Recombinant Liriodendron tulipifera ATP synthase subunit c, chloroplastic (atpH)

What is the molecular structure and basic function of ATP synthase subunit c in Liriodendron tulipifera?

ATP synthase subunit c (atpH) from Liriodendron tulipifera is a critical component of the F0 sector of chloroplastic ATP synthase. This protein functions as part of the membrane-embedded proton channel that facilitates proton movement through the thylakoid membrane, ultimately driving ATP synthesis. The protein consists of 81 amino acids (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) with a highly hydrophobic character, consistent with its membrane-embedded nature . The functional unit typically comprises multiple c-subunits arranged in a ring structure, with the number varying between different species. In chloroplasts, this c-ring plays a crucial role in converting the proton gradient generated during photosynthesis into mechanical energy that drives ATP synthesis.

The protein is alternatively known as ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, or lipid-binding protein, reflecting its various structural and functional aspects . The UniProt accession number for this protein is Q0G9N2, which provides a standardized reference for comparative studies .

How does the chloroplastic ATP synthase subunit c from L. tulipifera compare structurally with homologs from other plant species?

Comparative analysis reveals significant conservation of the atpH protein across different plant species, though with notable variations that reflect evolutionary adaptations. When aligned with the homologous protein from Atropa belladonna (Q8RU61), the L. tulipifera atpH shows high sequence similarity but contains distinct amino acid substitutions .

The sequence comparison below highlights the differences between L. tulipifera and A. belladonna ATP synthase subunit c:

L. tulipifera: MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV
A. belladonna: MNPLVFAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV

The L. tulipifera ATP synthase subunit c is particularly valuable for evolutionary studies due to the species' position within Magnoliids, which diverged early in flowering plant evolution. This makes it an excellent reference point for understanding the ancestral state of this protein in angiosperms .

What are the optimal methods for expression and purification of recombinant L. tulipifera ATP synthase subunit c?

Recombinant expression of L. tulipifera ATP synthase subunit c typically employs bacterial expression systems, with E. coli being the preferred host due to its high yield and established protocols. The procedure involves the following methodological steps:

• Gene Synthesis and Cloning: The atpH gene sequence (coding for amino acids 1-81) is optimized for E. coli codon usage and synthesized or amplified from L. tulipifera chloroplast DNA. The gene is then cloned into an appropriate expression vector with a histidine tag for purification purposes .

• Expression Conditions: Optimal expression is achieved in E. coli cultures induced with IPTG (typically 0.5-1.0 mM) at mid-log phase (OD600 = 0.6-0.8), followed by incubation at lower temperatures (16-25°C) for 12-18 hours to enhance protein folding and solubility. This approach helps minimize inclusion body formation that often occurs with membrane proteins .

• Protein Extraction and Purification: Due to the hydrophobic nature of ATP synthase subunit c, specialized extraction buffers containing detergents (e.g., 1% DDM or CHAPS) are required. Purification typically employs immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged protein, followed by size exclusion chromatography for higher purity .

• Storage and Stability: The purified protein should be stored in a Tris-based buffer containing 50% glycerol at -20°C for short-term storage or -80°C for extended storage. Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For working solutions, storage at 4°C for up to one week is recommended .

What are the critical considerations for designing experiments involving ATP synthase subunit c from L. tulipifera?

When designing experiments with L. tulipifera ATP synthase subunit c, researchers should consider several critical factors:

• Protein Stability and Buffer Conditions: Due to its hydrophobic nature, maintaining the protein in solution requires careful buffer optimization. A Tris-based buffer system (pH 7.5-8.0) with 50% glycerol helps maintain stability. For functional studies, the inclusion of appropriate lipids or detergents that mimic the native membrane environment is essential .

• Temperature Sensitivity: Experimental temperatures should be carefully controlled, as the protein's stability and activity can be significantly affected. While storage requires cold temperatures (-20°C to -80°C), functional assays are typically performed at 25-30°C to better represent physiological conditions .

• Reconstitution Methods for Functional Studies: To study the functional aspects of the c-subunit, reconstitution into liposomes is often necessary. This involves mixing the purified protein with lipids (typically phosphatidylcholine and phosphatidylglycerol at a 3:1 ratio) followed by detergent removal via dialysis or adsorption to Bio-Beads .

• Validation of Protein Folding: Circular dichroism (CD) spectroscopy should be employed to confirm that the recombinant protein maintains its predominantly α-helical secondary structure, which is crucial for its function. The expected CD spectrum should show characteristic minima at 208 and 222 nm, indicative of α-helical content .

• Control Experiments: When assessing functional properties, parallel experiments with well-characterized ATP synthase c-subunits from model organisms (e.g., Arabidopsis thaliana or Spinacia oleracea) provide important reference points for comparative analysis .

How can the atpH protein from L. tulipifera be used in evolutionary studies of plant mitochondrial and chloroplast genomes?

The ATP synthase subunit c from L. tulipifera represents a valuable resource for evolutionary studies due to several unique characteristics:

• Phylogenetic Position: Liriodendron tulipifera (tulip tree) belongs to the Magnoliids, a basal angiosperm lineage that diverged early in flowering plant evolution. This makes its atpH protein an excellent reference point for ancestral state reconstruction of ATP synthase components .

• Evolutionary Rate: The mitochondrial genome of L. tulipifera has evolved remarkably slowly, with an extraordinarily low genome-wide silent substitution rate. This slow evolutionary rate extends to chloroplast genes like atpH, making it a valuable molecular fossil for comparative genomics. Sequence analysis reveals that L. tulipifera proteins often retain ancestral features lost in faster-evolving lineages .

• Methodological Approach for Evolutionary Studies:

  a. Sequence Alignment: Multiple sequence alignment of atpH proteins from diverse plant lineages, including L. tulipifera, other magnoliids like Magnolia stellata, monocots, and eudicots, allows identification of conserved and variable regions.

  b. Phylogenetic Tree Construction: Maximum likelihood or Bayesian methods using aligned atpH sequences help reconstruct evolutionary relationships and estimate divergence times.

  c. Selection Analysis: Calculation of dN/dS ratios across the phylogeny identifies potential sites under positive or purifying selection.

• Ancestral Feature Retention: L. tulipifera atpH provides insights into the ancestral characteristics of this protein in flowering plants. Comparative analysis suggests that the ancestral angiosperm atpH likely had features similar to those observed in L. tulipifera, with subsequent modifications occurring in various lineages during angiosperm diversification .

What methodologies are most effective for studying the interaction between ATP synthase subunit c and other components of the chloroplast ATP synthase complex?

Investigating protein-protein interactions involving ATP synthase subunit c requires specialized techniques due to its hydrophobic nature and membrane-embedded context. The following methodologies have proven effective:

• Crosslinking Mass Spectrometry (XL-MS): This approach involves:

  a. Chemical crosslinking of purified ATP synthase complexes or reconstituted systems containing recombinant L. tulipifera atpH using bifunctional reagents like DSS or BS3.

  b. Enzymatic digestion of crosslinked proteins followed by LC-MS/MS analysis.

  c. Computational analysis of crosslinked peptides to identify interaction interfaces between atpH and other subunits.

• Co-immunoprecipitation with Specific Adaptations:

  a. Utilizing antibodies against the His-tag of recombinant atpH or against native protein.

  b. Including appropriate detergents (0.5-1% digitonin or 0.1% DDM) to maintain membrane protein complexes.

  c. Analyzing co-precipitated proteins by Western blotting or mass spectrometry.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  a. Exposing purified L. tulipifera ATP synthase complexes to D2O buffer.

  b. Quenching the exchange reaction at different time points.

  c. Analyzing deuterium incorporation patterns to identify regions involved in protein-protein interactions.

• Reconstitution Studies with Fluorescently Labeled Components:

  a. Site-specific labeling of recombinant atpH with fluorescent probes at non-critical residues.

  b. Reconstitution with other ATP synthase components in liposomes.

  c. Monitoring protein-protein interactions using Förster resonance energy transfer (FRET) when interacting partners are labeled with compatible fluorophores.

These approaches have revealed that the c-subunit ring interacts primarily with subunit a of the Fo sector and subunits γ and ε of the F1 sector, forming the rotary motor component of the ATP synthase complex .

What experimental approaches can determine the proton translocation efficiency of L. tulipifera ATP synthase subunit c in reconstituted systems?

Assessing proton translocation through the c-ring requires specialized methodologies to recreate the membrane environment and measure proton movement:

• Liposome Reconstitution Protocol:

  a. Preparation of unilamellar liposomes using a mixture of phosphatidylcholine and phosphatidylglycerol (typically 3:1 ratio).

  b. Incorporation of purified L. tulipifera ATP synthase c-subunits into liposomes via detergent-mediated reconstitution.

  c. Removal of detergent through dialysis or Bio-Bead adsorption.

  d. Verification of successful reconstitution using freeze-fracture electron microscopy or dynamic light scattering.

• pH Gradient Establishment and Measurement:

  a. Creation of a pH gradient across liposome membranes using either acid-base transitions or light-driven proton pumps co-reconstituted with the c-subunits.

  b. Monitoring the pH gradient using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine.

  c. Quantification of fluorescence changes over time to determine proton translocation rates.

• Patch-Clamp Electrophysiology:

  a. Formation of a "gigaseal" on liposomes containing reconstituted c-rings.

  b. Recording of ion currents at different membrane potentials.

  c. Analysis of current-voltage relationships to determine conductance properties.

• Comparison with Site-Directed Mutants:

  a. Generation of specific mutations in the L. tulipifera atpH gene, particularly targeting the conserved proton-binding glutamate residue.

  b. Parallel analysis of wild-type and mutant proteins using the above methods.

  c. Quantification of the impact of mutations on proton translocation efficiency.

These approaches have been used to determine that the c-ring structure of plant ATP synthases typically accommodates 14 protons per 360° rotation, contributing to the efficiency of ATP synthesis under varying environmental conditions .

How does the RNA editing profile of atpH in L. tulipifera compare with other plant species, and what methodologies are best for studying this phenomenon?

RNA editing is a critical post-transcriptional modification in plant organellar genes, including atpH. The analysis of L. tulipifera provides valuable insights into the evolution of this process:

• RNA Editing Profile Characteristics:

  The mitochondrial genome of L. tulipifera exhibits extensive RNA editing, with more editing sites than found in many other flowering plants. This high editing frequency extends to chloroplast genes, including atpH. Comparative analysis reveals that many of these editing sites are ancestral, having been lost in various angiosperm lineages during evolution .

• Methodological Approaches for RNA Editing Analysis:

  a. RT-PCR and Direct Sequencing:

  • Extraction of total RNA from L. tulipifera leaf tissue.

  • DNase treatment to eliminate DNA contamination.

  • Reverse transcription using atpH-specific primers.

  • PCR amplification of cDNA and genomic DNA.

  • Sanger sequencing of both products to identify C-to-U editing sites by comparison.

  b. High-Throughput RNA-Seq Analysis:

  • Preparation of RNA-seq libraries from total or organelle-enriched RNA.

  • Deep sequencing to generate high coverage of transcripts.

  • Bioinformatic analysis comparing RNA-seq reads to the reference genome sequence.

  • Identification of editing sites based on nucleotide discrepancies between genomic and transcript sequences.

  c. Targeted Approach Using STS-PCRseq:

  • Design of primers flanking potential editing sites in atpH.

  • Generation of amplicon libraries from cDNA.

  • Deep sequencing of amplicons to quantify editing efficiency at each site.

  • Comparative analysis across different tissues or developmental stages.

• Evolutionary Context and Comparative Analysis:

  L. tulipifera represents an important reference point for understanding the ancestral state of RNA editing in flowering plants. Research indicates that the common ancestor of angiosperms had extensive RNA editing, with over 700 sites across the mitochondrial genome. The atpH gene in L. tulipifera retains many ancestral editing sites that have been lost in other lineages, making it valuable for reconstructing the evolution of this process .

• Functional Implications:

  RNA editing in atpH often restores conserved amino acids that are critical for protein function. The editing pattern in L. tulipifera suggests that this process plays an important role in maintaining ATP synthase functionality despite genomic mutations. Experimental approaches comparing edited and unedited protein variants can quantify the impact of editing on protein stability and activity .

What protocols are most effective for studying the expression and activity changes of ATP synthase subunit c under various environmental stresses in L. tulipifera?

Investigating stress responses in ATP synthase components requires integrated approaches spanning from gene expression to protein function:

• Stress Treatment Experimental Design:

  a. Control Conditions: Growth of L. tulipifera seedlings under controlled conditions (22-25°C, 16/8h light/dark cycle, 50-60% relative humidity).

  b. Stress Treatments:

  • Cold stress: Exposure to 4°C for varying durations (6h, 12h, 24h, 48h).

  • Heat stress: Exposure to elevated temperatures (35-40°C).

  • Drought stress: Withholding water until predefined soil moisture levels are reached.

  • High light stress: Exposure to light intensities above normal growth conditions (>800 μmol m⁻² s⁻¹).

  c. Tissue Sampling: Collection of leaf tissue at multiple time points during stress treatment, with immediate freezing in liquid nitrogen.

• Gene Expression Analysis:

  a. RT-qPCR Protocol:

  • RNA extraction using specialized kits for woody plants.

  • cDNA synthesis with random hexamers or oligo(dT) primers.

  • qPCR targeting atpH with normalization to stable reference genes (e.g., actin, GAPDH).

  • Data analysis using the 2^(-ΔΔCt) method to quantify relative expression changes.

  b. RNA-Seq Approach:

  • Preparation of RNA-seq libraries from control and stressed plants.

  • Sequencing to generate 30-50 million reads per sample.

  • Bioinformatic analysis focusing on differential expression of atpH and other ATP synthase genes.

• Protein Level Assessment:

  a. Western Blot Analysis:

  • Protein extraction using buffers containing detergents suitable for membrane proteins.

  • SDS-PAGE separation followed by transfer to PVDF membranes.

  • Immunodetection using antibodies specific to ATP synthase subunit c or to the recombinant tag.

  • Quantification of band intensity to determine protein abundance changes.

  b. Proteomics Approach:

  • Extraction of thylakoid membranes from control and stressed plants.

  • Digestion of proteins and preparation for LC-MS/MS analysis.

  • Label-free quantification to determine changes in ATP synthase subunit abundance.

• Functional Activity Assessment:

  a. ATP Synthesis Measurement:

  • Isolation of intact chloroplasts from control and stressed plants.

  • Measurement of ATP synthesis rates using luciferin-luciferase assays.

  • Correlation of activity changes with expression data.

  b. Electrochromic Shift (ECS) Measurements:

  • Non-invasive spectroscopic technique to assess proton motive force in vivo.

  • Comparison between control and stressed plants to evaluate ATP synthase function.

How can researchers effectively compare the evolutionary conservation of ATP synthase stress responses between L. tulipifera and other plant species?

Comparative stress response studies provide insights into the evolutionary conservation of ATP synthase regulation across plant lineages:

Research using these approaches has revealed that while the core function of ATP synthase is highly conserved across plants, regulatory mechanisms have diversified during evolution. L. tulipifera, with its slow evolutionary rate, often retains ancestral regulatory patterns that have been modified in more derived lineages like Arabidopsis or rice .

What computational approaches are most effective for modeling the structure and dynamics of L. tulipifera ATP synthase subunit c in membrane environments?

Computational modeling provides valuable insights into the structure and dynamics of membrane-embedded proteins like ATP synthase subunit c:

• Homology Modeling Workflow:

  a. Template Selection:

  • Identification of experimentally determined structures of ATP synthase c-subunits from other species.

  • Assessment of sequence similarity to L. tulipifera atpH.

  • Selection of optimal templates (typically bacterial or algal ATP synthase structures).

  b. Model Building Protocol:

  • Sequence alignment between L. tulipifera atpH and template sequences.

  • Generation of multiple models using software like MODELLER or SWISS-MODEL.

  • Refinement focusing on transmembrane regions and functionally important sites.

  • Validation using metrics like DOPE score, QMEAN, and Ramachandran plot analysis.

• Molecular Dynamics Simulation Approach:

  a. System Preparation:

  • Embedding of the modeled c-subunit ring in a lipid bilayer mimicking thylakoid membrane composition.

  • Solvation with explicit water molecules and addition of counter-ions.

  • Energy minimization to remove steric clashes and unfavorable interactions.

  b. Simulation Parameters:

  • Utilization of appropriate force fields (e.g., CHARMM36 for proteins and lipids).

  • NPT ensemble conditions (constant particle number, pressure, and temperature).

  • Production runs of 100-500 ns to capture relevant dynamics.

  • Analysis of stability, conformational changes, and proton pathway dynamics.

• Proton Translocation Modeling:

  a. Quantum Mechanics/Molecular Mechanics (QM/MM) Approach:

  • Treatment of the proton-binding site with quantum mechanical methods.

  • Simulation of the rest of the system with molecular mechanics.

  • Investigation of protonation/deprotonation events at key glutamate residues.

  b. Constant pH Molecular Dynamics:

  • Simulation allowing protonation states to change dynamically.

  • Assessment of pKa shifts in the membrane environment.

  • Characterization of the complete proton transport pathway.

• Integration with Experimental Data:

  a. Validation Protocol:

  • Comparison of predicted structures with experimental data (e.g., CD spectroscopy results).

  • Testing of computational predictions through site-directed mutagenesis experiments.

  • Refinement of models based on experimental feedback.

These computational approaches have revealed that the c-ring structure from L. tulipifera likely forms a tight oligomeric assembly with specific amino acid residues facilitating both protein-protein interactions within the ring and proton translocation across the membrane .

What are the best methodological approaches for investigating post-translational modifications of ATP synthase subunit c in L. tulipifera and their functional significance?

Post-translational modifications (PTMs) play important roles in regulating ATP synthase function, requiring specialized techniques for their detection and functional characterization:

Research using these approaches has identified several key PTMs in plant ATP synthase components, including phosphorylation, acetylation, and oxidative modifications. These PTMs often serve as regulatory mechanisms that adjust ATP synthase activity in response to changing environmental conditions and metabolic demands .

What are the most promising future research directions involving L. tulipifera ATP synthase subunit c based on current scientific knowledge?

Based on current understanding of ATP synthase biology and the unique characteristics of L. tulipifera, several promising research directions emerge:

• Evolutionary and Comparative Studies:

  The position of L. tulipifera as a member of the early-diverging Magnoliids makes its ATP synthase components particularly valuable for evolutionary studies. Future research should focus on comprehensive comparative analyses across the plant kingdom to better understand the ancestral characteristics of ATP synthase and its evolutionary trajectory. The remarkably slow evolutionary rate of L. tulipifera mitochondrial and chloroplast genomes makes it an excellent reference point for such studies .

• Structural Biology Approaches:

  Despite advances in computational modeling, high-resolution structural data for plant ATP synthase components remains limited. Future efforts should focus on cryo-electron microscopy studies of the complete ATP synthase complex from L. tulipifera, which could provide unprecedented insights into the structural organization of this complex in basal angiosperms. These studies would be particularly valuable for understanding how the c-ring interfaces with other components of the ATP synthase complex .

• Engineering Applications:

  The unique properties of L. tulipifera ATP synthase components could be harnessed for biotechnological applications. Potential directions include:

  a. Bioengineering of c-subunits with modified proton/ATP ratios to enhance photosynthetic efficiency.

  b. Development of synthetic biology applications utilizing the robust structural characteristics of L. tulipifera ATP synthase components.

  c. Creation of hybrid ATP synthase complexes incorporating features from different species to optimize performance under specific conditions.

• Environmental Adaptation Mechanisms:

  Further research on how ATP synthase regulation contributes to environmental adaptation in L. tulipifera could provide valuable insights for climate change biology. This species' remarkable resilience and longevity (with some specimens living over 400 years) suggest effective stress response mechanisms that may involve ATP synthase regulation. Understanding these mechanisms could inform strategies for enhancing crop resilience to environmental stresses .

What methodological challenges remain in the study of chloroplast ATP synthase from L. tulipifera, and how might they be addressed?

Despite significant advances, several methodological challenges persist in the study of L. tulipifera ATP synthase:

• Tissue Availability and Manipulation:

  L. tulipifera is a large tree with slow growth, making it challenging to implement genetic manipulation techniques routinely used in model plants. Future approaches should focus on:

  a. Development of cell culture systems from L. tulipifera leaf tissue that maintain chloroplast integrity.

  b. Optimization of transformation protocols for transient expression in L. tulipifera tissues.

  c. Utilization of heterologous expression systems where L. tulipifera genes are expressed in tractable model organisms.

  d. Implementation of emerging technologies like CRISPR-Cas9 delivered via nanomaterials for non-traditional model organisms.

• Functional Reconstitution Challenges:

  The complete functional reconstitution of plant ATP synthase complexes remains technically challenging due to their complexity and membrane-embedded nature. Future methodological improvements should include:

  a. Development of native nanodiscs or other membrane mimetics specifically optimized for plant ATP synthase components.

  b. Implementation of cell-free expression systems capable of co-translational membrane insertion.

  c. Creation of hybrid systems where L. tulipifera components are integrated into well-characterized ATP synthase complexes from model organisms.

• In Vivo Monitoring Techniques:

  Current techniques for studying ATP synthase function in vivo have limited resolution. Future methodological directions should explore:

  a. Development of genetically encoded sensors for ATP synthesis and proton movement applicable to non-model plants.

  b. Adaptation of advanced microscopy techniques (like super-resolution microscopy) for visualizing ATP synthase dynamics in intact chloroplasts.

  c. Implementation of non-invasive spectroscopic methods with higher spatial and temporal resolution.

• Bioinformatic Challenges:

  The analysis of L. tulipifera genomic and proteomic data presents unique challenges due to limited reference information. Future efforts should focus on:

  a. Completion of a high-quality L. tulipifera genome assembly with comprehensive annotation.

  b. Development of specialized bioinformatic pipelines for analyzing RNA editing and other post-transcriptional modifications in non-model plants.

  c. Creation of improved structural prediction algorithms specifically optimized for membrane proteins from diverse plant lineages.

Addressing these methodological challenges will require interdisciplinary approaches combining expertise from plant biology, biochemistry, structural biology, and computational science. The resulting advances would not only benefit studies of L. tulipifera ATP synthase but would also contribute to broader understanding of energy metabolism across the plant kingdom .