Recombinant Pinus koraiensis ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in Pinus koraiensis chloroplasts?

ATP synthase subunit c (atpH) in Pinus koraiensis is a crucial component of the F0 sector of the chloroplastic ATP synthase complex. It forms a multimeric ring embedded in the thylakoid membrane that rotates during ATP synthesis. This rotation is mechanically coupled to ATP production and is driven by proton translocation across the membrane along an electrochemical gradient. The full amino acid sequence of this protein is: MDPLISAASVIAAGLSVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV .

Structurally, this protein adopts an alpha-helical secondary structure, which is essential for its proper function and assembly into the c-ring. The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits per oligomeric ring, which is organism-dependent and directly relates to the metabolic efficiency of the photosynthetic process .

How does recombinant Pinus koraiensis ATP synthase subunit c differ from native protein?

Recombinant Pinus koraiensis ATP synthase subunit c is produced through heterologous expression systems, typically using bacterial hosts such as Escherichia coli. While the amino acid sequence remains identical to the native protein, several differences may exist:

The recombinant protein may include expression tags determined during the production process, which can influence solubility and purification characteristics but might need to be removed for certain functional studies .

What are the optimal storage conditions for recombinant Pinus koraiensis ATP synthase subunit c?

For optimal stability and activity retention, recombinant Pinus koraiensis ATP synthase subunit c should be stored in Tris-based buffer containing 50% glycerol. The recommended storage temperature is -20°C, with extended storage preferably at -80°C. Working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing cycles should be avoided as they can lead to protein denaturation and functional loss .

For researchers conducting extended studies, it is advisable to prepare single-use aliquots that minimize the need for multiple freeze-thaw cycles. The protein should be handled carefully during thawing, ideally by gentle thawing on ice rather than rapid warming, to preserve its structural integrity and functional properties.

How can I investigate the stoichiometry of c-subunits in the ATP synthase c-ring of Pinus koraiensis?

Investigating c-subunit stoichiometry in ATP synthase c-rings requires a multifaceted approach. Based on methodologies developed for similar proteins such as spinach chloroplast ATP synthase, the following experimental strategy is recommended:

• Protein Expression and Purification: Express the recombinant c-subunit as a fusion protein with a solubility-enhancing partner such as maltose binding protein (MBP). This approach has been successful in producing the monomeric c₁ subunit of spinach chloroplast ATP synthase in E. coli BL21 derivative cells .

• Structural Analysis: Employ a combination of:

  • Cryo-electron microscopy to visualize the assembled c-ring

  • Cross-linking mass spectrometry to determine subunit interactions

  • Atomic force microscopy to examine the surface topology of reconstituted c-rings

• Functional Reconstitution: Reconstitute purified c-subunits into liposomes and measure proton translocation using pH-sensitive fluorescent dyes. The proton/ATP ratio can provide indirect evidence of c-ring stoichiometry.

• Comparative Analysis: Compare results with known c-ring stoichiometries from other species, considering evolutionary relationships and metabolic adaptations. The variability in c-ring stoichiometry is organism-dependent and relates to the specific metabolic requirements of the organism .

What role does ATP synthase subunit c play in pine resistance to pine wood nematode (PWN)?

While ATP synthase subunit c itself has not been directly implicated in pine wood nematode (PWN) resistance, it operates within the energetic machinery that supports defense responses. Research on transgenic Pinus koraiensis indicates that metabolic engineering of defense compounds can enhance resistance to PWN.

Specifically, the transcription factor PsbHLH1 from PWN-resistant Pinus strobus has been demonstrated to activate the production of pinosylvin stilbenoids in transgenic P. koraiensis calli. These compounds are highly toxic to PWN and contribute to resistance. The transgenic expression of PsbHLH1 increased the expression of genes involved in pinosylvin stilbene biosynthesis, including PkSTS (pinosylvin synthase), PkPMT (pinosylvin O-methyltransferase), and PkACC (acetyl-CoA carboxylase) .

The relationship between ATP synthase function and secondary metabolite production could involve:

• Energy provision for defense compound biosynthesis

• Potential regulatory crosstalk between bioenergetic status and defense responses

• Influence on redox homeostasis that may affect defense signaling pathways

Researchers interested in this relationship could investigate how alterations in ATP synthase activity affect the capacity of pine trees to produce defense compounds like pinosylvin stilbenoids.

How can I express and purify functional recombinant ATP synthase subunit c for structural studies?

Expressing and purifying functional recombinant ATP synthase subunit c for structural studies presents challenges due to its hydrophobic nature and tendency to aggregate. A methodology adapted from successful approaches with similar proteins includes:

• Codon-Optimized Gene Design: Design a codon-optimized synthetic gene for expression in E. coli, incorporating the full sequence: MDPLISAASVIAAGLSVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV .

• Fusion Protein Strategy: Express the subunit c as a fusion protein with a solubility-enhancing partner. For example, researchers have successfully used maltose binding protein (MBP) fusion for expression of spinach chloroplast ATP synthase subunit c .

• Expression Conditions:

  • Host: BL21 derivative E. coli strains

  • Temperature: Lower temperature (16-20°C) to enhance proper folding

  • Induction: Gentle induction with lower IPTG concentrations (0.1-0.5 mM)

• Purification Protocol:

  Step	Method	Buffer Conditions	Notes
  1	Affinity chromatography	Tris buffer with mild detergent	Captures fusion protein
  2	Protease cleavage	Optimized conditions for tag removal	Releases subunit c
  3	Reversed-phase chromatography	Acetonitrile gradient	Separates subunit c
  4	Circular dichroism analysis	Standard conditions	Confirms alpha-helical structure

• Quality Assessment: Confirm the correct alpha-helical secondary structure using circular dichroism spectroscopy before proceeding to structural studies .

What are the best methods for studying the interaction of ATP synthase subunit c with other subunits of the ATP synthase complex?

Studying the interactions between ATP synthase subunit c and other components of the ATP synthase complex requires techniques that can capture transient and stable protein-protein interactions within membrane environments. The following methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP): Using antibodies against the recombinant ATP synthase subunit c to pull down interacting partners, followed by mass spectrometry analysis for identification. This requires generating specific antibodies against the Pinus koraiensis ATP synthase subunit c or using epitope tags.

• Cross-linking Mass Spectrometry (XL-MS): Applying chemical cross-linkers to stabilize protein interactions, followed by digestion and mass spectrometry to identify cross-linked peptides. This technique can map interaction interfaces with amino acid-level resolution.

• Biolayer Interferometry (BLI) or Surface Plasmon Resonance (SPR): These techniques can measure binding kinetics between purified subunit c and other ATP synthase components in real-time, providing quantitative data on interaction affinities.

• Reconstitution Studies: Reconstituting purified subunits into liposomes and assessing functional assembly through ATP synthesis assays or proton translocation measurements.

• Cryo-electron Microscopy: For visualizing the assembled complex and determining the spatial arrangement of subunits within the native conformation.

When interpreting interaction data, it's important to consider the hydrophobic nature of subunit c and its natural membrane environment, which may require specialized approaches to maintain protein stability and native conformation during experiments.

How can I develop an efficient expression system for recombinant Pinus koraiensis ATP synthase subunit c?

Developing an efficient expression system for the hydrophobic ATP synthase subunit c requires careful optimization of multiple parameters:

• Vector Selection and Design:

  • Use vectors with strong but inducible promoters (e.g., T7)

  • Incorporate fusion partners that enhance solubility (MBP, SUMO, or TrxA)

  • Include precision protease cleavage sites for tag removal

  • Consider codon optimization for the expression host

• Host Strain Selection:

  • BL21(DE3) derivatives with enhanced membrane protein expression capabilities

  • C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

  • Strains with reduced proteolytic activity (e.g., BL21(DE3) pLysS)

• Expression Condition Optimization Matrix:

  Parameter	Variables to Test	Expected Impact
  Temperature	16°C, 20°C, 25°C, 30°C	Lower temperatures may improve folding
  Induction OD₆₀₀	0.4, 0.6, 0.8, 1.0	Cell density affects expression efficiency
  Inducer concentration	0.1, 0.25, 0.5, 1.0 mM IPTG	Lower concentrations may reduce toxicity
  Medium composition	LB, TB, 2YT, M9	Nutrient availability affects yield
  Additives	Glycerol, sorbitol, betaine	Osmolytes can improve folding

• Monitoring Expression:

  • Use Western blotting to track expression levels

  • Employ fluorescent fusion partners for real-time monitoring

  • Analyze cell fractions to determine localization (cytoplasmic, inclusion bodies, or membrane)

Research on spinach chloroplast ATP synthase subunit c demonstrated that expression as a fusion protein with MBP followed by protease cleavage and reversed-phase column purification yielded functional protein with correct alpha-helical structure . This approach could be adapted for Pinus koraiensis ATP synthase subunit c, with species-specific optimizations.

What techniques can be used to study the proton translocation mechanism of ATP synthase subunit c in artificial membrane systems?

Studying the proton translocation mechanism of ATP synthase subunit c requires reconstitution into artificial membrane systems that mimic the native environment. The following techniques can provide insights into this fundamental process:

• Liposome Reconstitution:

  • Purified subunit c can be reconstituted into liposomes composed of phospholipids mimicking the thylakoid membrane composition

  • The c-ring needs to be properly assembled with a uniform orientation to enable functional studies

  • Reconstitution with additional ATP synthase components (particularly subunit a) is necessary for proton translocation

• pH-Sensitive Fluorescent Probes:

  • Probes such as ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine can be entrapped within liposomes

  • Fluorescence quenching or enhancement in response to pH changes can be measured in real-time

  • This allows quantification of proton translocation rates under various conditions

• Patch-Clamp Electrophysiology:

  • Giant unilamellar vesicles (GUVs) containing reconstituted c-rings can be studied using patch-clamp techniques

  • Single-channel recordings can provide insights into proton conductance properties

  • The influence of membrane potential on proton translocation can be directly measured

• Solid-State NMR Spectroscopy:

  • Can provide atomic-level insights into structural changes during proton translocation

  • Selective isotopic labeling of key residues involved in proton binding can track conformational changes

  • Helps elucidate the molecular mechanism of the proton translocation pathway

• Molecular Dynamics Simulations:

  • Complement experimental approaches with computational modeling

  • Can simulate proton movement through the c-ring at nanosecond timescales

  • Allows testing of hypotheses about key residues and their roles in the proton translocation mechanism

When designing these experiments, researchers should consider that the c-subunit of ATP synthase typically works in concert with other subunits, particularly subunit a, which provides the proton access channel. Therefore, reconstitution of the complete proton translocation machinery may be necessary for fully functional studies.

How can I analyze differences in ATP synthase subunit c structure and function across different pine species?

Comparative analysis of ATP synthase subunit c across pine species requires a systematic approach that integrates structural, functional, and evolutionary perspectives:

• Sequence Alignment and Phylogenetic Analysis:

  • Collect ATP synthase subunit c sequences from multiple pine species

  • Perform multiple sequence alignment using tools like ClustalW or MUSCLE

  • Construct phylogenetic trees using methods like neighbor-joining with bootstrap validation

  • Identify conserved regions and species-specific variations

• Structural Comparison:

  • Generate homology models of ATP synthase subunit c from different species

  • Analyze structural conservation and divergence using structural alignment tools

  • Focus on key functional regions: proton-binding site, helical packing interfaces, lipid-interacting regions

• Functional Comparison Matrix:

  Parameter	Measurement Method	Expected Variations	Evolutionary Significance
  Proton binding affinity	pH-dependent spectroscopy	pKa differences	Adaptation to pH environment
  c-ring stoichiometry	Cryo-EM, AFM	Different numbers of c-subunits	Energy efficiency adaptation
  Thermal stability	Differential scanning calorimetry	Melting temperature variations	Climate adaptation
  Lipid interactions	Native mass spectrometry	Lipid specificity differences	Membrane adaptation

• Ecological Correlation Analysis:

  • Compare structural and functional properties with ecological parameters

  • Consider climate conditions, habitat, and evolutionary history

  • Assess whether variations correlate with environmental adaptations

For example, comparing Pinus koraiensis ATP synthase subunit c with subunit c from Pinus strobus (eastern white pine) and other pine species could reveal adaptations related to their different native environments. This approach has been utilized in comparing ATP synthase stoichiometry across different organisms, revealing that the ratio of protons translocated to ATP synthesized varies according to the number of c-subunits per oligomeric ring in an organism-dependent manner .

What bioinformatic tools are most appropriate for analyzing the evolutionary conservation of ATP synthase subunit c in conifers?

The evolutionary analysis of ATP synthase subunit c in conifers requires specialized bioinformatic tools that can handle the unique aspects of chloroplast-encoded proteins in gymnosperm lineages:

• Sequence Retrieval and Database Tools:

  • UniProt/Swiss-Prot for curated protein sequences (e.g., Q7GUD2 for Pinus koraiensis atpH)

  • Chloroplast genome databases and conifer-specific genomic resources

  • NCBI Genbank for nucleotide sequences of atpH genes

• Multiple Sequence Alignment Tools:

  • MAFFT: Particularly effective for sequences with conserved motifs and variable regions

  • T-Coffee: Provides high accuracy for small numbers of diverse sequences

  • ClustalW: Used successfully in previous studies of bHLH protein sequences in pines

• Phylogenetic Analysis Software:

  • MEGA5: Suitable for constructing phylogenetic trees using methods like neighbor-joining with bootstrap testing

  • MrBayes: For Bayesian phylogenetic inference

  • PHYLIP: Successfully used for phylogenetic analysis in pine species

  • IQ-TREE: For maximum likelihood phylogeny with model testing

• Selective Pressure Analysis:

  • PAML: To detect sites under positive or negative selection

  • HyPhy: For analyzing selective pressures across the protein sequence

  • SelectionMap: To visualize selection patterns in a structural context

• Structural Conservation Analysis:

  • ConSurf: Maps conservation scores onto protein structures

  • CODEML: Analyzes site-specific evolutionary rates

  • SNAP and SIFT: Predict functional effects of amino acid substitutions

• Domain Architecture Analysis:

  • SMART (Simple Modular Architecture Research Tool): For analyzing protein domain architecture, successfully used in studies of pine transcription factors

  • InterProScan: Integrates various protein signature databases

  • HMMER: For hidden Markov model-based sequence analysis

When applying these tools to conifer ATP synthase subunit c, researchers should account for the slower evolutionary rates of chloroplast genes in gymnosperms compared to angiosperms. This approach enables identification of conserved functional domains and species-specific adaptations that may relate to the varying environmental conditions experienced by different pine species.

How can I correlate ATP synthase structure and function with pine tree adaptation to different environmental conditions?

Correlating ATP synthase structure and function with pine adaptation to environmental conditions requires an integrative ecophysiological approach:

• Comparative Sampling Strategy:

  • Select pine species from diverse environmental gradients (altitude, latitude, temperature, precipitation)

  • Include Pinus koraiensis and closely related species adapted to different conditions

  • Collect both genomic/proteomic data and ecophysiological measurements

• Structural and Functional Analysis:

  • Sequence and analyze ATP synthase subunit c across selected species

  • Determine c-ring stoichiometry using structural biology techniques

  • Measure ATP synthesis rates and proton translocation efficiency under different conditions

  • Analyze thermal stability and pH optimum of the enzyme complex

• Environmental Correlation Framework:

  Environmental Factor	ATP Synthase Parameter	Analytical Method	Expected Adaptive Signature
  Temperature regime	Thermal stability	DSC, activity assays at various temperatures	Shifts in stability curves matching habitat temperature
  Light intensity	ATP synthesis capacity	Enzyme kinetics under different light conditions	Altered Vmax or Km in high/low light adapted species
  Drought stress	Proton gradient utilization efficiency	Proton/ATP ratio measurements	Changes in c-ring stoichiometry affecting energy efficiency
  Elevation	Oxygen sensitivity	Activity assays under varying O₂ concentrations	Adaptations to different oxygen partial pressures

• Metabolic Context Analysis:

  • Investigate relationships between ATP synthase function and production of defensive compounds

  • Consider how ATP synthase activity may support synthesis of pinosylvin stilbenoids that provide resistance to pine wood nematode (PWN)

  • Examine potential metabolic trade-offs between energy efficiency and stress response

• Statistical Analysis:

  • Principal Component Analysis (PCA) to identify patterns in multidimensional data

  • Phylogenetically Independent Contrasts to account for shared evolutionary history

  • Multiple regression models to test specific environment-function hypotheses

This approach would help identify whether variations in ATP synthase structure and function represent adaptive responses to environmental challenges or reflect phylogenetic constraints. For example, research on pinosylvin stilbenoid production in pine species has shown that transcription factors like PsbHLH1 can influence metabolic pathways that contribute to PWN resistance . Similar adaptive patterns might exist in the bioenergetic machinery, including ATP synthase.

What are the most significant knowledge gaps in our understanding of Pinus koraiensis ATP synthase subunit c?

Despite advances in our understanding of ATP synthase function in plant chloroplasts, several significant knowledge gaps remain specific to Pinus koraiensis ATP synthase subunit c:

• C-ring Stoichiometry: The exact number of c-subunits in the Pinus koraiensis ATP synthase c-ring remains undetermined. This stoichiometry directly affects the proton/ATP ratio and thus the bioenergetic efficiency of the enzyme .

• Species-Specific Adaptations: How the structure and function of ATP synthase subunit c in Pinus koraiensis differ from those in other pine species and how these differences relate to environmental adaptation remains poorly understood.

• Regulatory Mechanisms: The factors controlling expression, assembly, and activity regulation of ATP synthase in pine chloroplasts, particularly under stress conditions, require further investigation.

• Integration with Metabolism: The relationship between ATP synthase function and specialized metabolite production (such as pinosylvin stilbenoids) that contribute to stress resistance needs further elucidation .

• Post-translational Modifications: The presence and functional significance of post-translational modifications on the native protein in Pinus koraiensis remain largely unexplored.

Addressing these knowledge gaps would require interdisciplinary approaches combining structural biology, biochemistry, molecular genetics, and ecophysiology. The development of improved recombinant expression systems for the protein will facilitate many of these investigations .

What future research directions would advance our understanding of ATP synthase function in pine species?

Future research on ATP synthase in pine species should focus on several promising directions that integrate molecular mechanisms with ecological and evolutionary perspectives:

• Comparative Structural Biology: Determine the c-ring stoichiometry across multiple pine species using cryo-electron microscopy and assess whether variations correlate with environmental adaptations.

• Climate Adaptation Studies: Investigate how ATP synthase structure and function vary among pine populations from different climatic regions to identify potential adaptations to temperature, drought, or light conditions.

• Metabolic Integration: Explore the relationship between ATP synthase efficiency and specialized metabolism, particularly the production of defense compounds like pinosylvin stilbenoids that provide resistance to pathogens .

• Genetic Engineering Approaches:

  • Develop CRISPR/Cas9 systems for targeted modification of ATP synthase genes in pine species

  • Create transgenic lines with altered c-ring stoichiometry to test hypotheses about energy efficiency and stress tolerance

  • Apply the successful approaches used with transcription factors like PsbHLH1 to manipulate ATP synthase components

• Systems Biology Integration: Create comprehensive models linking photosynthetic electron transport, ATP synthesis, carbon fixation, and specialized metabolism in pine chloroplasts.

• Novel Analytical Techniques: Develop improved methods for studying membrane protein complexes in conifers, potentially adapting approaches that have been successful with spinach chloroplast ATP synthase .