Recombinant Cryptomeria japonica ATP synthase subunit c, chloroplastic (atpH)

What is the basic structure of Cryptomeria japonica ATP synthase subunit c, and how does it compare to other plant species?

ATP synthase subunit c (atpH) is a critical component of the F0 sector of the chloroplastic ATP synthase complex in Cryptomeria japonica. While specific structural data for the C. japonica atpH is limited, we can extrapolate from related research that this small, hydrophobic protein forms part of the membrane-embedded rotor ring in the F0 domain. The protein likely contains two transmembrane alpha-helices connected by a short hydrophilic loop, similar to other plant ATP synthase c subunits.

Comparison with other plant species suggests conservation of key functional residues, particularly the protonatable acidic residue (typically glutamate) that is essential for proton translocation. Based on the amino acid sequence patterns observed in the related atpF subunit (subunit b), we can expect high sequence conservation in functional domains with species-specific variations in non-critical regions .

What are the critical functional domains of the atpH protein, and what experimental approaches can confirm their roles?

The critical functional domains of ATP synthase subunit c include:

• The proton-binding site containing a conserved acidic residue

• Transmembrane helices that contribute to the formation of the c-ring

• Interaction surfaces with other ATP synthase subunits

To confirm their roles experimentally, researchers can employ:

• Site-directed mutagenesis targeting conserved residues followed by functional assays

• Crosslinking studies to identify protein-protein interaction sites

• CryoEM structural studies similar to those used for other ATP synthase complexes

• ATP hydrolysis assays under varying conditions (pH, temperature, ion concentrations)

The critical importance of structural integrity in the functional domains is demonstrated by studies on ATP synthase inhibitors such as cruentaren A, which binds to specific interfaces within the ATP synthase complex and disrupts its function .

How does the genomic organization of the atpH gene in Cryptomeria japonica compare with other conifers?

Cryptomeria japonica has a diploid chromosome complement of 2n = 2x = 22 , providing the genomic foundation for all its chloroplast genes including atpH. While specific details about the atpH gene organization in C. japonica are not well-documented, we can infer from related species that the chloroplast atpH gene is likely maintained under strong selective pressure due to its essential function in energy metabolism.

The gene likely resides in the chloroplast genome, as is typical for chloroplastic ATP synthase components. Comparative genomic analyses would be required to definitively establish the exact organizational features relative to other conifers. The atpH gene in most plants is relatively small (~240 bp) encoding approximately 80 amino acids.

What are the optimal expression systems and conditions for producing recombinant C. japonica atpH protein for structural studies?

Based on successful expression of the related ATP synthase subunit b (atpF) , the recommended expression system for recombinant C. japonica atpH would be an in vitro E. coli expression system. The following methodology is suggested:

• Vector Selection: A pET-based expression vector with an N-terminal His-tag to facilitate purification.

• E. coli Strain: BL21(DE3) or Rosetta strains are recommended for membrane protein expression.

• Induction Conditions: Expression at lower temperatures (16-20°C) with reduced IPTG concentration (0.1-0.5 mM) to enhance proper folding.

• Extraction Protocol: Use of specialized detergents for membrane protein solubilization.

For storage and handling of the purified protein:

• Store at -20°C/-80°C in buffer containing 50 mM HEPES-NaOH (pH 7.5), 50 mM KCl

• Add 6% Trehalose to stabilize the protein during freeze-thaw cycles

• Avoid repeated freeze-thaw cycles

What are the methodological considerations for measuring ATP hydrolysis activity of ATP synthase containing C. japonica atpH?

To accurately measure ATP hydrolysis activity, researchers should consider the following methodological approach:

• Buffer Composition: Use 50 mM HEPES-NaOH buffer with optimal pH (7.5-8.5) and 50 mM KCl .

• Cation Requirements: Test both Mg²⁺ and Ca²⁺ as cofactors, as they can significantly affect enzyme kinetics .

• pH Optimization: Evaluate activity across a pH range (6.5-8.5), as alkaline conditions may provide optimal activity for Ca²⁺-dependent ATPase activity .

• Quantification Methods:

  • HPLC analysis using a Cosmosil 5C18 column (4.6 × 250 mm)

  • Mobile phase: 50 mM potassium dihydrogenphosphate (pH 4.6), 25 mM tetrabutylammonium hydrogensulfate, 0.5% acetonitrile

  • Flow rate: 1 mL/min

  • Detection: UV absorption at 254 nm for ADP/ATP quantification

• Controls: Include negative controls (denatured protein) and positive controls (commercial ATP synthase).

• Kinetic Analysis: Determine KM and Vmax values under different conditions to characterize enzyme behavior. For reference, AtVIPP1 showed KM = 1.07 mM and Vmax = 0.39 μM Pi release/μg protein/min at pH 7.5 .

What purification strategies are most effective for isolating the recombinant C. japonica atpH protein while maintaining structural integrity?

Based on successful approaches with related proteins, the following purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to bind the His-tagged protein.

• Buffer Composition: Include appropriate detergents (0.03-0.05% DDM or 0.5-1% CHAPS) to maintain solubility of this hydrophobic membrane protein.

• Secondary Purification: Size exclusion chromatography using a sucrose density gradient (0.4-1.6 M) containing 50 mM HEPES-NaOH (pH 7.5) and 50 mM KCl .

• Centrifugation Conditions: Ultracentrifugation at 85,000 ×g to separate protein complexes.

• Protein Concentration: Use specialized concentration devices designed for membrane proteins to avoid aggregation.

• Quality Control: Assess protein purity using SDS-PAGE and Western blotting with anti-His antibodies, and verify structural integrity using circular dichroism.

How can cryoEM be optimized for structural determination of ATP synthase complexes containing C. japonica atpH?

CryoEM has proven valuable for determining ATP synthase structures, as demonstrated in studies with inhibitor binding . For optimal cryoEM studies of C. japonica ATP synthase:

• Sample Preparation:

  • Protein concentration: ~20 mg/mL of purified ATP synthase complex

  • Grid preparation: Optimize freezing conditions to prevent preferred orientation

  • Consider amphipathic additives to improve particle distribution

• Data Collection Strategy:

  • Collect images at multiple defocus values (typically -1.5 to -3.0 μm)

  • Implement dose-fractionation (movie mode) to mitigate beam-induced motion

  • Use energy filters to enhance contrast

• Image Processing:

  • Implement 3D classification to identify different conformational states

  • Apply focused refinement on the c-ring to enhance resolution in this region

  • Consider symmetry-based approaches based on the c-ring stoichiometry

• Resolution Enhancement:

  • Apply particle subtraction methods to focus on specific domains

  • Implement CTF refinement and Bayesian polishing

  • Consider multi-body refinement to account for domain flexibility

Previous cryoEM studies on ATP synthase achieved resolutions of 2.9 Å, allowing visualization of inhibitor binding sites and protein-inhibitor interactions .

What approaches can be used to investigate the interaction between atpH and other ATP synthase subunits in Cryptomeria japonica?

To investigate subunit interactions within the ATP synthase complex:

• Crosslinking Mass Spectrometry (XL-MS):

  • Apply chemical crosslinkers (BS3, DSS, or EDC) to stabilize protein-protein interactions

  • Perform enzymatic digestion followed by LC-MS/MS analysis

  • Identify crosslinked peptides using specialized software (pLink, xQuest)

  • Map interaction sites based on crosslinked residues

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

  • Monitor deuterium incorporation into protein backbones

  • Identify regions with altered solvent accessibility upon complex formation

  • Map binding interfaces based on protection patterns

• Co-immunoprecipitation with Targeted Mutations:

  • Introduce mutations in potential interaction surfaces

  • Assess impact on complex formation through co-IP experiments

  • Quantify binding affinities through surface plasmon resonance

• Computational Approaches:

  • Molecular docking of modeled subunit structures

  • Molecular dynamics simulations to assess stability of predicted interactions

  • Evolutionary coupling analysis to identify co-evolving residues

These methods can reveal the structural basis for ATP synthase assembly and function, similar to the insights gained from studying inhibitor binding at the αTPβTP and αDPβDP interfaces .

How should researchers interpret ATP hydrolysis kinetic data for recombinant C. japonica atpH in the context of physiological relevance?

When analyzing ATP hydrolysis kinetic data:

• Enzyme Kinetic Parameters:

  • Calculate KM and Vmax using Michaelis-Menten or Lineweaver-Burk plots

  • Compare values across different conditions (pH, temperature, ion concentrations)

  • Consider substrate inhibition effects at high ATP concentrations

• Physiological Context:

  • Relate in vitro activity to estimated chloroplast ATP concentrations (typically 1-5 mM)

  • Consider the influence of pH changes during photosynthesis (stromal pH can increase from ~7 to ~8 in light)

  • Account for physiological ion concentrations, particularly Mg²⁺ and Ca²⁺ fluctuations

• Comparative Analysis:

  • Construct a data table comparing kinetic parameters across conditions:

*Estimated values based on related ATP synthase components

• Functional Implications:

  • Higher affinity (lower KM) at alkaline pH suggests adaptation to function during active photosynthesis

  • Differences between Mg²⁺ and Ca²⁺ activation may indicate regulatory mechanisms

  • Substrate inhibition at high affinity conditions may represent a regulatory mechanism

What statistical approaches are most appropriate for analyzing variability in ATP synthase activity across different experimental preparations?

For robust statistical analysis of ATP synthase activity:

• Descriptive Statistics:

  • Calculate means, standard deviations, and coefficients of variation

  • Assess normality of data distribution using Shapiro-Wilk or Kolmogorov-Smirnov tests

• Comparative Statistics:

  • For normally distributed data: ANOVA followed by post-hoc tests (Tukey's HSD)

  • For non-normally distributed data: Kruskal-Wallis with Mann-Whitney U tests

  • For time-dependent measurements: Repeated measures ANOVA

• Regression Analysis:

  • Use non-linear regression for enzyme kinetic parameters

  • Consider mixed-effects models when combining data from multiple preparations

• Variance Component Analysis:

  • Partition variance into components (preparation-to-preparation, technical, biological)

  • Calculate intraclass correlation coefficients to assess consistency

• Statistical Power Considerations:

  • Conduct power analysis to determine adequate sample sizes

  • Report effect sizes alongside p-values

When interpreting p-values, consider the biological significance of differences. For example, in ATP hydrolysis studies, significant differences in Pi release rates between time points should be evaluated in the context of physiological ATP turnover rates .

What are the common challenges in expressing and purifying functional recombinant C. japonica atpH, and how can they be addressed?

Common challenges and their solutions include:

• Low Expression Levels:

  • Challenge: Hydrophobic membrane proteins often express poorly

  • Solutions:

    • Optimize codon usage for E. coli

    • Try fusion partners (MBP, SUMO) to enhance solubility

    • Test different E. coli strains (C41/C43 designed for membrane proteins)

    • Consider cell-free expression systems

• Protein Aggregation:

  • Challenge: Membrane proteins tend to aggregate during purification

  • Solutions:

    • Screen multiple detergents (DDM, CHAPS, LDAO)

    • Include stabilizing agents (glycerol, trehalose)

    • Maintain low temperature throughout purification

    • Consider purifying with lipid nanodiscs or amphipols

• Loss of Activity:

  • Challenge: Maintaining functionality during purification

  • Solutions:

    • Minimize exposure to harsh conditions

    • Include appropriate cofactors (Mg²⁺ or Ca²⁺)

    • Optimize buffer pH based on activity profiling

    • Consider co-expression with partner subunits

• Heterogeneity:

  • Challenge: Multiple conformational states affecting structural studies

  • Solutions:

    • Add nucleotides or inhibitors to stabilize specific conformations

    • Use gradient fixation methods

    • Implement extensive 3D classification in cryoEM processing

• Storage Stability:

  • Challenge: Activity loss during storage

  • Solutions:

    • Lyophilize with trehalose (6%) for extended shelf life

    • Store at -80°C with minimal freeze-thaw cycles

    • Consider flash-freezing small aliquots in liquid nitrogen

How can researchers troubleshoot inconsistent results in ATP hydrolysis assays with recombinant C. japonica atpH?

When encountering inconsistencies in ATP hydrolysis assays:

• Enzyme Quality Assessment:

  • Verify protein purity using SDS-PAGE

  • Confirm protein concentration using multiple methods (Bradford, BCA, A280)

  • Assess protein folding using circular dichroism

  • Check for proteolytic degradation during storage

• Assay Components Verification:

  • Test ATP quality using HPLC analysis

  • Prepare fresh buffer components and verify pH

  • Use high-purity divalent cations (Mg²⁺, Ca²⁺)

  • Implement positive controls with commercial ATP synthase

• Methodological Consistency:

  • Standardize reaction temperatures using water bath or heat block

  • Control reaction time precisely

  • Ensure consistent mixing during reactions

  • Implement internal standards in HPLC analysis

• Detection System Calibration:

  • Prepare fresh standard curves for each experiment

  • Verify linear range of the assay

  • Assess background signal from buffer components

  • Check for interfering substances in enzyme preparations

• Systematic Troubleshooting Approach:

  • Vary one parameter at a time to identify sources of variability

  • Document detailed protocols including lot numbers of reagents

  • Validate HPLC methods using spike recovery experiments

  • Consider alternative detection methods (coupled enzyme assays, radiolabeled ATP)

What are the emerging techniques that could advance our understanding of C. japonica atpH structure and function?

Several cutting-edge approaches show promise for advancing ATP synthase research:

• Cryo-Electron Tomography:

  • Enables visualization of ATP synthase in native membrane environments

  • Can reveal spatial organization and interactions with other complexes

  • Provides insights into structural heterogeneity

• Time-Resolved Structural Methods:

  • TR-FRET to monitor conformational changes during catalysis

  • Time-resolved cryoEM to capture different states of the catalytic cycle

  • Mixing-spraying cryoEM for millisecond time resolution

• Integrative Structural Biology:

  • Combining cryoEM with mass spectrometry and molecular dynamics

  • Cross-validation of structural models through multiple techniques

  • Building comprehensive models of the entire ATP synthase complex

• Single-Molecule Techniques:

  • Optical tweezers to measure torque generation

  • Single-molecule FRET to track conformational dynamics

  • Magnetic tweezers to study mechanochemical coupling

• Advanced Computational Approaches:

  • Enhanced molecular dynamics simulations

  • Machine learning for structure prediction and classification

  • Quantum mechanical calculations of proton transfer pathways

These approaches could help resolve outstanding questions about the c-ring stoichiometry, proton pathway, and regulatory mechanisms in C. japonica ATP synthase, building upon the structural insights already gained from ATP synthase-inhibitor complexes .

How might comparative studies between C. japonica atpH and other plant species inform evolutionary adaptations of ATP synthase?

Comparative studies of ATP synthase across plant species can reveal:

• Evolutionary Conservation and Divergence:

  • Identify conserved residues essential for function

  • Map species-specific variations to functional adaptations

  • Trace evolutionary history of key regulatory mechanisms

• Adaptation to Environmental Conditions:

  • Compare c-subunits from plants adapted to different temperatures

  • Analyze variations in proton-binding sites across species with different optimal pH

  • Correlate structural differences with environmental pressures

• Methodological Approach:

  • Sequence alignment and phylogenetic analysis of atpH across plant lineages

  • Homology modeling based on available structures

  • Functional characterization across temperature and pH ranges

  • Site-directed mutagenesis to convert species-specific residues

• Potential Research Questions:

  • Does the c-ring stoichiometry vary between species adapted to different environments?

  • Are there species-specific differences in ion selectivity (H⁺ vs. Na⁺)?

  • How do regulatory mechanisms of ATP synthase differ across plant lineages?

C. japonica, as a conifer with a diploid complement of 2n = 2x = 22 , represents an important evolutionary lineage for comparative studies with angiosperms and other plant groups.