Recombinant Panax ginseng ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, chloroplastic (atpH) in Panax ginseng?

ATP synthase subunit c, chloroplastic (atpH) in Panax ginseng is a critical component of the ATP synthase complex located in the chloroplast. This protein forms part of the F0 sector of ATP synthase, which functions as a proton channel through the membrane. The full-length protein consists of 81 amino acids and plays an essential role in the energy transduction process during photosynthesis. The protein is encoded by the atpH gene in the chloroplast genome and has several synonyms including ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, and Lipid-binding protein .

How does atpH differ from other ATP synthase subunits in plants?

The atpH protein (ATP synthase subunit c) differs from other ATP synthase subunits primarily in its location and function within the complex. While subunits like alpha and beta form the catalytic F1 portion that synthesizes ATP, subunit c forms part of the membrane-embedded F0 portion that facilitates proton translocation. In Panax ginseng chloroplasts, the atpH protein has a specific amino acid sequence (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) that allows it to form a ring structure in the membrane . This structure is crucial for the rotary mechanism of ATP synthesis. Unlike nuclear-encoded ATP synthase subunits, atpH is encoded in the chloroplast genome, specifically in regions with relatively low nucleotide sequence diversity compared to other regions of the chloroplast genome .

What expression systems are optimal for recombinant production of Panax ginseng atpH protein?

The E. coli expression system has been demonstrated to be effective for the recombinant production of Panax ginseng atpH protein. When expressing this chloroplastic protein, adding an N-terminal His-tag facilitates purification while maintaining protein function . For optimal expression, researchers should consider the following methodology:

• Gene synthesis or PCR amplification of the atpH coding sequence (243 bp encoding 81 amino acids)

• Cloning into a vector with a strong promoter (e.g., T7) and His-tag sequence

• Transformation into an E. coli strain optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3))

• Induction at lower temperatures (16-25°C) to enhance proper folding of membrane proteins

• Purification via nickel affinity chromatography under conditions that maintain the protein's native conformation

This approach yields protein with greater than 90% purity as determined by SDS-PAGE, suitable for downstream structural and functional studies .

What are the critical considerations for storage and handling of purified recombinant atpH protein?

Based on experimental data, there are several critical considerations for maintaining the stability and functionality of purified recombinant atpH protein:

• Storage buffer composition: The protein should be stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0. Trehalose acts as a stabilizing agent by preventing protein aggregation and protecting against freeze-thaw damage .

• Temperature conditions: Long-term storage should be at -20°C or preferably -80°C, with working aliquots maintained at 4°C for up to one week .

• Freeze-thaw cycles: Repeated freeze-thaw cycles dramatically reduce protein stability and activity. Therefore, the protein should be divided into single-use aliquots before freezing .

• Reconstitution protocol: Prior to reconstitution, centrifuge the vial briefly to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 50% before aliquoting .

• Handling considerations: As a membrane protein, atpH is susceptible to aggregation. Maintain the protein in solutions with appropriate detergents if native conformation is required for experiments.

How can researchers verify the authenticity and functional integrity of recombinant atpH protein?

Researchers should implement a multi-faceted approach to verify both the authenticity and functional integrity of recombinant atpH protein:

• Sequence verification: Confirm the amino acid sequence (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) using mass spectrometry and N-terminal sequencing .

• Purity assessment: Perform SDS-PAGE analysis to confirm >90% purity, with Western blotting using anti-His antibodies to verify the presence of the His-tag .

• Structural integrity: Circular dichroism (CD) spectroscopy can assess secondary structure components, which should show high alpha-helical content characteristic of membrane-spanning subunits of ATP synthase.

• Functional assays: Reconstitute the protein into liposomes to measure proton translocation activity. Additionally, incorporate the protein into ATP synthase complexes lacking the c-subunit to assess restoration of ATP synthesis activity.

• Interaction studies: Verify specific binding to other ATP synthase subunits using pull-down assays or surface plasmon resonance.

Implementing these verification steps ensures that experimental results obtained with the recombinant protein will accurately reflect the native biological properties of atpH.

What role does the atpH protein play in chloroplast energy metabolism?

The atpH protein (ATP synthase subunit c) plays a crucial role in chloroplast energy metabolism as an essential component of the ATP synthesis machinery. This protein forms an oligomeric ring structure within the thylakoid membrane that functions as a proton channel in the F0 portion of ATP synthase. During photosynthesis, light-driven electron transport establishes a proton gradient across the thylakoid membrane. The atpH protein ring facilitates proton movement down this gradient, which drives the rotation of the ATP synthase complex. This rotational energy is then converted into chemical energy through conformational changes in the F1 portion, leading to ATP synthesis.

How does the primary structure of atpH relate to its function in ATP synthesis?

The primary structure of Panax ginseng atpH protein (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) displays specific sequence characteristics that directly relate to its function in ATP synthesis :

• Hydrophobic amino acid clusters: The abundance of alanine, leucine, isoleucine, and valine residues creates hydrophobic regions that facilitate membrane integration. These regions form two membrane-spanning α-helices that are critical for establishing the proton channel structure.

• Conserved polar residues: Specific polar amino acids (particularly glutamate) within the transmembrane regions are positioned to participate in proton binding and translocation, functioning as the key sites for proton movement through the membrane.

• N-terminal processing site: The N-terminal region contains recognition sequences for proper processing and localization to the chloroplast thylakoid membrane.

• Oligomerization interfaces: Specific residues form the contacts between adjacent c-subunits, enabling the formation of the c-ring structure (typically containing 10-15 c-subunits in chloroplast ATP synthase).

This sequence-structure-function relationship is highly conserved across species, reflecting the fundamental importance of this protein in the evolutionarily conserved process of ATP synthesis.

What is the relationship between chloroplast genome organization and atpH expression in Panax ginseng?

The relationship between chloroplast genome organization and atpH expression in Panax ginseng reveals several important aspects of gene regulation in plastids:

• Genomic location: The atpH gene in Panax ginseng is located within the chloroplast genome, specifically in regions with lower nucleotide sequence diversity (the inverted repeat region or IR) compared to other regions like the large single copy region (LSC) and small single copy region (SSC) . This genomic context contributes to the gene's evolutionary stability.

• Operon structure: The atpH gene is typically part of a polycistronic transcription unit containing multiple ATP synthase-related genes, enabling coordinated expression of functionally related proteins.

• Promoter elements: Expression is driven by chloroplast-specific promoter elements recognized by plastid-encoded RNA polymerase (PEP) and/or nuclear-encoded RNA polymerase (NEP), allowing for both developmental and environmental regulation of expression.

• Post-transcriptional processing: The primary transcript undergoes extensive processing, including RNA editing and intergenic cleavage, to generate mature mRNA for translation.

Comparative analysis of four Chinese Panax ginseng strains (Damaya, Ermaya, Gaolishen, and Yeshanshen) shows conservation of these genomic features, indicating their importance for proper atpH expression and function in energy metabolism .

How can recombinant atpH protein be used to study energy metabolism in medicinal plants?

Recombinant atpH protein serves as a valuable tool for investigating energy metabolism in medicinal plants like Panax ginseng through several research applications:

• Comparative structural biology: Purified recombinant atpH protein can be used for structural studies (X-ray crystallography, cryo-EM) to compare the ATP synthase complex architecture between medicinal plants and model organisms. This reveals adaptations that may relate to the unique metabolic requirements of medicinal plants producing bioactive compounds.

• Protein-protein interaction studies: Using techniques such as pull-down assays or bimolecular fluorescence complementation with recombinant atpH, researchers can identify interaction partners within the ATP synthase complex and potentially uncover plant-specific regulatory mechanisms.

• Energy metabolism assays: Reconstituting recombinant atpH into liposomes allows for measurement of proton conductance and ATP synthesis rates. This provides a controlled system to study how specific bioactive compounds from Panax ginseng (like ginsenosides) might modulate energy production pathways .

• Antibody development: Purified recombinant atpH can be used to generate specific antibodies for immunolocalization studies, enabling visualization of ATP synthase distribution within chloroplasts during different developmental stages or under various stress conditions.

• Site-directed mutagenesis: Creating mutations in recombinant atpH allows for structure-function studies to determine how specific amino acid residues contribute to ATP synthesis efficiency, potentially identifying regions that could be modified to enhance energy production in medicinal plants.

What insights does atpH sequence analysis provide about chloroplast genome evolution in Panax species?

Sequence analysis of atpH in Panax species provides several key insights into chloroplast genome evolution:

• Conservation patterns: Comparative genomic analyses of four Chinese Panax ginseng strains (Damaya, Ermaya, Gaolishen, and Yeshanshen) reveal that the atpH gene shows high sequence conservation, reflecting strong selective pressure to maintain this essential component of energy metabolism .

• Phylogenetic markers: The atpH gene and its flanking regions serve as valuable phylogenetic markers for understanding evolutionary relationships within the Panax genus. Analysis shows that nucleotide sequence diversity in the inverted repeat region (IR) containing atpH is lower than that of large single copy region (LSC) and small single copy region (SSC), providing information about differential evolutionary rates across the chloroplast genome .

• Codon usage bias: Analysis of atpH codon usage patterns across Panax species reveals evolutionary adaptations to optimize translation efficiency in the chloroplast genetic system.

• RNA editing patterns: Sequence analysis has identified RNA editing sites in atpH transcripts, which differ between Panax species and represent another layer of evolutionary adaptation in chloroplast gene expression.

• Selection signatures: The ratio of non-synonymous to synonymous substitutions in atpH across Panax species indicates the strength and direction of selection acting on this gene, providing evidence for its evolutionary significance in adaptation to different environmental conditions.

How can researchers correlate atpH function with the medicinal properties of Panax ginseng?

Researchers can establish meaningful correlations between atpH function and the medicinal properties of Panax ginseng through several methodological approaches:

• Metabolic flux analysis: By manipulating atpH expression levels (through transgenic approaches or RNA interference) and measuring changes in ATP production, researchers can trace how altered energy metabolism affects biosynthetic pathways of medicinal compounds like ginsenosides.

• Stress response studies: Comparing atpH sequence, expression, and function across different Panax ginseng strains with varying medicinal potency can reveal connections between energy metabolism efficiency and adaptation to environmental stresses that trigger production of bioactive compounds.

• Bioactive compound interaction studies: Direct studies of how ginsenosides interact with ATP synthase components, including atpH, can identify potential feedback mechanisms. Research has shown that ginsenosides enhance mitochondrial respiration capacity and ATP production in aerobic respiration-dominated cells , suggesting a relationship between these compounds and energy-producing complexes.

• Comparative transcriptomics: Correlating atpH expression patterns with transcriptional changes in pathways producing medicinal compounds under various growth conditions can reveal regulatory connections.

• Isotope labeling experiments: Using stable isotope-labeled carbon sources combined with mass spectrometry analysis can trace carbon flow from photosynthesis (involving ATP produced via atpH function) into medicinal compound biosynthesis pathways.

This multi-faceted approach allows researchers to establish connections between fundamental energy metabolism processes involving atpH and the plant's ability to produce valuable medicinal compounds.

How might posttranslational modifications of atpH influence ATP synthesis efficiency in Panax ginseng?

Posttranslational modifications (PTMs) of atpH likely play a significant but underexplored role in regulating ATP synthesis efficiency in Panax ginseng. Advanced research should consider:

• Phosphorylation sites: Mass spectrometry analysis of recombinant and native atpH can identify phosphorylation sites that may modify the protein's proton-conducting properties. Research methodology should include comparative phosphoproteomic analysis of atpH under different physiological conditions to correlate modifications with ATP synthesis rates.

• Acetylation dynamics: The relationship between NAD+-dependent SIRT1 activation and mitochondrial biosynthesis observed with ginsenoside treatment suggests a potential role for protein deacetylation in regulating energy metabolism. Researchers should investigate whether atpH undergoes acetylation/deacetylation cycles and how these modifications affect protein function.

• Oxidative modifications: Redox-sensitive amino acid residues in atpH may serve as sensors of chloroplast redox state, potentially modulating ATP synthase activity in response to changing light conditions or oxidative stress. Targeted redox proteomics can identify these modifications.

• Lipid interactions: The specific lipid microenvironment may influence atpH function through direct interactions. Lipidomic analysis coupled with activity assays in reconstituted systems with defined lipid compositions would reveal these dependencies.

• Protein-protein interaction modifications: PTMs may alter atpH interactions with other ATP synthase subunits or regulatory proteins. Crosslinking mass spectrometry (XL-MS) can identify interaction interfaces and how they change with different modifications.

This research direction could reveal important regulatory mechanisms connecting environmental sensing to energy metabolism modulation in medicinal plants.

What role might atpH play in the adaptation of Panax ginseng to environmental stresses?

The adaptation of Panax ginseng to environmental stresses likely involves significant contributions from atpH through several mechanisms that merit advanced investigation:

• Stress-responsive expression patterns: Quantitative PCR and RNA-seq analyses should be employed to track changes in atpH expression under various environmental stresses (drought, temperature extremes, high light, pathogen exposure). Correlation with physiological parameters of stress tolerance would establish functional relationships.

• Structural adaptations: Comparative sequence analysis of atpH across Panax species from different environmental niches may reveal adaptive mutations. Functional characterization of these variants through recombinant expression and in vitro activity assays would determine their impact on ATP synthesis efficiency under stress conditions.

• Integration with stress signaling pathways: Chloroplast retrograde signaling pathways may involve ATP homeostasis sensors that monitor ATP synthase function. Researchers should investigate how atpH-dependent ATP production connects to nuclear gene expression responses during stress adaptation.

• Interaction with stress-protective compounds: Given that ginsenosides (stress-induced secondary metabolites) promote energy metabolism , researchers should examine whether these compounds directly interact with ATP synthase components as part of a feedback mechanism enhancing stress resilience.

• Role in reactive oxygen species (ROS) management: ATP synthase function influences proton gradient dissipation, which can affect ROS production in chloroplasts. Researchers should investigate how atpH variants or expression levels correlate with ROS management capacity during environmental stress.

Understanding these mechanisms could provide insights into breeding or engineering more stress-resilient medicinal plants with enhanced bioactive compound production.

How do interactions between nuclear and chloroplast genomes regulate atpH expression and function?

The regulation of atpH expression and function through nuclear-chloroplast genome interactions represents a complex area requiring sophisticated research approaches:

• Anterograde signaling pathways: Nuclear-encoded factors that regulate chloroplast gene expression, including atpH, should be identified through techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) and DNA affinity purification sequencing (DAP-seq). This would reveal transcription factors and other regulatory proteins involved in coordinating nuclear and chloroplast gene expression.

• Retrograde signaling mechanisms: Experimental manipulation of atpH expression or function (through site-directed mutagenesis or RNA interference) coupled with transcriptomic analysis can reveal how changes in chloroplast ATP synthesis capacity trigger retrograde signals affecting nuclear gene expression.

• RNA editing machinery: The nuclear-encoded components responsible for RNA editing of chloroplast transcripts, including atpH, should be characterized. CRISPR-Cas9 knockout of candidate editing factors followed by RNA sequencing can identify specific factors involved in atpH transcript maturation.

• Translational regulation: Polysome profiling and ribosome footprinting techniques can reveal how nuclear-encoded factors influence the translation efficiency of atpH mRNA in the chloroplast, potentially responding to environmental cues or developmental stages.

• Epigenetic regulation: Analysis of DNA methylation patterns and chromatin structure around nuclear genes encoding regulators of chloroplast function may reveal epigenetic mechanisms coordinating nuclear-chloroplast interactions during development and stress responses.

This research area is particularly relevant for understanding how medicinal plants like Panax ginseng coordinate primary metabolism (energy production via atpH) with secondary metabolism (production of bioactive compounds), potentially leading to strategies for enhancing medicinal compound yield.