Recombinant Huperzia lucidula ATP synthase subunit c, chloroplastic (atpH)

What is Huperzia lucidula ATP synthase subunit c, and what is its significance in plant biology?

Huperzia lucidula ATP synthase subunit c (atpH) is a chloroplast-encoded protein component of the ATP synthase complex in the lycophyte Huperzia lucidula. This protein plays a critical role in the energy conversion machinery of chloroplasts, where it functions as part of the membrane-embedded F₀ portion of the ATP synthase complex. The significance of this protein lies in its fundamental role in the process of oxidative phosphorylation and photophosphorylation, where it contributes to the formation of ATP through the utilization of the proton gradient generated during photosynthesis. The activity of ATP synthase is directly connected to the proton gradient formed during the light-dependent reactions of photosynthesis . As a component of the ATP synthase complex in a basal vascular plant lineage, atpH provides important evolutionary insights into the conservation and divergence of energy production mechanisms across plant phyla. Understanding the structure and function of this protein contributes to our broader knowledge of chloroplast bioenergetics and the evolution of photosynthetic processes in early land plants.

How does the atpH gene differ between Huperzia lucidula and other plant species?

The atpH gene in Huperzia lucidula represents an interesting case study in comparative chloroplast genomics due to its position in an early diverging vascular plant lineage. When comparing the atpH gene across different plant species, researchers have observed both conserved features and lineage-specific variations. The complete chloroplast genome sequence of Huperzia lucidula (154,373 bp) has revealed the genomic context of the atpH gene . Unlike some Selaginella species that have experienced substantial gene loss in their chloroplast genomes, Huperzia lucidula has retained most of its plastid genes, including atpH . The conservation of atpH in Huperzia lucidula contrasts with the extensive gene loss observed in some other lycophyte lineages, where many genes have been lost or pseudogenized, particularly in Selaginella species . This pattern suggests that the ATP synthase complex components have been under strong selective pressure throughout plant evolution. Sequence analysis of atpH across diverse plant lineages can provide important insights into the evolutionary constraints on this essential energy-producing complex, with the Huperzia sequence serving as an important reference point for primitive vascular plants.

What are the most effective methods for isolating and purifying Recombinant Huperzia lucidula ATP synthase subunit c?

The isolation and purification of Recombinant Huperzia lucidula ATP synthase subunit c requires a systematic approach combining molecular biology techniques with protein purification methods. Based on established protocols, the following methodological workflow can be effectively implemented:

• Gene Cloning and Expression Vector Construction:

  • Isolate genomic DNA from Huperzia lucidula tissue using standard extraction methods.

  • Amplify the atpH gene using PCR with specifically designed primers based on the known chloroplast genome sequence .

  • Clone the amplified gene into an appropriate expression vector (e.g., pET system) with a fusion tag (His-tag or GST-tag) to facilitate purification.

• Recombinant Protein Expression:

  • Transform the constructed expression vector into a suitable E. coli strain (BL21(DE3) or similar).

  • Optimize expression conditions including temperature (typically 16-30°C), IPTG concentration (0.1-1.0 mM), and induction time (4-24 hours).

  • Validate expression using SDS-PAGE and western blotting with antibodies specific to the fusion tag or atpH protein.

• Protein Purification Strategy:

  • Lyse bacterial cells using sonication or pressure-based methods in an appropriate buffer system (typically containing 20-50 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl, and protease inhibitors).

  • Perform affinity chromatography using Ni-NTA resin (for His-tagged protein) or glutathione-sepharose (for GST-tagged protein).

  • Further purify using ion-exchange chromatography and/or size exclusion chromatography to obtain highly pure protein.

  • Assess purity using SDS-PAGE and protein concentration using Bradford or BCA assay.

Commercial suppliers like CUSABIO TECHNOLOGY LLC have established protocols for producing this recombinant protein , which may serve as reference methodologies. For researchers requiring detailed structural and functional analysis, additional purification steps may be necessary to ensure the protein maintains its native conformation.

How can researchers effectively reconstitute ATP synthase containing Huperzia lucidula atpH into liposomes for functional studies?

Reconstitution of ATP synthase containing Huperzia lucidula atpH into liposomes provides a controlled system for studying its functional properties. This methodological approach requires careful consideration of lipid composition, protein-to-lipid ratios, and buffer conditions to maintain protein activity. The following protocol outlines a systematic approach based on established methodologies:

• Liposome Preparation:

  • Prepare lipid mixture (typically phosphatidylcholine, phosphatidylethanolamine, and cardiolipin at a ratio of 7:2:1) in chloroform.

  • Evaporate solvent under nitrogen gas to form a thin lipid film and remove residual solvent under vacuum.

  • Hydrate the lipid film with reconstitution buffer (typically 10-20 mM HEPES, pH 7.5, 100 mM KCl, 2.5 mM MgCl₂) to form multilamellar vesicles.

  • Subject vesicles to freeze-thaw cycles (5-10 cycles) followed by extrusion through polycarbonate filters (100-200 nm) to form unilamellar vesicles.

• Protein Incorporation:

  • Solubilize purified recombinant Huperzia lucidula ATP synthase complex or subunit c in mild detergent (e.g., n-dodecyl-β-D-maltoside at 0.05-0.1%).

  • Gradually add detergent-solubilized protein to preformed liposomes at a protein-to-lipid ratio of approximately 1:50 to 1:100 (w/w).

  • Remove detergent by dialysis or using detergent-absorbing beads (Bio-Beads SM-2) with gentle agitation at 4°C for 24-48 hours.

• Functional Assessment:

  • Verify protein incorporation by density gradient centrifugation and/or freeze-fracture electron microscopy.

  • Assess ATP synthase activity using established methods including:

    • ATP-dependent proton translocation measured by pH-sensitive fluorescent dyes (e.g., ACMA or pyranine)

    • ATP synthesis driven by artificially imposed proton gradients

    • ATP-Pi exchange reactions that indicate the reversibility of the ATP synthase mechanism

• Creating Proton Gradients for ATP Synthesis:

  • Incubate proteoliposomes in acidic buffer (e.g., malonate at pH 5.5) with valinomycin (K⁺ ionophore).

  • Rapidly transfer them to alkaline buffer (pH 8.4) containing high K⁺ concentration (approximately 150 mM) .

  • This creates both a pH gradient (ΔpH) and membrane potential (Δψ) that drives ATP synthesis.

This reconstitution system enables the study of ATP synthase function under controlled conditions, allowing researchers to investigate the specific contributions of atpH to enzyme activity and the effects of mutations or environmental factors on ATP production efficiency.

What techniques can be used to study the interaction between Huperzia lucidula atpH and other subunits of the ATP synthase complex?

Understanding the protein-protein interactions between Huperzia lucidula atpH and other ATP synthase subunits is crucial for elucidating the structure-function relationship of this complex. Several complementary techniques can be employed to investigate these interactions:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against the atpH subunit or use antibodies against fusion tags.

  • Incubate antibodies with solubilized ATP synthase complex to precipitate atpH and its interacting partners.

  • Identify co-precipitated proteins using western blotting or mass spectrometry.

  • This approach can identify stable interactions but may miss transient or weak interactions.

• Yeast Two-Hybrid (Y2H) Analysis:

  • Clone atpH and potential interacting partners into appropriate Y2H vectors.

  • Co-transform into yeast strains and screen for reporter gene activation.

  • Validate positive interactions using mutation analysis to identify critical residues.

  • Note that this method may be challenging for membrane proteins like atpH and may require using modified systems designed for membrane proteins.

• Chemical Cross-linking Coupled with Mass Spectrometry:

  • Treat purified ATP synthase complex with chemical crosslinkers (e.g., DSS, BS3, or EDC).

  • Digest the cross-linked complex with proteases and analyze by LC-MS/MS.

  • Identify cross-linked peptides using specialized software to map interaction interfaces.

  • This approach provides spatial information about protein proximities within the complex.

• Förster Resonance Energy Transfer (FRET):

  • Generate fusion constructs of atpH and other subunits with appropriate fluorescent proteins.

  • Express proteins in heterologous systems or reconstitute in liposomes.

  • Measure FRET efficiency to determine proximity and potential interactions.

  • This technique can detect interactions in real-time and in native-like environments.

• Surface Plasmon Resonance (SPR) or Biolayer Interferometry (BLI):

  • Immobilize purified atpH on a sensor chip or biosensor.

  • Flow solutions containing potential interaction partners over the immobilized protein.

  • Measure binding kinetics and affinity constants.

  • These methods provide quantitative data on binding parameters.

These techniques can be used in combination to build a comprehensive understanding of how atpH interacts with other subunits to form a functional ATP synthase complex. The integration of structural data with interaction maps can reveal important insights into the assembly, stability, and function of this essential enzyme complex in Huperzia lucidula.

How does the structure of Huperzia lucidula atpH compare to its homologs in other evolutionary lineages?

The structural comparison of Huperzia lucidula atpH with its homologs across evolutionary lineages provides valuable insights into the conservation and divergence of ATP synthase components. As a member of the lycophytes, one of the earliest diverging vascular plant lineages, Huperzia lucidula occupies a significant position in understanding the evolution of photosynthetic machinery. Structural analysis reveals several key patterns:

Sequence Conservation Analysis:

What are the methodological challenges in studying the function of Huperzia lucidula atpH in heterologous expression systems?

The study of Huperzia lucidula atpH in heterologous expression systems presents several methodological challenges that researchers must address to obtain meaningful functional data. These challenges span from initial expression to functional characterization and require careful experimental design:

• Codon Usage Optimization:

  • The evolutionary distance between Huperzia lucidula and common expression hosts (E. coli, yeast) results in different codon usage preferences.

  • Solution: Synthetic gene constructs with optimized codons based on the expression host's preferences while maintaining the amino acid sequence.

  • Validation: Compare expression levels between native and optimized gene sequences using quantitative RT-PCR and protein quantification.

• Membrane Protein Toxicity:

  • Expression of membrane proteins like atpH often results in toxicity to heterologous hosts due to membrane disruption or protein aggregation.

  • Solution: Use tightly controlled inducible promoters (e.g., PBAD or Tet-regulated systems) to minimize basal expression.

  • Implementation: Titrate inducer concentrations to balance protein expression with cell viability; consider lower temperature induction (16-20°C).

• Proper Membrane Insertion:

  • Ensuring correct folding and insertion of atpH into membranes is critical for functional studies.

  • Solution: Employ specialized expression hosts with enhanced membrane protein handling capabilities (e.g., C41/C43 E. coli strains).

  • Assessment: Use GFP fusion constructs to monitor membrane localization and folding status in real-time.

• Functional Assembly with Other ATP Synthase Subunits:

  • atpH functions as part of a multi-subunit complex, requiring proper assembly for activity assessment.

  • Solution: Co-express complementary subunits from the same source or use compatible subunits from the expression host.

  • Strategy: Develop a modular expression system allowing selective co-expression of various subunit combinations.

• Activity Measurement in Isolated Systems:

  • Isolating the protein while maintaining native-like lipid environment for functional studies is technically challenging.

  • Solution: Reconstitute purified protein into liposomes or nanodiscs that mimic the native membrane environment .

  • Measurement: Develop sensitive assays for proton translocation using pH-sensitive fluorescent dyes or electrochemical methods.

These methodological challenges highlight the complexity of studying membrane proteins from evolutionary distant organisms in heterologous systems. Addressing these challenges requires a multi-faceted approach combining molecular biology, protein biochemistry, and biophysical techniques. The successful expression and functional characterization of Huperzia lucidula atpH would provide valuable insights into the evolution and mechanism of this essential component of the ATP synthase complex.

How can researchers use Huperzia lucidula atpH as a model for understanding the evolution of chloroplast-encoded ATP synthase components?

Huperzia lucidula atpH represents an exceptional model for investigating the evolution of chloroplast-encoded ATP synthase components due to its position in an early-diverging vascular plant lineage. This evolutionary placement provides a unique window into the transitional stage between non-vascular plants and more complex vascular plants. Researchers can leverage this model through several strategic approaches:

• Phylogenomic Analysis Frameworks:

  • Construct comprehensive phylogenetic trees incorporating atpH sequences from diverse plant lineages including bryophytes, lycophytes, ferns, gymnosperms, and angiosperms.

  • Implement molecular clock analyses to estimate divergence times and evolutionary rates.

  • Calculate selection pressures (dN/dS ratios) across different lineages to identify conserved versus rapidly evolving regions.

  • This approach can reveal how selection pressures on ATP synthase components changed during key evolutionary transitions in plant history.

• Structural Evolution Analysis:

  • Compare the predicted or experimentally determined structures of atpH across evolutionary lineages.

  • Map conserved residues onto structural models to identify functionally critical regions.

  • Analyze co-evolution patterns between interacting subunits to understand how the ATP synthase complex evolved as a functional unit.

  • This structural perspective can identify how modifications in protein structure relate to functional adaptations across plant evolution.

• Chloroplast Genome Organization Comparison:

  • Analyze the genomic context of atpH in Huperzia lucidula (where the complete chloroplast genome is 154,373 bp) compared to other plant lineages.

  • Examine synteny relationships and gene order conservation to track genomic rearrangements.

  • Investigate the relationship between genome structure and gene expression regulation.

  • This genomic context provides insights into how chloroplast genome evolution influences ATP synthase component expression and function.

• Experimental Functional Substitution Studies:

  • Create chimeric ATP synthase complexes containing components from different evolutionary lineages.

  • Test functional compatibility between Huperzia lucidula atpH and ATP synthase components from other species.

  • Measure enzymatic activities of these hybrid complexes to assess functional conservation.

  • These experiments can reveal the degree of functional interchangeability and evolutionary constraints on ATP synthase components.

• Reconstruction of Ancestral Sequences:

  • Use maximum likelihood or Bayesian methods to infer ancestral atpH sequences at key nodes in plant evolution.

  • Synthesize and express these reconstructed ancestral proteins.

  • Compare the biochemical properties of ancestral and extant proteins.

  • This approach can provide direct evidence of functional changes that occurred during ATP synthase evolution.

By implementing these research strategies, scientists can use Huperzia lucidula atpH as a powerful model to understand how chloroplast-encoded ATP synthase components evolved throughout plant history. The insights gained from such studies contribute not only to our understanding of photosynthetic energy conversion mechanisms but also to broader questions about chloroplast genome evolution and the adaptation of photosynthetic machinery during plant terrestrialization.

What are the optimal conditions for analyzing Huperzia lucidula atpH expression and regulation in tissue samples?

The analysis of Huperzia lucidula atpH expression and regulation in tissue samples requires careful optimization of experimental conditions to account for the unique characteristics of lycophyte tissues and chloroplast gene expression. The following methodological framework outlines optimal conditions and technical considerations:

Sample Collection and Preservation Protocol:

• Collect fresh Huperzia lucidula tissue, preferably young photosynthetic stems, during mid-morning when photosynthetic activity is high.

• Immediately flash-freeze collected tissues in liquid nitrogen to prevent RNA degradation and preserve the native state of expression.

• Store samples at -80°C until processing, with storage time minimized to prevent degradation.

RNA Extraction Optimization:

• Use specialized RNA extraction protocols designed for recalcitrant plant tissues with high polyphenol and polysaccharide content.

• Include PVPP (polyvinylpolypyrrolidone) in the extraction buffer to absorb polyphenols.

• Implement a CTAB (cetyltrimethylammonium bromide)-based extraction method with high salt concentration to eliminate polysaccharide contamination.

• Validate RNA quality using spectrophotometry (A260/A280 ratio >1.8) and gel electrophoresis (intact rRNA bands).

Gene Expression Analysis Methods:

• RT-qPCR Analysis:

  • Design primers specifically for Huperzia lucidula atpH based on the complete chloroplast genome sequence .

  • Validate primer specificity through melting curve analysis and sequencing of amplicons.

  • Use multiple reference genes for normalization, preferably both nuclear and chloroplast-encoded genes.

  • Implement a relative quantification method with efficiency correction for accurate expression measurement.

• RNA-Seq Approach:

  • Enrich for chloroplast transcripts using rRNA depletion rather than poly(A) selection since chloroplast transcripts lack poly(A) tails.

  • Sequence at a minimum depth of 30 million reads per sample to ensure adequate coverage of chloroplast transcripts.

  • Apply specialized bioinformatic pipelines designed for organellar transcriptome analysis.

• Northern Blot Validation:

  • Use DIG-labeled or radiolabeled probes specific to atpH.

  • Include controls to distinguish mature transcripts from precursors and processing intermediates.

  • This method provides valuable information on transcript size and processing that complement qPCR and RNA-Seq data.

Protein Expression Analysis:

• Develop specific antibodies against Huperzia lucidula atpH or use cross-reactive antibodies validated for this species.

• Optimize protein extraction by including non-ionic detergents (e.g., 1% Triton X-100) to solubilize membrane-associated proteins.

• Perform Western blot analysis using appropriate loading controls for chloroplast proteins.

Environmental and Developmental Variables:

• Systematically analyze atpH expression across:

  • Different developmental stages (gametophyte vs. sporophyte)

  • Various tissue types (photosynthetic vs. non-photosynthetic)

  • Controlled environmental conditions (light intensity, photoperiod, temperature)

• Document all environmental parameters during sample collection to account for natural variation.

By implementing these optimized methods, researchers can obtain reliable data on Huperzia lucidula atpH expression patterns under various conditions, providing insights into the regulation of this important component of the photosynthetic apparatus in an evolutionary significant plant lineage.

How can researchers effectively design site-directed mutagenesis experiments to study critical residues in Huperzia lucidula atpH?

Site-directed mutagenesis provides a powerful approach to investigate structure-function relationships in the Huperzia lucidula atpH protein. Successfully designing and implementing these experiments requires careful planning and execution across multiple stages:

Stage 1: Identification of Critical Residues for Mutagenesis

• Sequence-Based Identification:

  • Perform multiple sequence alignments of atpH from diverse species to identify highly conserved residues.

  • Calculate conservation scores for each position to prioritize residues under strong evolutionary constraints.

  • Identify lineage-specific residues that might confer unique properties to Huperzia lucidula atpH.

• Structure-Based Selection:

  • Generate structural models of Huperzia lucidula atpH using homology modeling or AlphaFold predictions.

  • Identify residues involved in:

    • Proton translocation pathway

    • Subunit-subunit interfaces

    • Membrane-spanning regions

    • Catalytic sites or conformational hinges

• Functional Domain Analysis:

  • Target residues within known functional motifs based on annotation of homologous proteins.

  • Focus on residues implicated in proton binding, oligomerization, or conformational changes during catalysis.

Stage 2: Mutagenesis Strategy Design

• Mutation Types to Consider:

  • Conservative substitutions: Replace with amino acids of similar chemical properties to test specific chemical requirements.

  • Non-conservative substitutions: Replace with amino acids of different properties to test tolerance to major changes.

  • Alanine scanning: Systematically replace residues with alanine to eliminate side chain contributions.

  • Cysteine scanning: Introduce cysteines at strategic positions for subsequent chemical modification or crosslinking.

• Primer Design Guidelines:

  • Design primers with the mutation centered within the primer sequence.

  • Ensure primers have:

    • Appropriate length (25-45 nucleotides)

    • Sufficient GC content (40-60%)

    • Melting temperatures of 78-82°C

    • No strong secondary structures or self-complementarity

  • Validate primers using primer design software to avoid unintended mutations.

• Technical Approach Selection:

  • QuikChange site-directed mutagenesis for single mutations.

  • Gibson Assembly or Golden Gate cloning for multiple simultaneous mutations.

  • Megaprimer approach for mutations in difficult templates with high GC content.

Stage 3: Expression and Functional Assessment Systems

• Expression Strategy:

  • Clone wild-type and mutant atpH into expression vectors with appropriate tags for detection and purification.

  • Consider using chloroplast-specific expression systems when applicable.

  • Express in heterologous systems optimized for membrane protein expression (C41/C43 E. coli strains).

• Functional Assays:

  • Proton translocation assays using pH-sensitive fluorescent dyes.

  • ATP synthesis activity measurements in reconstituted liposomes .

  • Protein-protein interaction assays to assess impacts on complex assembly.

  • Thermal stability assays to determine effects on protein stability.

• Structural Impact Assessment:

  • Circular dichroism spectroscopy to evaluate secondary structure changes.

  • Tryptophan fluorescence to assess tertiary structure alterations.

  • Crosslinking experiments to examine subunit-subunit interactions.

Example Mutagenesis Target Table for H. lucidula atpH:

*Note: Residue positions are hypothetical for illustration, as the exact sequence of Huperzia lucidula atpH is not provided in the search results.

By systematically designing and implementing these mutagenesis experiments, researchers can gain valuable insights into the structure-function relationships of the Huperzia lucidula atpH protein and its role in ATP synthesis in this evolutionarily significant plant lineage.

What are the potential applications of comparative studies between Huperzia lucidula atpH and agricultural crop ATP synthase components?

Comparative studies between Huperzia lucidula atpH and agricultural crop ATP synthase components offer promising research directions with significant implications for crop improvement. These comparative analyses can leverage the evolutionary insights from a basal vascular plant to enhance our understanding of energy metabolism in economically important crops:

These comparative studies represent a promising frontier in agricultural research, where evolutionary insights from basal plant lineages like Huperzia lucidula can inform targeted approaches to enhance crop performance through optimizing fundamental bioenergetic processes.

How might advanced structural biology techniques contribute to our understanding of Huperzia lucidula atpH?

Advanced structural biology techniques offer unprecedented opportunities to elucidate the detailed molecular architecture and dynamic behavior of Huperzia lucidula atpH. These methodological approaches can reveal critical insights that traditional techniques cannot capture:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle cryo-EM can resolve the structure of the entire ATP synthase complex, placing atpH in its native context.

  • Recent advances in sample preparation and detection technology now allow near-atomic resolution (2-3 Å) for membrane protein complexes.

  • This approach could reveal the precise arrangement of atpH subunits within the c-ring and their interactions with other ATP synthase components.

  • Time-resolved cryo-EM may capture different conformational states during the catalytic cycle, providing insights into the dynamic aspects of ATP synthesis.

• Integrated Structural Proteomics:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map the solvent accessibility and conformational dynamics of atpH regions.

  • Cross-linking mass spectrometry (XL-MS) can identify interaction interfaces between atpH and neighboring subunits with residue-level precision.

  • These approaches provide complementary information to static structural methods, highlighting dynamic regions that may be critical for function.

• Solid-State Nuclear Magnetic Resonance (ssNMR):

  • ssNMR is particularly well-suited for membrane proteins like atpH and can provide atomic-resolution information in a lipid environment.

  • This technique can measure precise distances between specific atoms, providing constraints for structural modeling.

  • ssNMR can detect subtle conformational changes induced by protonation/deprotonation events, directly relevant to atpH's role in proton translocation.

• Molecular Dynamics Simulations:

  • Using structural data as input, MD simulations can model the behavior of atpH within a lipid bilayer over microsecond timescales.

  • These simulations can reveal proton pathways, lipid-protein interactions, and conformational changes not easily observed experimentally.

  • Integrating quantum mechanical calculations can provide insights into the energetics of proton transfer through the c-ring.

• Native Mass Spectrometry:

  • This emerging technique can analyze intact membrane protein complexes, providing information on subunit stoichiometry and complex stability.

  • It can detect small molecules or lipids that co-purify with the complex and may be essential for function.

  • Comparing the stability and assembly of Huperzia lucidula ATP synthase with those from other species could reveal evolutionary adaptations.

• Serial Femtosecond Crystallography (SFX) at X-ray Free Electron Lasers:

  • This cutting-edge approach can obtain high-resolution structures from microcrystals of membrane proteins.

  • Time-resolved SFX can potentially capture short-lived intermediates in the catalytic cycle with unprecedented temporal resolution.

  • Although technically challenging, this approach could provide transformative insights into the mechanism of ATP synthesis.

The integration of these advanced structural biology techniques would provide a comprehensive understanding of Huperzia lucidula atpH structure, dynamics, and function within the ATP synthase complex. This multi-dimensional view would contribute significantly to our fundamental understanding of bioenergetics in this evolutionarily significant plant lineage.