Recombinant Marchantia polymorpha ATP synthase subunit c, chloroplastic (atpH)

What is Marchantia polymorpha ATP synthase subunit c and why is it significant for research?

Marchantia polymorpha ATP synthase subunit c (atpH) is a critical component of the chloroplastic ATP synthase complex in liverworts. This protein is particularly significant as Marchantia polymorpha represents one of the most basal lineages of extant land plants, making it an excellent model for evolutionary studies of energy metabolism in plants. The protein functions as part of the F0 sector of ATP synthase, creating a proton channel through the membrane that drives ATP synthesis. Its structure consists of 81 amino acids with the sequence: MNPLISAASVIAAGLAVGLASIGPGIGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . Understanding this protein contributes to our knowledge of chloroplast evolution and energy production mechanisms across plant lineages.

What expression systems are used for recombinant production of Marchantia polymorpha atpH?

The most commonly utilized expression system for recombinant Marchantia polymorpha ATP synthase subunit c is E. coli, due to its high yield and relative simplicity. When expressing the protein in E. coli, researchers typically fuse the atpH gene to an N-terminal His tag to facilitate subsequent purification . For higher expression levels in plant-based systems, researchers have developed optimized vectors using specific promoters such as the MpEF1α promoter, which can yield significant amounts of recombinant proteins in Marchantia polymorpha . Chloroplast transformation systems have also been developed, which can yield up to 400-500 μg/g FW (approximately 15% of total soluble protein) of recombinant proteins . For membrane proteins like ATP synthase subunit c, special considerations for membrane insertion and proper folding are necessary during the expression process.

How should recombinant Marchantia polymorpha atpH be stored and reconstituted?

For optimal preservation of recombinant Marchantia polymorpha ATP synthase subunit c activity, the lyophilized protein powder should be stored at -20°C to -80°C upon receipt. Working aliquots can be kept at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and function .

For reconstitution, the vial containing the protein should first be briefly centrifuged to ensure all contents are at the bottom. The protein should then be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard practice) and aliquot before storing at -20°C or -80°C . This glycerol addition helps prevent protein denaturation during freezing by disrupting ice crystal formation that could damage protein structure.

What are the comparative structural features of ATP synthase subunit c across bryophytes and vascular plants?

The protein contains conserved functional motifs, particularly the key proton-binding site that typically involves a conserved carboxylate residue (aspartate or glutamate) essential for proton translocation. Comparative analysis suggests that while the core functional regions show high conservation, terminal regions and certain loop structures may exhibit lineage-specific adaptations. These differences might reflect evolutionary adaptations to different environmental pressures experienced by non-vascular versus vascular plants, potentially affecting the efficiency of ATP synthesis under various conditions.

How does the regulation of ATP synthase activity differ in Marchantia polymorpha compared to vascular plants?

Regulation of ATP synthase activity in Marchantia polymorpha shows both conservation and divergence compared to vascular plants. Research on related membrane proteins in Marchantia indicates that phosphorylation plays a crucial role in protein regulation. For instance, plasma membrane H+-ATPase in Marchantia polymorpha is regulated by phosphorylation of its penultimate threonine residue in response to physiological signals such as light, sucrose, and osmotic shock . This mechanism is similar to the regulation observed in vascular plants.

What approaches should be used to investigate protein-protein interactions involving ATP synthase subunit c in Marchantia polymorpha?

To investigate protein-protein interactions involving ATP synthase subunit c in Marchantia polymorpha, researchers should employ a multi-faceted approach combining both in vivo and in vitro methods. The following methodology is recommended:

• Co-immunoprecipitation (Co-IP): Using antibodies against the His-tagged recombinant ATP synthase subunit c to pull down the protein complex from Marchantia chloroplast extracts, followed by mass spectrometry to identify interacting partners.

• Yeast two-hybrid screening: Modified to accommodate membrane proteins by using split-ubiquitin systems, with the atpH sequence as bait to screen for potential interacting proteins.

• Bimolecular Fluorescence Complementation (BiFC): For visualizing interactions in vivo, fuse potential interacting proteins with complementary fragments of a fluorescent protein and express them in Marchantia using optimized promoters such as proMp35S or proMpEF1α .

• Proximity-based labeling: Using techniques such as BioID or APEX2 fused to ATP synthase subunit c to identify proximal proteins in the native chloroplast environment.

• Cryo-electron microscopy: For structural analysis of the entire ATP synthase complex, providing insights into the spatial arrangement and interactions of subunit c with other components.

When performing these experiments, it's critical to include appropriate controls and validate interactions through multiple independent methods. Comparative analysis with interaction data from other plant species can provide evolutionary context to the findings.

How can researchers optimize expression of recombinant Marchantia polymorpha atpH in heterologous systems?

To optimize expression of recombinant Marchantia polymorpha ATP synthase subunit c in heterologous systems, researchers should consider several key factors:

For E. coli expression systems:

• Codon optimization: Adjust the codon usage of the atpH gene to match the preferences of E. coli to improve translation efficiency.

• Expression temperature: Lower temperatures (16-20°C) can enhance proper folding of membrane proteins like ATP synthase subunit c.

• Induction conditions: Optimize IPTG concentration (typically 0.1-0.5 mM) and induction time (4-16 hours) for maximal protein yield while maintaining proper folding.

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

For expression in plant systems:

• Promoter selection: Strong constitutive promoters such as proMp35S×2 can yield high expression levels, though they may reduce plant growth. For controlled expression, inducible promoters like proMpHSP17.8A1 can be used .

• Subcellular targeting: Direct the protein to the chloroplast using appropriate transit peptides. The native chloroplast targeting sequence of the atpH gene can be used for this purpose.

• Vector design: For nuclear transformation, ensure the vector contains appropriate selection markers and regulatory elements. For chloroplast transformation, include homologous flanking sequences for proper integration into the chloroplast genome .

• Transformation method: Agrobacterium-mediated transformation is effective for nuclear integration, while biolistic methods are preferred for chloroplast transformation.

What purification strategies are most effective for recombinant Marchantia polymorpha atpH?

For efficient purification of recombinant Marchantia polymorpha ATP synthase subunit c, researchers should implement a multi-step purification strategy tailored to this highly hydrophobic membrane protein:

Step 1: Protein Extraction and Solubilization

• Harvest E. coli cells expressing His-tagged atpH protein and disrupt by sonication or French press in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and protease inhibitors .

• Solubilize membrane fractions containing the protein using appropriate detergents (e.g., n-dodecyl-β-D-maltoside (DDM) at 1% w/v or digitonin at 2% w/v).

Step 2: Affinity Chromatography

• Apply the solubilized extract to a Ni-NTA affinity column equilibrated with buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% detergent.

• Wash with increasing concentrations of imidazole (20-50 mM) to remove non-specifically bound proteins.

• Elute the His-tagged atpH protein with buffer containing 250-300 mM imidazole .

Step 3: Size Exclusion Chromatography

• Further purify the protein by size exclusion chromatography using a Superdex 200 column equilibrated with buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.05% detergent.

• Collect fractions and analyze by SDS-PAGE to identify those containing pure atpH protein.

Step 4: Validation and Storage

• Confirm protein identity by western blot using anti-His antibodies and/or mass spectrometry.

• Assess purity by SDS-PAGE (should be >90%) .

• Store purified protein in storage buffer containing 6% trehalose at pH 8.0 as recommended , with the addition of 5-50% glycerol for long-term storage.

How can functional activity of recombinant Marchantia polymorpha atpH be assessed?

Assessing the functional activity of recombinant Marchantia polymorpha ATP synthase subunit c requires specialized techniques that evaluate its role in proton translocation and ATP synthesis. The following methodological approaches are recommended:

Proton Translocation Assays:

• Reconstitution into liposomes: Incorporate purified recombinant atpH protein into liposomes containing pH-sensitive fluorescent dyes such as ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine.

• Establishing a proton gradient: Initiate proton translocation by creating an artificial proton gradient across the liposome membrane.

• Fluorescence measurements: Monitor changes in fluorescence that indicate proton movement through the ATP synthase c-ring.

ATP Synthesis Activity:

• Co-reconstitution assay: Reconstitute recombinant atpH with other ATP synthase subunits in liposomes.

• Energization: Establish a proton motive force (PMF) across the liposome membrane using pH gradient and/or electrical potential.

• ATP synthesis measurement: Detect ATP production using luciferase-based assays or NADP+ reduction coupled to ATP-dependent glucose phosphorylation.

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy: Evaluate the secondary structure composition of recombinant atpH protein to confirm proper folding.

• Proteolytic digestion patterns: Compare trypsin or chymotrypsin digestion patterns between native and recombinant proteins to assess structural similarity.

Interaction Studies:

• Binding assays with known partners: Test the ability of recombinant atpH to interact with other ATP synthase subunits using techniques like surface plasmon resonance (SPR).

• Assembly into c-rings: Analyze the ability of recombinant atpH to form oligomeric c-rings using blue native PAGE or analytical ultracentrifugation.

What are common challenges in expressing recombinant Marchantia polymorpha atpH and how can they be addressed?

Challenge 1: Low expression levels in heterologous systems

• Solution: Optimize codon usage for the expression host, try different growth temperatures (16-25°C), and test various induction conditions. For E. coli, specialized strains like C41(DE3) designed for membrane protein expression can improve yields .

• Alternative approach: Consider using Marchantia as an expression system with strong promoters like proMp35S×2, which can yield high expression levels for recombinant proteins .

Challenge 2: Protein misfolding and aggregation

• Solution: Express the protein at lower temperatures (16-20°C) and reduce inducer concentration. Adding chemical chaperones like glycerol (5-10%) or betaine (1 mM) to the culture medium can improve folding.

• Alternative approach: Co-express molecular chaperones or fuse the protein with solubility-enhancing tags like MBP (maltose-binding protein) while retaining the His-tag for purification .

Challenge 3: Difficulty in extracting and purifying the membrane protein

• Solution: Optimize detergent selection for solubilization by screening multiple detergents (DDM, digitonin, LMNG). Incorporate a stabilizing mixture of lipids during extraction.

• Alternative approach: Use protein fusion strategies that facilitate extraction, such as incorporating a removable fusion partner that enhances membrane extraction.

Challenge 4: Reduced growth of transgenic Marchantia expressing high levels of recombinant proteins

• Solution: Utilize inducible promoters like proMpHSP17.8A1 that allow plant growth before protein expression is triggered, as demonstrated in betanin production systems .

• Alternative approach: Target the protein to specific subcellular compartments that might better accommodate high expression levels, such as the chloroplast, which has shown tolerance for up to 15% of total soluble protein being recombinant protein .

How can researchers address discrepancies in experimental results when working with ATP synthase subunit c?

When encountering discrepancies in experimental results related to ATP synthase subunit c from Marchantia polymorpha, researchers should implement a systematic troubleshooting approach:

Step 1: Validate protein identity and integrity

• Confirm protein sequence using mass spectrometry to verify that the correct protein is being studied.

• Assess protein purity using multiple methods (SDS-PAGE, western blot, size exclusion chromatography) to ensure no contaminating proteins are affecting results .

• Check for protein degradation using N- and C-terminal antibody detection or mass spectrometry.

Step 2: Analyze experimental conditions

• Create a detailed comparison table of buffer compositions, pH values, and ionic strengths used across experiments.

• Standardize protein concentrations using multiple protein quantification methods (Bradford, BCA, and direct spectrophotometric measurement at 280 nm).

• Carefully control temperature and incubation times, as membrane proteins are often sensitive to these parameters.

Step 3: Examine specific variables that affect membrane proteins

• Compare detergent types and concentrations used for protein solubilization and storage.

• Assess lipid compositions in reconstitution experiments, as lipid environment significantly affects membrane protein function.

• Consider post-translational modifications that might differ between native and recombinant proteins.

Step 4: Implement rigorous controls

• Include positive controls using well-characterized ATP synthase subunits from model organisms.

• Run parallel experiments with commercially available ATP synthase components as reference points.

• Utilize negative controls with inactive protein variants (e.g., site-directed mutants affecting key functional residues).

Step 5: Cross-validate with complementary techniques

• If discrepancies appear in functional assays, validate with structural assessments (CD spectroscopy, thermal stability assays).

• Confirm protein-protein interaction results using multiple methods (pull-down assays, cross-linking studies, and analytical ultracentrifugation).

What statistical approaches are appropriate for analyzing ATP synthase activity data?

When analyzing ATP synthase activity data involving Marchantia polymorpha atpH, researchers should employ robust statistical approaches tailored to biochemical activity measurements:

Descriptive Statistics and Data Visualization:

• Calculate means, standard deviations, and standard errors for activity measurements across technical and biological replicates.

• Create box plots or violin plots to visualize data distribution and identify potential outliers.

• Use scatter plots with regression lines to examine relationships between activity and experimental variables (e.g., substrate concentration, pH, temperature).

Inferential Statistics for Hypothesis Testing:

• Analysis of Variance (ANOVA): For comparing activity across multiple experimental conditions, followed by appropriate post-hoc tests (Tukey's HSD or Bonferroni correction) to identify specific differences.

• Student's t-test: For direct comparison between two conditions (e.g., wild-type vs. mutant protein activity).

• Non-parametric alternatives: Use Mann-Whitney U test or Kruskal-Wallis test when data do not meet assumptions of normality.

Enzyme Kinetics Analysis:

• Fit activity data to appropriate enzyme kinetic models (Michaelis-Menten, Hill equation) using non-linear regression.

• Calculate and compare kinetic parameters (Km, Vmax, Hill coefficient) across experimental conditions.

• Use statistical tests (F-test or Akaike Information Criterion) to determine which kinetic model best fits the data.

Advanced Statistical Approaches:

• Principal Component Analysis (PCA): To identify patterns in multivariate data sets combining activity measurements with other protein characteristics.

• Hierarchical clustering: To identify groups of conditions with similar activity profiles.

• Mixed-effects models: When dealing with repeated measurements or nested experimental designs.

Reporting Standards:

• Always report sample sizes, p-values, and effect sizes.

• Include confidence intervals around parameter estimates.

• Use clear graphical representations with appropriate error bars (standard deviation or standard error of the mean).

How can genetic modifications of Marchantia polymorpha atpH advance our understanding of ATP synthase function?

Genetic modifications of Marchantia polymorpha ATP synthase subunit c offer powerful approaches to elucidate fundamental aspects of ATP synthase function and regulation. Several methodological strategies can be implemented:

Site-Directed Mutagenesis Approaches:

• Proton-binding site modifications: Introducing mutations in the conserved carboxylate residue responsible for proton binding can provide insights into the proton translocation mechanism.

• Interface residue alterations: Modifying amino acids involved in subunit-subunit interactions can reveal critical contact points for c-ring assembly and stability.

• Transmembrane domain mutations: Systematic alteration of residues in transmembrane helices can identify regions essential for proton channel formation and membrane anchoring.

Domain Swapping Experiments:

• Replace specific regions of Marchantia atpH with corresponding sequences from other species to identify domains responsible for species-specific functional properties.

• Create chimeric proteins combining domains from different ATP synthase subunits to investigate subunit cooperation.

Expression System Development:

• Develop chloroplast transformation vectors specifically designed for atpH modifications, building upon existing Marchantia chloroplast engineering tools that have achieved expression levels of up to 15% of total soluble protein .

• Create inducible expression systems using heat-shock promoters like proMpHSP17.8A1 to control the timing of modified atpH expression.

Integration with Structural Biology:

• Combine genetic modifications with cryo-electron microscopy to visualize structural changes in the ATP synthase complex resulting from specific mutations.

• Use cross-linking studies with genetically incorporated unnatural amino acids to map precise interaction networks within the ATP synthase complex.

These approaches can provide insights into fundamental questions such as the stoichiometry of proton translocation, the mechanism of rotary catalysis, and the evolutionary adaptations of ATP synthase in early land plants.

What role might Marchantia polymorpha ATP synthase research play in understanding chloroplast evolution?

Marchantia polymorpha occupies a unique position in plant evolution as a member of liverworts, which represent one of the earliest diverging lineages of land plants. Research on its ATP synthase subunit c provides valuable insights into chloroplast evolution:

Evolutionary Conservation Analysis:

• Comparative studies between Marchantia polymorpha atpH and its counterparts in algae, cyanobacteria, and vascular plants can identify core conserved features that have remained unchanged through hundreds of millions of years of evolution.

• Alignment of atpH sequences across diverse plant lineages reveals selective pressures on specific residues, indicating functional constraints that have shaped ATP synthase evolution.

Regulatory Mechanism Evolution:

• Investigation of ATP synthase regulation in Marchantia provides insights into ancestral regulatory mechanisms. For example, studies on related membrane proteins in Marchantia have shown that phosphorylation of the penultimate threonine residue and subsequent 14-3-3 protein binding - a key regulatory mechanism in vascular plants - is also present in this basal land plant .

• The presence of both phosphorylated and non-phosphorylated forms of related ATPases in Marchantia suggests potential evolutionary intermediates in regulatory mechanism development .

Genome Organization and Gene Transfer:

• Analysis of the atpH gene location and context in the Marchantia chloroplast genome provides evidence for genome rearrangements and gene transfers that occurred during chloroplast evolution.

• Comparison with nuclear-encoded ATP synthase components can shed light on the evolutionary process of gene transfer from chloroplast to nucleus.

Structural Adaptations:

• Structural studies of Marchantia ATP synthase can reveal adaptations that occurred during the transition from aquatic to terrestrial environments, particularly in relation to altered energy demands and environmental stressors.

• Investigation of protein-protein interactions in the Marchantia ATP synthase complex might identify ancestral interaction patterns that preceded the more complex regulatory networks seen in vascular plants.

How can research on Marchantia polymorpha atpH inform synthetic biology applications?

Research on Marchantia polymorpha ATP synthase subunit c has significant implications for synthetic biology applications, particularly in the following areas:

Chloroplast Engineering Platforms:

• Marchantia has emerged as an excellent model system for chloroplast engineering, with transformation methods yielding expression levels of up to 400-500 μg/g fresh weight (approximately 15% of total soluble protein) . Understanding atpH structure and function can inform the development of optimized chloroplast expression cassettes.

• The compact genome and rapid growth cycle of Marchantia make it an attractive platform for synthetic biology applications requiring chloroplast modification.

Biomimetic Energy Systems:

• Detailed understanding of the structure and function of ATP synthase subunit c can inspire the design of artificial molecular motors and energy-converting nanomachines.

• The proton translocation mechanism of ATP synthase can inform the development of synthetic membrane systems for energy conversion and storage.

Protein Production Optimization:

• Insights from the expression and assembly of atpH can guide the development of improved heterologous expression systems for membrane proteins.

• The comparison of different promoters (e.g., proMp35S×2, proMpEF1α) and subcellular targeting strategies in Marchantia has demonstrated that expression levels can be significantly optimized through appropriate vector design .

Metabolic Engineering:

• Understanding ATP synthase function and regulation in Marchantia can inform strategies for manipulating energy metabolism in engineered organisms.

• The successful expression of betanin in Marchantia using inducible heat-shock promoters (proMpHSP17.8A1) demonstrates the potential for controlled metabolic engineering in this system .

Evolutionary-Inspired Design:

• The study of ATP synthase in a basal land plant like Marchantia provides insights into evolutionarily optimized protein design principles that can be applied to synthetic protein engineering.

• Comparative analysis between Marchantia and other organisms can identify minimal functional units and critical design features for synthetic ATP synthase components.

How does Marchantia polymorpha ATP synthase subunit c compare to other model organisms?

The comparison demonstrates that Marchantia polymorpha ATP synthase subunit c shares high sequence identity with other plant chloroplast atpH proteins, particularly those from vascular plants like Arabidopsis and spinach. This conservation reflects the fundamental importance of this protein in chloroplast function across plant evolution. The slightly lower identity with Chlamydomonas and Synechocystis reflects their more distant evolutionary relationships, while the significantly lower identity with E. coli highlights the divergence between chloroplast and bacterial ATP synthases .

What expression systems and yields have been reported for recombinant proteins in Marchantia polymorpha?

This comparative analysis demonstrates that Marchantia polymorpha offers versatile expression platforms for recombinant proteins, with chloroplast transformation providing particularly high yields. The proMp35S×2 promoter delivers strong expression but may impact plant growth, while inducible systems like proMpHSP17.8A1 offer a balanced approach that minimizes growth effects . For membrane proteins like ATP synthase subunit c, chloroplast transformation may be particularly advantageous as it provides the native membrane environment for proper folding and assembly .

What methods are available for genetic modification of Marchantia polymorpha?

Marchantia polymorpha offers numerous advantages as a model system for studying ATP synthase subunit c, including its simple genome, basal evolutionary position, and diverse available genetic modification techniques. The direct modification of chloroplast genes through biolistics and homologous recombination is particularly relevant for atpH research, allowing precise manipulation of the native gene . The availability of efficient nuclear transformation methods also enables studies of nuclear factors regulating chloroplast gene expression, including PPR proteins that may interact with atpH transcripts .