Recombinant Zea mays ATP synthase subunit c, chloroplastic (atpH)
Description
Structure and Function of Chloroplastic ATP Synthase
ATP synthase in chloroplasts is a multimeric enzyme complex embedded in the thylakoid membrane. This remarkable molecular machine harnesses the proton gradient established during photosynthesis to synthesize adenosine triphosphate (ATP), which fuels various metabolic processes essential for plant growth and development . The enzyme consists of two major components: the membrane-embedded F₀ portion and the soluble F₁ portion that protrudes into the stroma.
The c-subunit forms a critical cylindrical oligomer within the F₀ portion of ATP synthase. This c-ring structure is embedded in the thylakoid membrane and functions as a rotor that mechanically couples proton translocation to ATP synthesis . As protons move through channels formed between the c-ring and the adjacent a-subunit, they drive the rotation of the c-ring. This rotational force is then transmitted to the central stalk of the F₁ portion, ultimately powering the synthesis of ATP from ADP and inorganic phosphate.
Evolutionary Conservation and Diversity
The c-subunit of ATP synthase demonstrates remarkable evolutionary conservation across species while exhibiting organism-specific adaptations. In plants like Zea mays, the chloroplastic ATP synthase subunit c is encoded by the nuclear genome and transported to chloroplasts via an N-terminal targeting sequence, unlike mitochondrial isoforms that show multiple variants with different targeting peptides .
Bacterial Expression Systems
Escherichia coli remains the preferred host for recombinant expression of the chloroplastic ATP synthase subunit c due to its simplicity, cost-effectiveness, and high yield potential. Similar to strategies employed for spinach ATP synthase subunit c, the Zea mays homolog can be expressed using a fusion protein approach . In this method, the hydrophobic c-subunit is initially expressed as a soluble fusion protein with partners such as maltose-binding protein (MBP).
The bacterial expression system typically involves:
-
Codon optimization of the maize atpH gene for efficient expression in E. coli
-
Fusion with solubility-enhancing proteins like MBP
-
Controlled expression conditions to minimize toxicity
-
Specific protease cleavage to release the mature c-subunit
Expression Challenges and Solutions
Table 1: Challenges and Solutions in Recombinant Expression of ATP Synthase Subunit c
| Challenge | Solution | Rationale |
|---|---|---|
| Protein hydrophobicity | Fusion with solubility partners (MBP) | Increases solubility and prevents aggregation |
| Membrane integration | Controlled induction conditions | Prevents overwhelming cellular membrane systems |
| Protein toxicity | Tight expression regulation | Minimizes detrimental effects on host cells |
| Codon bias | Gene optimization for E. coli | Enhances translation efficiency |
| Correct folding | Addition of chaperones | Facilitates proper protein folding |
Functional Significance of the c-Subunit
The c-subunit plays a pivotal role in determining the bioenergetic efficiency of ATP synthesis in chloroplasts. The number of c-subunits per oligomeric ring (c₍ₙ₎) varies among different organisms and directly influences the ratio of protons translocated to ATP molecules synthesized .
Stoichiometric Variation
The c-ring stoichiometry is organism-dependent and has significant metabolic implications. This variation is inherently related to the organism's metabolic requirements, though the exact factors determining this stoichiometry remain incompletely understood . Research on recombinant Zea mays ATP synthase subunit c enables investigation into these factors, potentially revealing adaptations specific to maize metabolism.
Contribution to ATP Synthesis Regulation
The c-subunit not only participates in the mechanical aspects of ATP synthesis but also contributes to the regulation of this process. The structure and arrangement of c-subunits within the ring affect the rotation kinetics and, consequently, the rate of ATP production. Understanding these mechanisms through recombinant protein studies provides insights into how plants like maize optimize energy production under varying environmental conditions.
Experimental Approaches to Studying Recombinant Zea mays ATP Synthase Subunit c
Several experimental techniques have been employed to investigate the structural and functional properties of recombinant ATP synthase subunit c from chloroplasts.
Purification Strategies
Purification of recombinant Zea mays ATP synthase subunit c typically follows protocols similar to those developed for other plant species. After expression as a fusion protein, the MBP-c₁ fusion is cleaved by specific proteases, and the liberated c-subunit is purified using reversed-phase column chromatography . This approach has successfully yielded significant quantities of highly purified c-subunit with preserved α-helical secondary structure.
Functional Reconstitution
Recombinant c-subunits can be reconstituted into liposomes to study their functional properties, including proton translocation efficiency and oligomerization capacity. These reconstitution experiments provide valuable insights into the c-subunit's role in ATP synthesis and its interactions with other components of the ATP synthase complex.
Genomic Organization and Expression Patterns
The gene encoding ATP synthase subunit c in Zea mays chloroplasts (atpH) is located within the nuclear genome, unlike some other ATP synthase components that are chloroplast-encoded. This nuclear localization necessitates coordinated expression and transport mechanisms to ensure proper assembly of the ATP synthase complex.
Targeting and Transport
Like other nuclear-encoded chloroplast proteins, the ATP synthase subunit c requires an N-terminal targeting peptide to direct it to chloroplasts following its synthesis in the cytosol. This targeting sequence is cleaved by matrix peptidases upon import, yielding the mature protein that integrates into the thylakoid membrane . While research on mitochondrial ATP synthase has identified multiple isoforms with different targeting peptides that serve non-redundant functions , less is known about potential diversity in chloroplastic targeting sequences.
Expression Regulation
The expression of atpH in Zea mays is regulated in coordination with other ATP synthase components and photosynthetic machinery. This regulation ensures appropriate stoichiometric relationships among the various subunits, facilitating efficient assembly of the complete ATP synthase complex.
Biotechnological Applications
Recombinant Zea mays ATP synthase subunit c has potential applications in various biotechnological fields:
Bioenergetic Research
The recombinant production of ATP synthase subunit c facilitates detailed studies of the biophysical and biochemical properties of this protein, contributing to our understanding of photosynthetic energy conversion in crop plants like maize. This research can reveal mechanisms underlying energy efficiency in different plant species and under various environmental conditions.
Agricultural Improvements
Understanding the structure-function relationships of ATP synthase components could lead to strategies for enhancing photosynthetic efficiency in crop plants. Potential applications include:
-
Engineering plants with optimized ATP synthase c-ring stoichiometry for improved energy conversion
-
Developing crop varieties with enhanced tolerance to environmental stresses
-
Creating plants with increased biomass production through improved photosynthetic efficiency
Biopharmaceutical Applications
The established protocols for recombinant expression of membrane proteins like ATP synthase subunit c have broader applications in pharmaceutical research. These methods can be adapted for the production of other challenging membrane proteins of therapeutic interest, such as receptors and transporters.
Future Research Directions
Several important areas warrant further investigation regarding recombinant Zea mays ATP synthase subunit c:
Stoichiometric Determination
A critical area for future research involves determining the exact number of c-subunits in the native Zea mays chloroplastic ATP synthase c-ring and investigating the factors that influence this stoichiometry. This knowledge would contribute to our understanding of the bioenergetic adaptations specific to maize metabolism .
Protein-Protein Interactions
Detailed analysis of interactions between the c-subunit and other components of the ATP synthase complex would provide insights into assembly mechanisms and regulatory processes. Studies examining binding domains similar to those identified in maize atpB could reveal how specific regions of the c-subunit contribute to complex formation.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is specifically requested and approved in advance. Additional charges apply for dry ice shipping.
The tag type is determined during the production process. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Q&A
What is ATP synthase subunit c, chloroplastic (atpH) in Zea mays?
ATP synthase subunit c, chloroplastic (atpH) is a critical component of the ATP synthase complex located in maize chloroplasts. It's also known as ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, or Lipid-binding protein. The protein contains large hydrophobic domains characteristic of membrane-bound proteins, which are essential for its function in the ATP synthase complex that produces adenosine triphosphate (ATP) required for photosynthetic metabolism .
How does the structure of Zea mays ATP synthase subunit c compare to homologous proteins in other species?
The ATP synthase subunit c in Zea mays shares structural similarities with homologous proteins in other species, particularly in terms of hydrophobicity profiles. For instance, comparisons between ATP synthase subunits in different species reveal conserved functional domains. In maize mitochondrial ATP synthase (atp6), there is a 44.6% nucleotide homology and 33.2% amino acid homology with the yeast counterpart. Hydropathy profiles generated for these polypeptides show similar patterns of large hydrophobic domains, which is characteristic of membrane-bound proteins involved in ATP synthesis . Similar structural conservation would be expected in the chloroplastic variant (atpH), though with specific adaptations for chloroplastic function.
Which vector constructs are recommended for cloning atpH genes?
Based on methodologies used for homologous proteins, several vector constructs can be employed for cloning atpH genes. These include:
| Vector | Manufacturer | Special Features |
|---|---|---|
| pMAL-c2x | New England Biolabs | Fusion with maltose-binding protein |
| pET-32a(+) | Novagen | T7 promoter, His-tag options |
| pFLAG-MAC | Sigma-Aldrich | FLAG-tag for detection/purification |
These vectors offer different advantages depending on experimental goals, such as improved solubility (pMAL-c2x), high-level expression (pET-32a+), or simplified purification (pFLAG-MAC) . The selection should be based on specific research requirements, including downstream applications and purification strategies.
What are the optimal conditions for purifying recombinant Zea mays atpH protein?
For optimal purification of recombinant Zea mays ATP synthase subunit c, chloroplastic (atpH), researchers should consider several key factors. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended for long-term storage. Purification typically requires centrifugation steps to bring contents to the bottom of storage vials prior to opening . The purity of commercially available recombinant atpH is typically >85% as verified by SDS-PAGE, which serves as a benchmark for laboratory purifications .
How can researchers assess the functional activity of recombinant atpH protein?
The functional activity of recombinant ATP synthase subunit c can be assessed using several complementary approaches:
-
In-gel ATPase assays: These generate white precipitates in regions with ATPase activity when run on blue native (BN) polyacrylamide gel electrophoresis (PAGE) .
-
Coupled spectrophotometric assays: These can measure ATP hydrolase activity in preparations containing the ATP synthase complex .
-
Reconstitution experiments: The recombinant c1 subunit can be used in reconstitution experiments of the multimeric ring (cn) to test assembly capability and function .
-
Oxygen consumption measurements: When incorporated into functional complexes, activity can be measured through succinate-dependent oxygen consumption, which is stimulated by ADP in oxidative phosphorylation systems .
How can researchers investigate the oligomeric state of ATP synthase complexes containing atpH?
The oligomeric state of ATP synthase complexes containing atpH can be investigated using a combination of techniques:
-
Blue Native (BN) Polyacrylamide Gel Electrophoresis (PAGE): This technique allows separation of high molecular weight complexes while maintaining their native structure. A 3%-10% gradient BN gel is typically used to resolve these complexes .
-
Electron Microscopy: Single particle electron microscopy can reveal structural details of dimeric and higher oligomeric ATP synthase complexes. This technique has been successfully applied to visualize ATP synthase complexes from various species .
-
In-gel Activity Assays: After BN-PAGE separation, in-gel ATPase activity assays can identify functional complexes, though it's worth noting that dimeric complexes may exhibit weak activity compared to monomers .
-
Crosslinking Studies: Chemical crosslinking followed by mass spectrometry can identify interaction interfaces between subunits within the complex.
What structural differences exist between monomeric and oligomeric forms of ATP synthase complexes?
Significant structural differences exist between monomeric and oligomeric forms of ATP synthase complexes, which likely apply to complexes containing Zea mays atpH:
-
In dimeric complexes, the F1 headpieces (containing α3β3 subunits) are typically separated by at least 2 nm .
-
Dimeric interfaces often contain specific protein densities between the two Fo parts that facilitate dimerization .
-
The angle between monomers in a dimer can vary significantly between species, with some forming acute angles and others appearing more parallel .
-
Some species exhibit unique structural domains at the dimer interface that may consist of protein complexes as large as 100-200 kDa .
-
Membrane-bound densities may appear at specific positions of the c subunit rotors in dimeric forms .
How does the nucleotide sequence of Zea mays atpH compare to homologs in other plant species?
While the search results don't provide direct sequence comparisons for chloroplastic atpH between Zea mays and other plant species, we can infer from related data that significant homology likely exists. In the case of mitochondrial ATP synthase subunit 6 (atp6) in maize, sequence comparisons revealed 44.6% nucleotide homology and 33.2% amino acid homology with the yeast counterpart . For chloroplastic ATP synthase subunits, conservation patterns would be expected to follow phylogenetic relationships among plant species, with higher conservation among closely related grass species and decreasing homology with evolutionary distance.
What evidence suggests recombination events in the evolution of ATP synthase genes?
Intriguing evidence for recombination in the evolution of ATP synthase genes comes from studies of the maize mitochondrial atp6 gene. Researchers have discovered that 122 base pairs of nucleotide sequence interior to the atp6 gene have extensive homology with the 5′ end of the cytochrome oxidase subunit II gene of maize mitochondria . This suggests historical recombination events between these two genes, providing insight into the evolutionary dynamics of ATP synthase complex components. Such recombination events may contribute to the diversity and functional adaptation of ATP synthase complexes across species and organelles.
How can recombinant Zea mays atpH be used in reconstitution studies?
Recombinant Zea mays ATP synthase subunit c can be valuable for reconstitution studies aimed at understanding the assembly and function of the ATP synthase complex. Specifically:
-
The recombinant protein can be used to reconstitute the multimeric c-ring (cn) in vitro, which is a critical step in understanding the structure-function relationship of the ATP synthase complex .
-
If reconstitution is successful, researchers can apply molecular biology techniques that cannot be used with native c-rings, enabling more detailed investigations of factors influencing the stoichiometric variation of the intact ring .
-
Reconstitution experiments can help elucidate the assembly process of the ATP synthase complex and identify critical interactions between subunits.
-
Such studies can also provide insights into the proton translocation mechanism, which is essential for ATP synthesis.
What methodological challenges arise in studying recombinant atpH in cross-species functional assays?
Several methodological challenges arise when studying recombinant atpH in cross-species functional assays:
-
Inhibitor sensitivity variations: Different species show varying sensitivities to classical F0F1 ATP synthase inhibitors like oligomycin and sodium azide . For example, Tetrahymena thermophila exhibits unusual resistance to these inhibitors compared to yeast. Researchers must consider these species-specific differences when designing functional assays.
-
ATPase activity variation: The specific ATPase activity can vary significantly between species, with some exhibiting time- and ATP-dependent activation . This variability necessitates careful control design and interpretation of results.
-
Oligomeric state considerations: Dimeric and higher oligomeric forms may exhibit different activity levels compared to monomers. In some species, dimeric forms show negligible hydrolase activity despite being structurally intact .
-
Protein-protein interactions: Species-specific accessory subunits may be required for full functionality, potentially limiting the functional reconstitution of recombinant subunits in heterologous systems.
How does recombinant expression affect the functional properties of atpH compared to native protein?
Recombinant expression can potentially affect several functional properties of the atpH protein compared to its native form:
-
Post-translational modifications: The recombinant expression system may not reproduce the exact pattern of post-translational modifications present in the native protein, potentially affecting function.
-
Protein folding: The hydrophobic nature of ATP synthase subunit c makes proper folding particularly challenging in heterologous expression systems, potentially resulting in structural differences from the native protein .
-
Protein-protein interactions: The recombinant protein may lack necessary interaction partners present in the native environment, affecting assembly into functional complexes.
-
Activity levels: Differences in lipid environment, accessory subunits, or structural integrity can lead to altered activity levels between recombinant and native proteins.
What techniques can be used to assess the membrane integration of recombinant atpH?
Several techniques can assess the membrane integration of recombinant atpH:
-
Hydropathy profile analysis: Computational analysis of the amino acid sequence can predict hydrophobic domains likely to interact with membranes. ATP synthase subunit c proteins typically contain large hydrophobic domains characteristic of membrane-bound proteins .
-
Membrane reconstitution assays: The recombinant protein can be incorporated into liposomes or nanodiscs to assess membrane integration capability.
-
Protease protection assays: Limited proteolysis of membrane-incorporated protein can identify protected regions, providing information about membrane topology.
-
Fluorescence-based techniques: Labeling the protein with environment-sensitive fluorophores can report on membrane interaction and insertion.
-
Electron microscopy: Visualization of protein-membrane complexes can provide direct evidence of membrane integration.
What are the most promising applications of recombinant Zea mays atpH in current research?
The most promising applications of recombinant Zea mays ATP synthase subunit c, chloroplastic (atpH) in current research include:
-
Structure-function studies of ATP synthase complexes to understand the fundamental mechanisms of energy conversion in photosynthetic organisms.
-
Reconstitution experiments to elucidate the factors influencing c-ring stoichiometry and assembly .
-
Comparative studies across species to understand evolutionary adaptation of ATP synthase complexes.
-
Investigation of protein-protein interactions within the ATP synthase complex using modified recombinant proteins.
-
Development of novel inhibitors or modulators of ATP synthase function for potential agricultural applications.
What unanswered questions remain about the structure and function of Zea mays atpH?
Several important questions remain unanswered regarding the structure and function of Zea mays atpH:
-
What is the exact stoichiometry of the c-subunit ring in Zea mays chloroplastic ATP synthase, and how does it compare to other species?
-
How do specific amino acid residues contribute to proton translocation and energy conversion efficiency?
-
What is the detailed assembly pathway of the c-ring, and what chaperones or assembly factors are involved?
-
How do environmental factors, particularly those relevant to agricultural conditions, affect the expression and function of atpH in maize?
-
What structural adaptations in Zea mays atpH contribute to the optimization of photosynthetic efficiency in this important crop species?
-
How might genetic modifications of atpH potentially contribute to improved photosynthetic efficiency and crop yield?
