Recombinant Pinus thunbergii ATP synthase subunit c, chloroplastic (atpH)
Product Specs
Please note that we prioritize shipping the format currently in stock. However, if you have specific format requirements, kindly indicate them during order placement, and we will fulfill your request.
All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please communicate with us in advance, as additional fees will apply.
We determine the tag type during production. If you have a specific tag type requirement, please communicate it to us, and we will prioritize its development.
Target Background
Q&A
What is the function of ATP synthase subunit c in Pinus thunbergii chloroplasts?
ATP synthase subunit c (atpH) in Pinus thunbergii chloroplasts functions as a critical component of the CF₀ complex within the complete ATP synthase enzyme. This subunit forms part of the membrane-embedded proton channel that facilitates proton movement across the thylakoid membrane. The organized assembly of multiple subunit c proteins creates a ring structure that rotates in response to the proton gradient, driving conformational changes in the CF₁ portion of the complex and enabling ATP synthesis. In Pinus thunbergii, as in other plants, this process is essential for converting light energy captured during photosynthesis into chemical energy stored as ATP .
How is the atpH gene organized within the Pinus thunbergii chloroplast genome?
The atpH gene in Pinus thunbergii is part of a gene cluster within the chloroplast genome. Although the specific organization in Pinus thunbergii is not fully detailed in the available research, comparative studies with other plant species like Chlamydomonas reinhardtii suggest that atpH may be part of a polycistronic transcription unit. In C. reinhardtii, the atpA gene cluster includes atpA, psbI, cemA, and atpH genes, encoding the α-subunit of CF₁ ATP synthase, a photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF₀ ATP synthase (atpH), respectively . The organization likely includes promoters preceding the atpH gene, allowing for both co-transcription with other genes and independent transcription, resulting in a complex pattern of transcripts that undergo post-transcriptional processing.
What is the structural relationship between recombinant atpH and the complete ATP synthase complex?
Recombinant Pinus thunbergii ATP synthase subunit c (atpH) represents one component of the larger ATP synthase complex, which consists of two major portions: the membrane-embedded CF₀ and the catalytic CF₁. The atpH protein forms part of the c-ring within the CF₀ portion, which spans the membrane and creates a proton channel. This ring typically contains multiple copies of the subunit c protein arranged in a circular structure. Studies on ATP synthase structure using cryo-electron microscopy (cryo-EM) have demonstrated that the c-ring interfaces with other subunits to create a complete rotary motor mechanism . When produced as a recombinant protein, atpH must be properly reconstituted into membrane environments (such as nanodiscs) to study its structural relationships within the complete complex, as its natural hydrophobic properties make it challenging to work with in isolation.
What conservation patterns exist in atpH sequences across conifer species compared to Pinus thunbergii?
The ATP synthase subunit c protein sequences show remarkable conservation across plant species due to the fundamental importance of their function. While specific conservation patterns between Pinus thunbergii and other conifers are not explicitly detailed in the search results, comparative genomic analyses of chloroplast genes typically reveal high conservation in the functional domains of ATP synthase components. The membrane-spanning regions and residues involved in proton translocation would be expected to show the highest degree of conservation. Variation is more likely to occur in regions not directly involved in the core functions of proton transport or subunit interactions. Phylogenetic analyses of atpH sequences can provide valuable information for understanding evolutionary relationships among conifer species and could potentially serve as molecular markers for taxonomic studies.
How do structural modifications of recombinant Pinus thunbergii atpH affect its integration into functional ATP synthase complexes?
Structural modifications of recombinant Pinus thunbergii atpH can significantly impact its ability to integrate into functional ATP synthase complexes. Site-directed mutagenesis of conserved residues, particularly those involved in proton translocation or subunit interactions, can disrupt the protein's ability to assemble properly within the c-ring structure. Research approaches similar to those used with other ATP synthase components involve engineering specific amino acid substitutions, followed by reconstitution experiments in lipid bilayers or nanodiscs to evaluate structural integrity . Functional assays measuring proton conductance or ATP synthesis rates when the modified atpH is incorporated into reconstituted complexes can reveal the impact of structural alterations. Advanced structural analysis techniques such as cryo-EM can then be employed to visualize precise molecular changes in the assembled complex, offering insights into structure-function relationships that cannot be obtained through sequence analysis alone.
What methodological challenges exist in distinguishing contradictory experimental results regarding Pinus thunbergii atpH function?
Researchers face several methodological challenges when addressing contradictory results regarding Pinus thunbergii atpH function. First, variations in experimental conditions, including protein expression systems, purification methods, and reconstitution approaches, can lead to apparently contradictory findings . Topological analysis approaches can help identify the source of contradictions in published literature by categorizing them as negation contradictions, antonym contradictions, or more complex replacement and switch contradictions .
To address these challenges, researchers should:
-
Implement standardized protocols for protein expression and purification
-
Carefully document all experimental conditions
-
Employ multiple complementary techniques to verify results
-
Use statistical approaches to distinguish biological variation from technical artifacts
| Contradiction Type | Example in atpH Research | Resolution Approach |
|---|---|---|
| Negation | Study A: "atpH is essential for ATP synthesis" vs. Study B: "atpH is not essential" | Examine experimental conditions, genetic backgrounds, and definition of "essential" |
| Antonym | Study A: "atpH enhances" vs. Study B: "atpH inhibits" | Investigate concentration-dependent effects and interaction partners |
| Replacement | Study A: "atpH functions in proton transport" vs. Study B: "atpH functions in complex assembly" | Consider multifunctional nature of proteins and context-dependent functions |
| Switch | Reversal of cause-effect relationships between studies | Analyze temporal sequences and control experiments |
How does the oligomeric state of recombinant Pinus thunbergii atpH affect its functional properties in vitro?
The oligomeric state of recombinant Pinus thunbergii atpH is critical to its functional properties in vitro, as the protein naturally exists as part of a multi-subunit c-ring in the ATP synthase complex. When expressed recombinantly, atpH may form various oligomeric states depending on experimental conditions, and these different states can exhibit distinct functional properties. Research approaches to investigate this relationship include size-exclusion chromatography, analytical ultracentrifugation, and native gel electrophoresis to characterize oligomeric forms .
Functional studies comparing different oligomeric states might reveal that only certain configurations support proton translocation or proper interaction with other ATP synthase subunits. Reconstitution experiments in which defined oligomeric states are incorporated into liposomes or nanodiscs, followed by proton transport assays, can establish structure-function relationships. Additionally, cross-linking studies combined with mass spectrometry can identify specific interaction interfaces that stabilize functional oligomers. Understanding these relationships is essential for interpreting in vitro studies and extrapolating results to the native complex's behavior.
What are the effects of post-translational modifications on Pinus thunbergii atpH function and stability?
Post-translational modifications (PTMs) of Pinus thunbergii atpH likely play significant roles in regulating its function, stability, and interactions within the ATP synthase complex. Although specific PTMs for Pinus thunbergii atpH are not detailed in the search results, research on ATP synthase components in other organisms suggests several potential modifications:
| Post-translational Modification | Potential Effect on atpH | Detection Method |
|---|---|---|
| Phosphorylation | Regulation of proton conductance or c-ring assembly | Mass spectrometry, phospho-specific antibodies |
| Acetylation | Protein stability and turnover | Acetylation-specific antibodies, mass spectrometry |
| Oxidative modifications | Response to stress conditions, altered function | Redox proteomics, mass spectrometry |
| Lipid modifications | Membrane association and stability | Lipidomics, specialized extraction protocols |
Researchers investigating PTMs should employ a combination of mass spectrometry-based proteomics, site-directed mutagenesis of potentially modified residues, and functional assays to determine how these modifications affect the protein's properties. The reversible nature of many PTMs suggests they might play important roles in dynamic regulation of ATP synthase activity in response to changing environmental conditions or developmental stages in Pinus thunbergii.
What are the optimal conditions for expressing recombinant Pinus thunbergii atpH in heterologous systems?
The optimal expression of recombinant Pinus thunbergii ATP synthase subunit c (atpH) in heterologous systems requires careful optimization of several parameters. For bacterial expression systems (such as E. coli), using specialized strains designed for membrane protein expression (like C41(DE3) or C43(DE3)) typically yields better results than standard strains. Temperature modulation is critical—lowering the expression temperature to 18-20°C after induction can significantly improve proper folding. The use of fusion tags such as maltose-binding protein (MBP) or SUMO can enhance solubility, while inclusion of a C-terminal His-tag facilitates purification without disrupting function.
For eukaryotic expression systems, yeast platforms like Pichia pastoris offer advantages for membrane protein expression . Key parameters to optimize include:
| Expression Parameter | Optimization Approach | Expected Outcome |
|---|---|---|
| Induction timing | Harvest cells at OD600 of 0.6-0.8 | Maximizes protein yield while minimizing toxicity |
| Inducer concentration | IPTG at 0.1-0.5 mM for bacteria | Balances expression level with proper folding |
| Media composition | Supplementation with specific lipids | Promotes proper membrane integration |
| Codon optimization | Adjust codons to match expression host | Improves translation efficiency |
| Co-expression partners | Include chaperones or other ATP synthase subunits | Enhances proper folding and complex assembly |
Post-expression, using specialized membrane protein extraction buffers containing mild detergents like DDM (n-dodecyl-β-D-maltopyranoside) or LMNG (lauryl maltose neopentyl glycol) at concentrations just above their critical micelle concentration helps maintain protein stability during purification.
How can researchers effectively reconstitute purified recombinant atpH into functional membrane systems?
Effective reconstitution of purified recombinant Pinus thunbergii atpH into functional membrane systems requires a methodical approach to maintain protein stability and function. The process involves transitioning the purified protein from detergent-solubilized state into a lipid environment that mimics its native membrane context. Researchers have successfully used nanodisc technology to create a controlled membrane environment for ATP synthase components . This approach involves mixing the purified protein with appropriate phospholipids and membrane scaffold proteins (MSPs), followed by controlled detergent removal.
The reconstitution protocol typically follows these steps:
-
Selection of appropriate lipid composition (often a mixture of POPC, POPE, and cardiolipin for chloroplast proteins)
-
Determination of optimal protein-to-lipid and lipid-to-MSP ratios through small-scale optimization experiments
-
Controlled detergent removal using bio-beads, dialysis, or cyclodextrin-based methods
-
Verification of successful reconstitution through techniques such as:
-
Negative-stain electron microscopy to confirm incorporation
-
Dynamic light scattering to assess nanodisc size and homogeneity
-
Functional assays such as proton translocation measurements
-
Alternative approaches include reconstitution into liposomes or lipid cubic phases, each offering distinct advantages for different experimental objectives. Functionalizing these membrane systems with fluorescent probes sensitive to proton concentration or membrane potential can provide direct readouts of atpH activity within the reconstituted system.
What techniques are most effective for analyzing the structure of Pinus thunbergii atpH in its native conformation?
Determining the structure of Pinus thunbergii atpH in its native conformation requires specialized techniques that can accommodate its hydrophobic nature and membrane environment. Cryo-electron microscopy (cryo-EM) has emerged as a particularly powerful method for membrane protein structural analysis, enabling near-atomic resolution structures of complete ATP synthase complexes . For this approach, samples are rapidly frozen in vitreous ice, preserving the protein in a near-native state without the need for crystallization.
Additional structural analysis techniques include:
| Technique | Information Provided | Advantages | Limitations |
|---|---|---|---|
| Cryo-EM | High-resolution 3D structure | Works with heterogeneous samples, preserves native state | Requires specialized equipment, challenging for small proteins |
| X-ray crystallography | Atomic-resolution structure | Highest possible resolution | Difficult to crystallize membrane proteins, potential crystal packing artifacts |
| NMR spectroscopy | Structural dynamics, ligand interactions | Provides information on protein motion | Size limitations, requires isotope labeling |
| Hydrogen-deuterium exchange MS | Conformational dynamics, solvent exposure | Works with minimal sample, no size limitation | Lower resolution than other methods |
| Cross-linking mass spectrometry | Protein-protein interaction interfaces | Captures transient interactions | Provides distance constraints rather than complete structure |
Researchers studying atpH often combine multiple techniques—for example, using molecular dynamics simulations based on cryo-EM structures to explore conformational changes during proton translocation. Site-directed spin labeling coupled with electron paramagnetic resonance (EPR) spectroscopy can provide valuable information about the dynamics of specific regions within the protein in different functional states.
How can researchers assess the impact of Pinus thunbergii atpH mutations on ATP synthase function?
Assessing the impact of Pinus thunbergii atpH mutations on ATP synthase function requires a multi-faceted approach combining molecular biology, biochemistry, and biophysical techniques. Initially, researchers should identify conserved residues through sequence alignment with well-characterized ATP synthase subunit c proteins from other species. Targeted mutations can then be introduced using site-directed mutagenesis, focusing on residues involved in proton binding (such as the essential glutamate residue in the middle of the second transmembrane helix), subunit interactions, or ring formation.
A comprehensive functional assessment workflow includes:
-
Expression and purification of wild-type and mutant proteins under identical conditions
-
Biochemical characterization:
-
Circular dichroism spectroscopy to verify proper secondary structure
-
Thermal stability assays to assess structural integrity
-
Detergent micelle size analysis to evaluate oligomerization properties
-
-
Reconstitution into membrane systems (nanodiscs or liposomes)
-
Functional assays:
-
Proton translocation measurements using pH-sensitive fluorescent dyes
-
ATP synthesis/hydrolysis activity when co-reconstituted with other ATP synthase components
-
Measurement of proton-motive force dissipation rates
-
How does recombinant Pinus thunbergii atpH research contribute to understanding evolutionary adaptations in conifer bioenergetics?
Research on recombinant Pinus thunbergii atpH provides valuable insights into evolutionary adaptations in conifer bioenergetics. By comparing the structure, function, and regulation of atpH across different plant lineages, researchers can identify conifer-specific adaptations that may have evolved in response to environmental pressures. Conifers like Pinus thunbergii have adapted to diverse ecological niches, often in challenging environments that require specialized energy management strategies. The ATP synthase complex, as a central component of energy production, likely reflects these adaptations.
Comparative analysis of atpH sequences, expression patterns, and functional properties across plant phylogeny can reveal signatures of selection that point to adaptive changes. For instance, variations in proton-binding residues might reflect adaptations to different pH environments within chloroplasts, while alterations in subunit interfaces could modify the coupling efficiency between proton movement and ATP synthesis. These adaptations could contribute to the remarkable resilience of conifers in environments with variable light, temperature, and water availability.
Future research directions should include reconstructing ancestral atpH sequences to experimentally test hypotheses about the functional consequences of evolutionary changes. Additionally, investigating variation within Pinus species growing in different environmental conditions could provide insights into ongoing selective pressures on bioenergetic systems.
What are the potential applications of understanding Pinus thunbergii atpH in enhancing photosynthetic efficiency?
-
Engineering optimized ATP synthase components with improved energy conversion efficiency
-
Developing stress-tolerant variants that maintain function under suboptimal conditions
-
Creating regulatory systems that better balance ATP production with metabolic demands
-
Designing synthetic biology approaches to incorporate conifer-specific adaptations into crop plants
Research suggests that ATP synthase can be a limiting factor in photosynthesis under certain conditions, particularly during rapid transitions in light availability or under stress. By understanding the specific properties of Pinus thunbergii atpH and its regulation, researchers could develop interventions that address these limitations. For example, incorporating specific residues or structural elements from conifer atpH into crop plants might enhance their performance under fluctuating environmental conditions.
The potential improvements in photosynthetic efficiency resulting from atpH optimization would have significant implications for both natural ecosystem productivity and agricultural yields, making this research area particularly valuable in the context of climate change and food security challenges.
How can contradictions in experimental results be leveraged to advance our understanding of atpH function?
Rather than viewing contradictions in experimental results as obstacles, researchers can leverage them as opportunities to deepen understanding of atpH function. Contradictions often reveal context-dependent behaviors or previously unknown regulatory mechanisms. By systematically analyzing contradictory findings through a topological approach to contradictions in scientific literature, researchers can identify patterns that lead to new hypotheses about atpH function .
For effective contradiction analysis, researchers should:
-
Classify contradictions according to their type (negation, antonym, replacement, switch, scope, or latent)
-
Examine experimental conditions in detail to identify variables that might explain different outcomes
-
Consider whether contradictions reflect genuine biological complexity rather than experimental artifacts
-
Design experiments specifically to test competing hypotheses derived from contradictory results
| Contradiction Analysis Approach | Implementation Strategy | Potential Outcome |
|---|---|---|
| Systematic literature review | Use structural analysis of contradictions in published findings | Identify patterns in conditions leading to different results |
| Meta-analysis | Quantitatively combine results across studies | Determine effect sizes and sources of heterogeneity |
| Replication with parameter variation | Systematically vary experimental conditions | Identify specific variables driving different outcomes |
| Computational modeling | Develop models that accommodate seemingly contradictory data | Generate unified hypotheses explaining diverse findings |
By embracing contradictions and systematically investigating their causes, researchers can develop more nuanced and comprehensive models of atpH function that account for its behavior across diverse contexts and conditions.
What are the key unresolved questions regarding Pinus thunbergii atpH that warrant further investigation?
Despite advances in our understanding of ATP synthase structure and function, several key questions about Pinus thunbergii atpH remain unresolved. The specific adaptations of this protein in conifers compared to other plant lineages are not fully characterized, particularly in relation to environmental stress responses. The regulatory mechanisms controlling atpH expression, assembly, and activity in response to changing environmental conditions require further investigation. Additionally, the potential role of atpH in coordinating energy production with other metabolic processes in conifer chloroplasts remains poorly understood.
Future research should address these knowledge gaps through integrative approaches combining genomics, structural biology, and physiological studies. Particularly valuable would be investigations examining atpH function under conditions that mimic environmental stresses relevant to conifer habitats, such as drought, temperature extremes, and variable light conditions. The development of conifer-specific experimental tools, including genetic manipulation systems for Pinus species, would greatly accelerate progress in this field.
