Recombinant Lepidium virginicum ATP synthase subunit c, chloroplastic (atpH)
Description
Introduction
The recombinant Lepidium virginicum ATP synthase subunit c, chloroplastic (atpH), is a hydrophobic membrane protein critical for proton translocation in the F₀ sector of chloroplast ATP synthase. This subunit forms a multimeric c-ring that drives ATP synthesis through rotational catalysis. Produced via recombinant expression systems, it serves as a model for studying the structural and functional mechanisms of ATP production in photosynthesis .
Amino Acid Sequence and Structure
The Lepidium virginicum atpH subunit consists of 81 amino acids (AA 1–81) with a predicted molecular weight of ~9 kDa. Its sequence includes hydrophobic residues critical for membrane integration and proton translocation . Key structural features include:
Expression Systems
The subunit is typically expressed in E. coli as a fusion protein with maltose-binding protein (MBP) to enhance solubility . Host systems include:
Purification Workflow
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Affinity Chromatography: MBP-c fusion protein purified via maltose-binding columns .
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Protease Cleavage: MBP tag removed using thrombin or factor Xa .
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Reversed-Phase Chromatography: Final purification with ethanol eluent .
Mechanistic Overview
The atpH subunit participates in:
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Proton Translocation: Each subunit binds and transports one H⁺ across the thylakoid membrane during c-ring rotation .
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ATP Synthesis: Rotation of the c-ring drives γ-stalk rotation in the F₁ sector, catalyzing ATP synthesis (3 ATP per full rotation) .
Stoichiometric Variability
The number of c-subunits (n) in the ring varies across species, directly influencing the H⁺/ATP ratio. For example:
| Organism | c-Subunit Count (n) | H⁺/ATP Ratio |
|---|---|---|
| Spinach | 14 | 4.67 |
| Lepidium virginicum | Not explicitly reported (inferred from structural homologs) . |
Table 1: Production Parameters for Recombinant atpH
| Parameter | Value (E. coli) | Source |
|---|---|---|
| Gene Optimization | Codon-optimized for E. coli expression | |
| Tag | N-terminal His-tag | |
| Purification | Affinity chromatography → RP-HPLC |
Table 2: Functional Analogs in Other Species
| Species | c-Subunit Count (n) | H⁺/ATP Ratio |
|---|---|---|
| Spinacia oleracea | 14 | 4.67 |
| Synechococcus | 9 | 3.33 |
| Rhodomonas salina | 12 | 4.00 |
Product Specs
Please note that we will prioritize shipping the format currently in stock. However, if you have specific format requirements, kindly specify them when placing your order, and we will prepare the product accordingly.
All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C, while lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is Lepidium virginicum ATP synthase subunit c, chloroplastic (atpH)?
ATP synthase subunit c, chloroplastic (atpH) from Lepidium virginicum is a component of the F0 sector of the ATP synthase complex located in the chloroplast. It functions as part of the proton channel that enables proton flow across the thylakoid membrane, which is essential for ATP synthesis in photosynthetic organisms. This protein is also known by several other names including ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, and F-ATPase subunit c Lipid-binding protein .
What are the structural characteristics of Lepidium virginicum atpH?
The recombinant Lepidium virginicum ATP synthase subunit c, chloroplastic is typically produced with greater than 85% purity as determined by SDS-PAGE analysis . While the search results don't specify the exact molecular weight or tertiary structure, ATP synthase subunit c proteins generally feature a hydrophobic domain that spans the membrane multiple times, creating part of the proton channel essential for ATP synthesis. The protein's structure is optimized for its role in facilitating proton movement across the thylakoid membrane during photophosphorylation.
How does Lepidium virginicum atpH compare to ATP synthase components in other species?
Lepidium virginicum atpH shares functional similarities with ATP synthase subunit c proteins from other plants like Nasturtium officinale and photosynthetic organisms like Chloranthus spicatus, Pavlova lutherii, and various cyanobacteria including Synechococcus sp. and Cyanothece sp. . While the core function remains consistent across species, evolutionary adaptations may result in sequence variations that optimize performance under different environmental conditions specific to each organism's ecological niche.
What expression systems are suitable for producing recombinant Lepidium virginicum atpH?
Recombinant Lepidium virginicum ATP synthase subunit c can be effectively expressed in multiple host systems including E. coli, yeast, baculovirus-infected insect cells, or mammalian cell cultures . Each expression system offers distinct advantages: E. coli provides high protein yields and cost-effectiveness, yeast systems offer eukaryotic post-translational modifications, baculovirus systems are excellent for membrane proteins, and mammalian cells provide the most authentic eukaryotic processing. Selection should be based on specific experimental requirements, particularly considering the hydrophobic nature of this membrane protein.
What purification techniques yield the highest purity for recombinant Lepidium virginicum atpH?
Standard purification protocols typically achieve greater than 85% purity as determined by SDS-PAGE . For membrane proteins like ATP synthase subunit c, effective purification commonly employs a multi-step approach beginning with cell lysis using detergents to solubilize membrane proteins, followed by affinity chromatography (if a tag was incorporated), ion-exchange chromatography to separate based on charge properties, and size exclusion chromatography as a polishing step. The choice of detergents is critical for maintaining protein stability and native conformation throughout the purification process.
How can researchers verify the structural integrity of purified Lepidium virginicum atpH?
Beyond the standard SDS-PAGE analysis that confirms size and initial purity , researchers should implement multiple analytical techniques to verify structural integrity. Circular dichroism spectroscopy can assess secondary structure elements, particularly important for confirming proper folding. Mass spectrometry provides precise molecular weight confirmation and can identify post-translational modifications. For functional validation, reconstitution into liposomes followed by proton transport assays would confirm that the protein maintains its native functionality.
How can Lepidium virginicum atpH be used in photosynthesis research studies?
Researchers can utilize recombinant Lepidium virginicum atpH in reconstitution experiments to study ATP synthesis mechanisms in chloroplasts. By incorporating the purified protein into artificial membrane systems, investigators can manipulate components systematically to examine how subunit c contributes to proton transport efficiency and ATP production. This approach is particularly valuable for understanding species-specific adaptations in photosynthetic energy conversion. Comparative studies with other plant ATP synthase components, such as the ATP synthase subunit beta (ATPB) also found in Lepidium virginicum , can provide insights into the integrated function of the complete ATP synthase complex.
What are effective protocols for studying protein-lipid interactions involving Lepidium virginicum atpH?
Given its lipid-binding properties , protocols for studying protein-lipid interactions should employ techniques such as differential scanning calorimetry to measure thermodynamic parameters of binding, surface plasmon resonance for binding kinetics, and fluorescence spectroscopy with lipid probes to monitor interaction dynamics. Additionally, reconstitution into nanodiscs or liposomes of defined lipid composition allows researchers to systematically investigate how specific lipids affect protein structure and function. These approaches can reveal how the lipid environment modulates atpH function within the chloroplast membrane.
How can researchers investigate the role of Lepidium virginicum atpH in ATP synthase assembly?
To study ATP synthase assembly processes, researchers can implement co-expression systems where atpH is expressed alongside other ATP synthase components, particularly those from Lepidium virginicum like ATP synthase subunit beta . Using fluorescently-tagged proteins combined with Förster resonance energy transfer (FRET) analysis allows real-time monitoring of protein-protein interactions. Complementary approaches include pulse-chase experiments to track assembly kinetics, crosslinking studies to identify interaction interfaces, and cryo-electron microscopy to visualize intermediate assembly states.
How can site-directed mutagenesis of Lepidium virginicum atpH advance understanding of proton transport mechanisms?
Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in atpH. By systematically modifying conserved residues, particularly those in transmembrane domains, researchers can identify amino acids essential for proton binding, channel formation, and rotational torque generation. Mutated constructs should be functionally characterized using proton transport assays in reconstituted systems, complemented by structural studies to determine how mutations affect protein conformation. This approach has proven invaluable for elucidating molecular mechanisms in other ATP synthase systems and would likely yield similar insights for the Lepidium virginicum protein.
What approaches can be used to study the integration of Lepidium virginicum atpH with photosystem components?
To study integration with other photosynthetic complexes, researchers should consider multi-protein reconstitution systems incorporating both ATP synthase components and photosystem proteins from Lepidium virginicum. Techniques such as blue native electrophoresis can identify stable super-complexes, while functional coupling can be assessed by measuring electron transport rates alongside ATP synthesis in reconstituted thylakoid membrane systems. Proximity labeling techniques using engineered peroxidases can identify neighboring proteins in the native environment, providing insights into the spatial organization of these complexes in the chloroplast.
How can comparative genomics enhance our understanding of Lepidium virginicum atpH evolution?
Comparative genomic approaches should align atpH sequences across diverse plant species, particularly focusing on other members of the Brassicaceae family like Nasturtium officinale, which also has an characterized ATP synthase subunit c . Analysis of sequence conservation, selection pressures, and evolutionary rates can identify functionally critical regions versus adaptable domains. Correlation with ecological factors may reveal adaptive traits related to photosynthetic efficiency under different environmental conditions. This approach provides evolutionary context for functional studies and may identify unique features of the Lepidium virginicum protein with biotechnological potential.
What spectroscopic methods are most suitable for studying Lepidium virginicum atpH structure and dynamics?
Multiple spectroscopic approaches can provide complementary structural information about Lepidium virginicum atpH. Fourier-transform infrared spectroscopy is particularly valuable for membrane proteins, providing insights into secondary structure elements within the lipid environment. Nuclear magnetic resonance spectroscopy, though challenging with membrane proteins, can provide atomic-level details about protein dynamics when isotope labeling is employed. Hydrogen-deuterium exchange mass spectrometry offers information about solvent-accessible regions and conformational flexibility. Together, these techniques provide a comprehensive view of protein structure and dynamics not achievable through any single method.
How should researchers interpret functional data from recombinant Lepidium virginicum atpH versus native protein?
When interpreting functional data, researchers must carefully consider differences between recombinant and native systems. The recombinant Lepidium virginicum atpH, typically produced in heterologous expression systems , may lack specific post-translational modifications or proper assembly with partner proteins found in the native chloroplast environment. Control experiments should include comparison with chloroplast membrane preparations from Lepidium virginicum when possible. Activity measurements should be normalized and conducted under conditions mimicking the chloroplast environment, including appropriate pH gradients, ion concentrations, and lipid compositions to obtain physiologically relevant data.
What are the best approaches for quantifying proton translocation efficiency of Lepidium virginicum atpH?
Proton translocation efficiency can be quantified using reconstituted proteoliposomes loaded with pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine. By establishing a pH gradient and monitoring fluorescence changes upon activation, researchers can measure proton transport rates under various conditions. Complementary electrophysiological techniques, such as patch-clamp analysis of enlarged liposomes or planar lipid bilayers containing purified atpH, can provide direct measurements of proton currents with high temporal resolution. These approaches should be calibrated using ionophores to establish maximum response benchmarks.
Can Lepidium virginicum atpH research inform applications in synthetic biology?
The structural and functional characterization of Lepidium virginicum atpH can significantly contribute to synthetic biology applications. Detailed knowledge of how this protein functions in proton transport and ATP synthesis could inform the design of artificial photosynthetic systems or bio-inspired energy conversion devices. Additionally, understanding the specific properties of this protein compared to homologs from other species may reveal design principles for engineering ATP synthase components with enhanced efficiency or novel functionalities. The recombinant production systems already established for this protein provide a foundation for such biotechnological applications.
How might research on Lepidium virginicum atpH intersect with studies on the plant's water-soluble chlorophyll-binding protein?
Recent research has identified a water-soluble chlorophyll-binding protein (WSCP) from Lepidium virginicum that, when reconstituted with chlorophyll a, serves as a novel contrast agent for photoacoustic imaging . Studies on atpH could complement this research by providing insights into how photosynthetic energy generation supports chlorophyll synthesis and maintenance. Potential collaborative research could investigate whether environmental factors that affect ATP synthase efficiency also influence WSCP expression or chlorophyll binding capacity, revealing integrated responses to environmental stressors.
What strategies can overcome protein aggregation issues during recombinant Lepidium virginicum atpH expression?
Membrane proteins like ATP synthase subunit c are prone to aggregation during heterologous expression. To mitigate this challenge, researchers should optimize several parameters: (1) reduce expression temperature to slow synthesis rate and improve folding; (2) test multiple detergents for solubilization, particularly mild non-ionic detergents like DDM (n-dodecyl β-D-maltoside); (3) co-express with molecular chaperones that facilitate proper folding; (4) consider fusion tags like SUMO that enhance solubility; and (5) explore expression as inclusion bodies followed by controlled refolding protocols if traditional approaches fail. Implementation of high-throughput screening of these conditions can efficiently identify optimal expression parameters.
How can researchers address challenges in functional reconstitution of Lepidium virginicum atpH?
Functional reconstitution faces several challenges including maintaining protein stability during the reconstitution process and achieving proper orientation in artificial membranes. Successful approaches include: (1) testing multiple reconstitution methods (direct incorporation, detergent removal by dialysis, or adsorption to preformed liposomes); (2) optimizing lipid composition to include chloroplast-specific lipids like monogalactosyldiacylglycerol; (3) controlling protein:lipid ratios to prevent overcrowding; and (4) verifying reconstitution success using freeze-fracture electron microscopy or dynamic light scattering. Activity assays should include positive controls using well-characterized ATP synthase preparations to benchmark reconstitution efficiency.
What approaches can resolve contradictory functional data when studying Lepidium virginicum atpH?
When faced with contradictory functional data, researchers should implement a systematic troubleshooting approach: (1) standardize protein preparation protocols to eliminate batch-to-batch variability; (2) verify protein integrity before each experiment using techniques like circular dichroism; (3) control environmental parameters (pH, temperature, ionic strength) precisely across experiments; (4) employ multiple complementary functional assays to cross-validate findings; and (5) consider the influence of experimental system (detergent micelles versus liposomes versus nanodiscs) on protein behavior. Collaborative cross-laboratory testing using standardized protocols can help resolve persistent contradictions and establish reproducible results.
