Recombinant Populus trichocarpa ATP synthase subunit c, chloroplastic (atpH)

What is the molecular structure of Populus trichocarpa ATP synthase subunit c, chloroplastic (atpH)?

Populus trichocarpa ATP synthase subunit c, chloroplastic (atpH) is a small membrane protein consisting of 81 amino acids with the sequence: MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . The protein is highly hydrophobic, containing primarily non-polar amino acids that facilitate its insertion into the membrane. As part of the F0 sector of ATP synthase, subunit c forms an oligomeric ring structure embedded in the membrane. This c-ring plays a crucial role in the rotary mechanism of ATP synthase, converting the proton gradient energy into mechanical rotation that drives ATP synthesis.

What expression systems are suitable for producing recombinant atpH protein?

Escherichia coli is the most commonly used expression system for recombinant Populus trichocarpa atpH protein production . When designing an expression system, researchers should consider:

• Vector selection: pET vectors with T7 promoters are effective for membrane protein expression

• E. coli strain optimization: BL21(DE3) or C41(DE3) strains are often used for membrane proteins

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations (0.1-0.5 mM) can improve proper folding

• Addition of solubility tags: His-tags facilitate purification while potentially enhancing solubility

For optimal protein yields, expression parameters should be systematically tested using an experimental design approach with controlled variables (temperature, induction time, media composition) while measuring the dependent variable (protein yield) .

What purification methods are most effective for recombinant atpH protein?

Purification of recombinant His-tagged atpH protein typically involves:

• Cell lysis: Mechanical disruption or detergent-based methods optimized for membrane proteins

• Membrane isolation: Differential centrifugation to isolate membrane fractions

• Solubilization: Use of appropriate detergents (DDM, LDAO, or Triton X-100)

• Affinity chromatography: Ni-NTA or TALON resin binding to the His-tag

• Size exclusion chromatography: For further purification and buffer exchange

Similar to the approach by Sugiura et al. for other membrane proteins, researchers may consider ion exchange chromatography using DEAE-cellulose followed by ammonium sulfate fractionation for increased purity . Protein purity should be verified using SDS-PAGE analysis, with expected purity greater than 90% for most research applications .

What are the optimal storage conditions for maintaining atpH protein stability?

Recombinant atpH protein stability depends on appropriate storage conditions:

• Short-term storage: Keep working aliquots at 4°C for up to one week

• Long-term storage: Store at -20°C/-80°C in aliquots to avoid repeated freeze-thaw cycles

• Lyophilization: The protein can be supplied as lyophilized powder

• Reconstitution: Use deionized sterile water to reconstitute to 0.1-1.0 mg/mL

• Cryoprotectant addition: Add glycerol to a final concentration of 5-50% (recommended 50%)

The storage buffer should be Tris/PBS-based with 6% trehalose at pH 8.0 to maintain protein integrity . Stability should be monitored periodically using activity assays or structural analysis techniques.

How does the assembly process of ATP synthase differ between Populus trichocarpa and other model organisms?

The assembly of ATP synthase in plants like Populus trichocarpa shares fundamental similarities with other organisms but exhibits important distinctions:

• Modular assembly: Similar to yeast, ATP synthase assembly in plants likely involves distinct modules: the c-ring, F1, and the membrane subunits

• Convergent pathways: Evidence suggests two separate pathways (F1/c-subunit assembly and membrane subunit/stator assembly) that converge at the final stages

• Chloroplast-specific factors: Unlike mitochondrial ATP synthase, chloroplastic assembly requires chloroplast-specific chaperones and assembly factors

• Translational regulation: Expression of plastid-encoded subunits may be translationally regulated by nuclear-encoded components, similar to the regulation observed in yeast where F1 regulates the translation of subunits 6 and 8

ATP synthase assembly in Populus trichocarpa likely follows this sequence: assembly of the c-ring → binding of F1 → attachment of the stator arm → incorporation of additional membrane subunits . This differs from bacterial systems where the assembly process is less compartmentalized.

What experimental approaches can resolve contradictions in atpH function data?

When addressing contradictory results regarding atpH function, researchers should employ:

• Topological data analysis (TDA): Use computational approaches to identify patterns in contradictory datasets. TDA can reveal structural relationships between seemingly disparate experimental results by identifying topological features that remain consistent despite data variations

• Multi-method validation: Design experiments using complementary techniques:

  Technique	Application	Resolution	Limitations
  Site-directed mutagenesis	Structure-function	Amino acid level	Limited to conserved residues
  Cryo-EM	Structural analysis	2-4Å	Static representation
  MD simulation	Dynamic behavior	Femtosecond	Computational constraints
  FRET analysis	Rotary mechanics	Nanometer	Requires fluorophore attachment
  Electrophysiology	Proton translocation	Single channel	Technical complexity

• Statistical rigor: Implement robust statistical analyses to distinguish biological variations from technical artifacts

• Cross-species comparison: Compare function across evolutionary diverse organisms to identify conserved mechanisms versus species-specific adaptations

When analyzing contradictory results, researchers should consider that protein function may be context-dependent, varying with lipid environment, pH, temperature, or interaction partners .

What is the role of atpH in the proton translocation mechanism of chloroplastic ATP synthase?

The c-subunit (atpH) forms an oligomeric ring in the membrane domain (F0) that functions as the primary proton translocation apparatus:

• Proton binding site: Each c-subunit contains a conserved carboxylate residue (typically glutamate or aspartate) that becomes protonated/deprotonated during the catalytic cycle

• Rotation mechanism: Protonation changes drive rotation of the c-ring relative to subunit a, which contains the proton half-channels

• Stoichiometry impact: The number of c-subunits per ring (typically 8-15) determines the H+/ATP ratio and thermodynamic efficiency

The direction of rotation in chloroplastic ATP synthase is clockwise (viewed from the membrane), opposite to that of mitochondrial ATP synthase, reflecting the reversed proton gradient direction . The precise mechanism involves:

• Proton entry through a half-channel in subunit a

• Protonation of the c-subunit carboxylate at the a/c interface

• Rotation of the c-ring, moving the protonated site into the hydrophobic membrane environment

• Deprotonation at another a/c interface, releasing the proton to the opposite side

• Coupling of c-ring rotation to the central stalk (γ subunit), driving conformational changes in F1 that catalyze ATP synthesis

This mechanism represents a remarkable example of energy conversion from the electrochemical gradient to mechanical rotation to chemical synthesis.

How can researchers design experiments to study the role of lipid interactions with atpH in ATP synthase function?

Designing experiments to study lipid-atpH interactions requires a systematic approach:

• Experimental Design Framework:

  • Define independent variables: lipid composition, membrane thickness, fluidity

  • Control extraneous variables: temperature, pH, ionic strength

  • Measure dependent variables: ATP synthesis rate, proton translocation, c-ring stability

• Reconstitution Approaches:

  • Prepare liposomes with defined lipid compositions

  • Reconstitute purified atpH or complete ATP synthase

  • Compare activity in different lipid environments

• Advanced Biophysical Techniques:

  Technique	Application	Measurement	Advantage
  Solid-state NMR	Lipid-protein interface	Atomic resolution	Native-like environment
  Hydrogen-deuterium exchange	Membrane exposure	Peptide level	Conformational dynamics
  Fluorescence anisotropy	Lipid fluidity effects	Nanosecond	Real-time measurements
  Native mass spectrometry	Bound lipids	Exact mass	Direct detection of interactions
  Molecular dynamics	Interaction prediction	Atomistic	Parameter modification

• Mutational Analysis:

  • Generate atpH mutants with altered lipid-facing residues

  • Assess functional consequences in different lipid environments

  • Correlate structural changes with functional outcomes

This multi-faceted approach allows researchers to distinguish between specific lipid interactions that are essential for function versus those that modulate activity or provide structural support .

What are the current challenges in expressing and purifying functional atpH for structural studies?

Several challenges exist when producing atpH for structural studies:

• Membrane protein expression barriers:

  • Toxicity to host cells due to membrane disruption

  • Inclusion body formation requiring refolding

  • Low expression yields due to limited membrane surface area

  • Proper insertion into membranes requiring specialized machinery

• Purification challenges:

  • Selecting detergents that maintain native structure

  • Preventing aggregation during concentration

  • Removing lipids while maintaining stability

  • Achieving homogeneity required for crystallography

• Structural study limitations:

  • Difficulty obtaining crystals suitable for X-ray diffraction

  • Challenges in cryo-EM sample preparation for small membrane proteins

  • NMR signal overlap due to repetitive sequences

  • Native oligomeric state determination

Current methodological approaches to address these challenges include:

• Fusion with solubility-enhancing partners

• Green fluorescent protein fusions to monitor expression and folding

• Nanodiscs or amphipols as detergent alternatives

• Antibody fragment co-crystallization to increase polar surface area

Success in structural studies requires careful optimization of each experimental stage from expression to final analysis, often necessitating iterative refinement of conditions .

How can atpH be used as a model to study membrane protein assembly processes?

The c-subunit (atpH) serves as an excellent model system for studying fundamental aspects of membrane protein assembly:

• Sequential assembly monitoring:

  • Using pulse-chase experiments to track the incorporation of labeled atpH into the c-ring

  • Employing BN-PAGE to identify assembly intermediates

  • Applying cross-linking techniques to capture transient interactions

• Assembly factor identification:

  • Genetic screens for assembly-deficient mutants

  • Proximity labeling to identify proteins in the assembly environment

  • Co-immunoprecipitation to isolate assembly complexes

• Compartmentalization studies:

  • Investigating the coordination between chloroplast and nuclear genomes

  • Examining protein import and membrane insertion pathways

  • Studying spatiotemporal aspects of complex assembly

The atpH assembly process can be experimentally manipulated by:

• Depleting specific assembly factors

• Introducing mutations in interface residues

• Altering membrane composition

• Modifying environmental conditions (temperature, pH)

This systematic approach allows researchers to elucidate general principles of membrane protein assembly that may apply across diverse biological systems.

What computational methods can predict the impact of mutations in atpH on ATP synthase function?

Computational approaches for predicting mutation effects include:

• Sequence-based methods:

  • Conservation analysis across species

  • Coevolution detection to identify functionally linked residues

  • Machine learning algorithms trained on known mutations

• Structure-based predictions:

  • Molecular dynamics simulations to assess structural stability

  • Free energy calculations for mutation impact

  • Electrostatic analysis for charged residue mutations

  • Protein-protein interface analysis for assembly effects

• Integrated approaches:

  Method	Strengths	Limitations	Application
  Homology modeling	Uses related structures	Accuracy depends on template	Initial structural assessment
  Rosetta ddG	Fast energy calculations	Limited conformational sampling	Stability predictions
  FoldX	Empirical force field	Static structures	Rapid screening
  MD simulations	Dynamic behavior	Computationally intensive	Detailed mechanism analysis
  QM/MM methods	Chemical reaction analysis	Limited system size	Proton transfer modeling

• Validation approaches:

  • Retrospective analysis of known mutations

  • Cross-validation with experimental data

  • Statistical assessment of prediction accuracy

These computational methods can guide experimental design by identifying high-priority mutations for functional testing and providing mechanistic hypotheses for experimental validation .

How can researchers analyze contradictions in experimental data related to atpH structure and function?

When confronted with contradictory data regarding atpH structure and function, researchers should implement a systematic analysis approach:

• Data characterization and classification:

  • Categorize contradictions by experimental technique

  • Identify variables that differ between studies

  • Evaluate statistical robustness of conflicting results

• Topological data analysis approach:

  • Apply computational methods to identify patterns in seemingly contradictory datasets

  • Use persistent homology to detect structural similarities despite apparent differences

  • Visualize data relationships to identify potential reconciliation points

• Meta-analysis framework:

  Analysis Step	Method	Expected Outcome
  Data extraction	Systematic literature review	Comprehensive dataset
  Quality assessment	GRADE criteria	Reliability ranking
  Effect size calculation	Standardized mean difference	Comparable results
  Heterogeneity analysis	I² statistic, forest plots	Variation quantification
  Publication bias evaluation	Funnel plots, Egger's test	Bias detection
  Sensitivity analysis	Leave-one-out method	Robustness assessment

• Experimental reconciliation:

  • Design targeted experiments addressing specific contradictions

  • Use multiple complementary techniques on identical samples

  • Systematically vary experimental conditions to identify context-dependent effects

This comprehensive approach can transform apparent contradictions into deeper insights about context-dependent behavior of atpH under different experimental conditions.

What emerging technologies show promise for studying the dynamic behavior of atpH during ATP synthesis?

Several cutting-edge technologies are transforming our ability to study atpH dynamics:

• Time-resolved structural methods:

  • Time-resolved cryo-EM with millisecond capture capability

  • Serial femtosecond crystallography using X-ray free-electron lasers

  • High-speed atomic force microscopy for direct observation of conformational changes

• Single-molecule techniques:

  • FRET sensors positioned on rotating elements

  • Gold nanorod imaging for rotational dynamics

  • Magnetic tweezers to measure torque generation

  • Optical trapping to quantify mechanical forces

• Advanced spectroscopic approaches:

  • 2D infrared spectroscopy for protonation state detection

  • Site-specific vibrational probes to track local environment changes

  • EPR with spin labels at strategic positions

• In-cell methodologies:

  • Genetically encoded sensors for conformational changes

  • Proximity labeling to map dynamic interactions

  • Super-resolution microscopy for in vivo visualization

These technologies will enable researchers to move beyond static structural models to understand how atpH functions dynamically within the complete ATP synthase complex during catalysis .

What are the key considerations for designing experiments to compare atpH function across different plant species?

Designing comparative studies of atpH across plant species requires careful experimental planning:

• Experimental design framework:

  • Independent variable: plant species with varying evolutionary relationships

  • Controlled variables: expression systems, purification methods, assay conditions

  • Dependent variables: ATP synthesis rates, proton translocation efficiency, assembly kinetics

• Sample selection considerations:

  • Include species representing major plant lineages

  • Consider plants adapted to different environmental conditions

  • Include species with sequenced genomes for accurate comparisons

• Standardization requirements:

  Parameter	Standardization Approach	Importance
  Protein expression	Same vector and host system	Eliminates expression bias
  Purification protocol	Identical methods and buffers	Ensures comparable purity
  Functional assays	Standardized conditions and reagents	Enables direct comparison
  Data analysis	Consistent statistical methods	Prevents analytical artifacts
  Controls	Common reference species	Provides normalization benchmark

• Complementary approaches:

  • Sequence analysis for evolutionary patterns

  • Structural comparisons to identify conserved features

  • Chimeric proteins to map functional domains

  • Reciprocal complementation of mutants

This systematic approach allows researchers to distinguish between conserved mechanisms essential for atpH function versus species-specific adaptations that may reflect environmental or metabolic specialization .

What quality control methods should be applied to verify the integrity and activity of purified recombinant atpH?

A comprehensive quality control approach for recombinant atpH should include:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (expected >90% purity)

  • Western blotting with anti-His or specific antibodies

  • Mass spectrometry for precise molecular weight determination

  • Size-exclusion chromatography for aggregation analysis

• Structural integrity verification:

  • Circular dichroism for secondary structure content

  • Tryptophan fluorescence for tertiary structure assessment

  • Limited proteolysis to probe folding quality

  • Thermal shift assays for stability measurement

• Functional validation:

  • Reconstitution into liposomes for proton translocation assays

  • Assembly with complementary subunits to form functional complexes

  • ATP synthesis activity when incorporated into the holoenzyme

  • Proton gradient formation/dissipation measurements

• Oligomeric state analysis:

  • Blue native PAGE for native complex detection

  • Analytical ultracentrifugation for stoichiometry determination

  • Crosslinking studies for interaction mapping

  • Light scattering for molecular weight verification

Researchers should establish acceptance criteria for each quality parameter based on the specific experimental requirements, and maintain detailed documentation of quality control results for reproducible research .

How can researchers optimize the experimental design for studying atpH interactions with other ATP synthase subunits?

Optimizing experiments to study atpH interactions requires a systematic approach:

• Experimental design considerations:

  • Define clear hypotheses about specific interactions

  • Select appropriate independent variables (protein concentrations, buffer conditions)

  • Control extraneous variables (temperature, pH, ionic strength)

  • Choose sensitive dependent variables (binding affinity, complex formation)

• In vitro interaction studies:

  Technique	Information Provided	Sensitivity	Limitations
  Surface plasmon resonance	Real-time kinetics	nM-μM	Requires surface immobilization
  Isothermal titration calorimetry	Thermodynamic parameters	μM	Sample intensive
  Microscale thermophoresis	Binding in solution	nM	Requires fluorescent labeling
  Biolayer interferometry	Association/dissociation rates	nM	Surface effects possible
  Native mass spectrometry	Complex stoichiometry	-	Limited by ionization efficiency

• In vivo interaction approaches:

  • Split-reporter complementation assays

  • FRET/BRET-based interaction monitoring

  • Proximity-dependent labeling (BioID, APEX)

  • Co-immunoprecipitation from native membranes

• Structural studies of complexes:

  • Cross-linking mass spectrometry for interface mapping

  • Cryo-EM of reconstituted complexes

  • Hydrogen-deuterium exchange to identify protected regions

  • Computational docking validated by experimental constraints

By combining multiple complementary approaches and carefully controlling experimental variables, researchers can generate robust data on atpH interactions with other ATP synthase components .