Recombinant Campylobacter jejuni subsp. doylei ATP synthase subunit c (atpE)
Description
Functional Role in ATP Synthesis
ATP synthase subunit c, also known as the F0 sector subunit c, plays a critical role in cellular bioenergetics. This protein functions as part of the membrane-embedded portion of the ATP synthase complex that forms the proton channel . The protein is alternatively named F-type ATPase subunit c or lipid-binding protein, reflecting its structural and functional properties .
Within the complex machinery of ATP synthase, the c subunit contributes to the creation of the proton gradient necessary for ATP synthesis. The F1F0 ATP synthase produces ATP from ADP in the presence of a proton or sodium gradient. During catalysis, ATP synthesis in the catalytic domain of F1 is coupled via a rotary mechanism of the central stalk subunits to proton translocation across the membrane .
Expression Systems
Cell-free expression systems have also been utilized for producing this transmembrane protein, which can sometimes be challenging to express in cellular systems due to potential toxicity or improper folding . The flexibility in expression systems allows researchers to select the most appropriate method based on their specific experimental requirements and downstream applications.
Immunological Research and Vaccine Development
Recombinant proteins from Campylobacter jejuni have significant potential in vaccine development strategies. While the search results do not specifically detail the use of atpE in vaccine development, the approaches used with other Campylobacter proteins could be applicable.
Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis worldwide and a major concern in public health . Infections with this pathogen are particularly prevalent among young children in low-resource settings, contributing to significant mortality, stunted growth, and lifelong physical and cognitive impairments . The rising threat of antimicrobial resistance further highlights the urgent need for new interventions to curb Campylobacter infections .
Recent research on Campylobacter vaccine development has explored approaches such as:
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Recombinant protein-based subunit vaccines
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Mucosal delivery systems using chitosan-based nanoparticles
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Production of both systemic (IgY) and local intestinal (sIgA) antibody responses
Similar approaches could potentially be applied to atpE, especially if it proves to be antigenic or surface-exposed in Campylobacter jejuni.
Diagnostic Applications
Recombinant proteins are valuable tools for developing diagnostic assays. The atpE protein could potentially be used in the development of:
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ELISA-based detection systems for Campylobacter jejuni
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Antibody production for immunological detection methods
Pathogenicity Mechanisms
Campylobacter jejuni is a leading cause of bacterial foodborne gastroenteritis worldwide . The pathogen colonizes the mucus layer of the intestinal epithelium, leading to symptoms ranging from mild, watery diarrhea to bloody diarrhea with fever and severe abdominal pain . In severe cases, autoimmune reactions to the infection can lead to post-infectious sequelae such as Guillain-Barré syndrome .
ATP Synthesis and Energy Metabolism in Campylobacter
ATP synthesis is a critical process for bacterial survival and virulence. In Campylobacter jejuni, the ATP synthase complex, including the atpE subunit, plays an essential role in energy production under the microaerophilic conditions preferred by this pathogen .
Energy metabolism in Campylobacter jejuni involves multiple specialized systems:
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Periplasmic nitrate reductase (Nap) for nitrate utilization under microaerophilic conditions
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Amino acid metabolic pathways for utilizing serine, aspartate, glutamate, and proline
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In some strains, pathways for metabolizing specific carbohydrates like L-fucose
The ATP synthase complex integrates with these metabolic pathways to ensure efficient energy production under the diverse conditions encountered by the bacterium during transmission and infection.
Comparative Analysis with ATP Synthases from Other Bacteria
The ATP synthase complex is highly conserved across various bacterial species, but with significant variations that can impact function and potential targeting strategies. Comparative analysis of Campylobacter jejuni subsp. doylei ATP synthase subunit c with homologous proteins from other bacteria provides insights into both conserved functional domains and unique features that could be exploited for species-specific interventions.
A systematic genomic investigation has shown varying degrees of protein conservation across Campylobacter species. For example, when comparing Campylobacter ureolyticus with Campylobacter jejuni subsp. doylei, approximately 10-11% of proteins are highly conserved (≥70% amino acid identity), while 34-37% are considered unique (<25% identity) . While the search results do not specifically analyze atpE conservation, this general pattern of both conservation and diversity within the genus is likely applicable.
Implications for Antimicrobial Development
The distinctive features of Campylobacter jejuni ATP synthase components could potentially be exploited for the development of targeted antimicrobial strategies. ATP synthesis is critical for bacterial survival, and compounds that specifically inhibit this process in Campylobacter without affecting host ATP synthesis could have therapeutic potential.
The rising concern of antimicrobial resistance in Campylobacter jejuni, including high levels of erythromycin resistance reported in both human and chicken isolates in Africa (51.0% and 54.0%, respectively), emphasizes the need for novel antimicrobial targets . Proteins essential for bacterial energy metabolism, like atpE, could represent such targets.
Role in Bacterial Survival and Virulence
Investigating the specific contributions of atpE to Campylobacter jejuni survival under various environmental conditions could reveal its importance in pathogenesis. This could involve generating defined mutants with alterations in the atpE gene and assessing their growth, survival, and virulence in various models.
Similar approaches have been successful in characterizing other genes in Campylobacter jejuni. For example, mutants in the lon and clpP genes, which encode ATP-dependent proteases, showed impaired growth at high temperatures and reduced virulence characteristics such as decreased motility, autoagglutination, and epithelial cell invasion .
Immunogenicity and Vaccine Potential
Evaluating the immunogenicity of recombinant atpE protein could determine its potential as a vaccine component. This would involve assessing antibody responses to the protein in various animal models and determining whether these responses confer protection against Campylobacter jejuni colonization or infection.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it during order placement, and we will fulfill your request.
Note: Our standard shipping method includes blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: cjd:JJD26997_0878
Q&A
What is Campylobacter jejuni subsp. doylei ATP synthase subunit c (atpE)?
Campylobacter jejuni subsp. doylei ATP synthase subunit c (atpE) is a critical component of the ATP synthase complex in this pathogenic ε-proteobacteria. It functions as part of the F₀ sector of ATP synthase, which is responsible for harnessing the energy from proton translocation across the membrane to drive ATP synthesis. This protein is also known by several alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c (short name: F-ATPase subunit c), and Lipid-binding protein . The gene coding for this protein is designated as atpE, with the ordered locus name JJD26997_0878 in the Campylobacter jejuni subsp. doylei genome .
ATP synthase is a multi-subunit enzyme complex that produces ATP from ADP and inorganic phosphate using energy derived from a transmembrane proton motive force. The complete ATP synthase consists of two major regions: the F₁ region composed of subunits α₃β₃γδε, and the F₀ region typically formed by three subunits with the stoichiometry ab₂c₁₀-₁₅ . The c-subunit (atpE) forms part of the rotor mechanism that is critical for the function of ATP synthase.
How are bacterial ATP synthases structured compared to mitochondrial versions?
Bacterial ATP synthases represent the simplest form of this enzyme and have been studied extensively due to the relative ease of genetic manipulation . While they perform the same core functions as the more complex mitochondrial ATP synthases, there are notable structural differences:
In bacterial systems like C. jejuni, the architecture of the membrane region allows the ATP synthase to perform the same core functions as the equivalent, but more complicated, mitochondrial complex . The simplicity of bacterial ATP synthases makes them excellent models for understanding the fundamental mechanisms of ATP synthesis.
What are the optimal storage and handling conditions for recombinant C. jejuni atpE?
Based on established protocols for recombinant proteins of this nature, the following storage and handling conditions are recommended for maintaining optimal activity of C. jejuni atpE:
Storage Conditions:
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Store at -20°C for regular use
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For extended storage, conserve at -20°C or -80°C
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The protein is typically supplied in a Tris-based buffer with 50% glycerol, optimized for stability
Handling Recommendations:
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Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity
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For ongoing experiments, store working aliquots at 4°C for up to one week
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Minimize exposure to room temperature during experimental procedures
These recommendations ensure the structural integrity and functional activity of the recombinant protein during storage and experimental use. Proper handling is crucial for obtaining reliable and reproducible research results when working with this protein.
How does the C. jejuni ATP synthase compare to other bacterial ATP synthases?
While the general architecture of ATP synthases is conserved across bacterial species, there are notable differences in structure and function:
| Feature | C. jejuni ATP-PRT | Other Bacterial ATP Synthases |
|---|---|---|
| Regulatory mechanism | Allosterically inhibited by histidine, which binds to a remote regulatory domain | Similar allosteric regulation in many species, but with structural variations |
| Inhibition | Competitively inhibited by AMP | Various inhibition profiles depending on species |
| Quaternary structure | Adopts hexameric quaternary structure in solution | Some bacterial ATP-PRTs show oligomeric equilibrium between different states |
| Conformational states | Two distinct hexameric conformations: open structure with ATP bound, compact closed form with histidine bound | Conformational changes vary between species |
The ATP-phosphoribosyltransferase (ATP-PRT) from C. jejuni belongs to the long form (HisGL) family and has been functionally characterized . Unlike previous hypotheses that suggested an allosteric mechanism driven by an oligomer equilibrium between hexameric and dimeric forms, the C. jejuni enzyme maintains a hexameric structure even when inhibited by histidine . This suggests that the allosteric mechanism involves conformational changes within the hexamer rather than changes in oligomeric state.
What conformational changes occur in C. jejuni ATP synthase during catalysis?
ATP synthase undergoes significant conformational changes during catalysis, which are essential for its function. In C. jejuni and other bacterial ATP synthases:
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Rotational States: The enzyme adopts multiple distinct rotational states during catalysis. In Bacillus PS3 (which serves as a model system), three rotational states have been observed, with the most striking difference being the angular position of the rotor (subunits γεc₁₀) .
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Symmetry Mismatch: The structure of bacterial ATP synthase, with three αβ pairs in the F₁ region and 10 c-subunits in the F₀ region (in Bacillus PS3), results in a symmetry mismatch between the 120° steps of the F₁ motor and 36° steps of the F₀ motor .
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Step Size: The 120° steps of the F₁ motor give an average rotational step of 3.3 c-subunits, with the closest integer steps being 3, 4, and 3 c-subunits in sequence. This results in slightly uneven rotation, which may be compensated by flexibility in the enzyme structure .
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Structural Flexibility: The C-terminal water-soluble part of subunit b displays significant conformational variability between states, which may help accommodate the symmetry mismatch between F₀ and F₁ regions .
These conformational changes are critical for coupling proton translocation to ATP synthesis and represent important targets for further research into the mechanistic details of ATP synthase function.
What techniques are most effective for structural studies of C. jejuni ATP synthase?
Multiple complementary techniques have been successfully employed to elucidate the structure and function of bacterial ATP synthases, which can be applied to C. jejuni research:
| Technique | Application | Advantages |
|---|---|---|
| Cryo-electron microscopy (Cryo-EM) | Determination of 3D structure | Allows visualization of multiple conformational states without crystallization |
| X-ray crystallography | High-resolution structural determination | Provides atomic-level details of protein structure |
| Isothermal titration calorimetry (ITC) | Binding studies for inhibitors and substrates | Quantifies thermodynamic parameters of molecular interactions |
| Site-directed mutagenesis | Functional analysis of specific residues | Identifies key amino acids involved in catalysis or regulation |
| Biochemical assays | Kinetic and regulatory characterization | Determines functional parameters under various conditions |
For example, cryo-EM has been successfully used to image bacterial ATP synthases and build atomic models in different rotational states . This approach revealed how subunit ε is positioned to inhibit ATP hydrolysis while allowing ATP synthesis and provided insights into the path of proton translocation .
When studying C. jejuni ATP synthase specifically, researchers have employed a combination of structural and biochemical approaches. The ATP-PRT enzyme from C. jejuni has been characterized through crystal structures showing binding of ligands at both active and allosteric sites, complemented by solution-state observations to illuminate the allosteric mechanism .
How can researchers investigate the inhibition mechanisms of C. jejuni ATP synthase?
Understanding inhibition mechanisms is crucial for both basic research and potential therapeutic applications. For C. jejuni ATP synthase, several approaches can be employed:
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Allosteric Inhibition Studies: C. jejuni ATP-PRT is allosterically inhibited by histidine, which binds to a remote regulatory domain . Researchers can investigate this mechanism by:
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Crystallography or cryo-EM to capture inhibited states
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Mutagenesis of residues in the histidine-binding regulatory domain
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Isothermal titration calorimetry to quantify binding parameters
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Competitive Inhibition Analysis: The enzyme is competitively inhibited by AMP . Studies can focus on:
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Kinetic analyses to determine inhibition constants
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Structural studies of AMP binding
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Design of AMP analogs with enhanced inhibitory properties
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Combined Inhibition Effects: Research shows that C. jejuni ATP-PRT is more potently inhibited by a combination of AMP and histidine than by either inhibitor alone . This synergistic effect can be studied through:
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Dose-response matrices combining both inhibitors
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Structural studies of the enzyme bound to both inhibitors simultaneously
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Molecular dynamics simulations to understand cooperative binding effects
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The inhibited enzyme adopts a more compact closed form when histidine is bound, compared to an open homohexameric structure observed with substrate ATP . This structural compression provides molecular details about how the enzyme responds to inhibitory signals and can guide the design of new inhibitors.
What methodologies can elucidate the proton translocation pathway in C. jejuni ATP synthase?
The proton translocation pathway is fundamental to ATP synthase function. Based on studies of bacterial ATP synthases, researchers can employ several approaches to investigate this pathway in C. jejuni:
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High-resolution structural studies: Cryo-EM or X-ray crystallography can reveal the architecture of the membrane region and the positioning of key residues involved in proton translocation .
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Site-directed mutagenesis: Systematic mutation of conserved residues in the F₀ region, particularly in subunits a and c, can identify amino acids critical for proton translocation.
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Functional assays: Measurement of ATP synthesis rates, proton pumping, and ATP hydrolysis under various conditions can correlate structural features with functional outcomes.
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Molecular dynamics simulations: Computational approaches can model proton movement through the enzyme complex and predict the effects of mutations or inhibitors.
Studies of bacterial ATP synthases have revealed that the transmembrane proton translocation pathway involves interactions between subunit a and the c-ring . The structures provide a model for understanding decades of biochemical analyses that have interrogated the roles of specific residues in the enzyme .
How does the c-ring rotation mechanism in C. jejuni ATP synthase contribute to energy conversion efficiency?
The c-ring rotation mechanism is central to the energy conversion process in ATP synthase. Research approaches to investigate this mechanism include:
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Rotational step analysis: Studies of bacterial ATP synthases have shown that the symmetry mismatch between the F₁ and F₀ regions leads to unequal step sizes during rotation. In systems with 10 c-subunits (like Bacillus PS3), the rotational steps appear to be almost exactly 3, 4, and 3 c-subunits in sequence .
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Energy storage and transmission: The unequal number of c-subunit steps between rotational states could lead to variable rotation speed for the c-ring in the active enzyme, similar to kinetic limping in kinesin motors. Alternatively, flexibility in the enzyme could maintain constant rotational velocity .
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Torsional energy storage: Research suggests that the central stalk (subunits γ and ε in bacteria) is the main region responsible for transient storage of torsional energy in rotary ATPases . This elastic coupling is likely important for smooth energy conversion.
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Structural stability analysis: Studies of yeast ATP synthase F₀ dimers indicate that the c-ring and subunit a are held together by hydrophobic interactions rather than by the peripheral stalk . This arrangement may be similar in bacterial systems like C. jejuni and could influence the efficiency of energy conversion.
Understanding these mechanisms is critical for comprehending how C. jejuni ATP synthase achieves efficient energy conversion and may lead to insights applicable to bioenergetics research and the development of novel antimicrobials targeting this essential bacterial enzyme.
