-type protein

  • Assess effects on protein stability, complex assembly, and ATP synthesis activity

• In vitro Modification Assays:

  • Expose purified recombinant atpH to relevant kinases, acetylases, or other modifying enzymes

  • Assess functional consequences through activity assays

  • Use phospho-specific or acetyl-specific antibodies to detect modifications

How does atpH functionally interact with atpI in the chloroplast ATP synthase complex, and what methods can be used to study these interactions?

The atpH (subunit c) and atpI (subunit a) proteins are critical components of the ATP synthase F0 sector in chloroplasts. Their interaction is essential for proton translocation and subsequent ATP synthesis.

Functional Relationship:

• atpH forms a ring structure (c-ring) within the membrane

• atpI (subunit a) interacts with the c-ring to create the proton channel

• This interaction couples proton flow with rotational movement of the c-ring, driving ATP synthesis

Methodological Approaches to Study Interactions:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using antibodies against atpH or atpI

  • FRET (Förster Resonance Energy Transfer) with fluorescently labeled subunits

  • Cross-linking studies followed by mass spectrometry identification

• Structural Studies:

  • Cryo-electron microscopy of reconstituted ATP synthase complexes

  • X-ray crystallography of the F0 sector

  • NMR studies of specific interaction domains

• Functional Reconstitution:

  • Co-expression of recombinant atpH (as described in ) and atpI (described in )

  • Reconstitution into liposomes to measure proton translocation

  • Site-directed mutagenesis of interface residues to map critical interaction points

What is the significance of studying the recombinant atpH protein from Adiantum capillus-veneris compared to other model organisms for understanding chloroplast bioenergetics?

Studying recombinant atpH protein from Adiantum capillus-veneris offers several unique advantages for understanding chloroplast bioenergetics:

Evolutionary Perspective:
Ferns like A. capillus-veneris occupy a critical evolutionary position between bryophytes and seed plants. The atpH protein from this organism provides insights into the evolutionary adaptation of the ATP synthase complex during the transition to vascular plants .

Structural Uniqueness:
The atpH protein from A. capillus-veneris may contain structural adaptations specific to fern chloroplasts, which have evolved under distinct environmental pressures.

Methodological Benefits:

• The complete chloroplast genome sequencing of A. capillus-veneris provides comprehensive genomic context for atpH

• The availability of recombinant expression systems for both atpH and atpI allows for detailed functional studies

• Comparative analysis with atpH from other organisms enables identification of conserved vs. lineage-specific features

By studying atpH from diverse evolutionary lineages, including A. capillus-veneris, researchers can develop a more complete understanding of ATP synthase function and adaptation across plant evolution, informing both basic research in bioenergetics and potential biotechnological applications.