Recombinant Clostridium botulinum ATP synthase subunit c (atpE)

What is Clostridium botulinum ATP synthase subunit c (atpE) and what is its function?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the F-type ATP synthase complex in Clostridium botulinum. The protein functions as part of the c-ring structure embedded in the membrane, which is essential for proton translocation across the membrane during ATP synthesis. The recombinant full-length protein consists of 79 amino acids with the sequence MDPKAFVSGMAALGAGIAALACIGAGIGTGNATGKAVEGVSRQPEASGKIMSTLVIGSAFSEATAIYGLIIALFLIFKI .

In C. botulinum, atpE is also known by several synonyms including ATP synthase F0 sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein . The protein plays a crucial role in energy production, and alterations in its expression have been observed under various stress conditions, including heat shock .

How is recombinant C. botulinum atpE typically expressed and purified for research purposes?

Recombinant C. botulinum atpE is typically expressed in heterologous systems, with E. coli being the most common expression host. The protein can be produced with fusion tags (commonly His-tag) to facilitate purification . The general methodology includes:

• Cloning the atpE gene into an appropriate expression vector with a His-tag or other affinity tag

• Transforming the construct into E. coli expression strains

• Inducing protein expression under optimized conditions

• Cell lysis and initial clarification of the lysate

• Affinity chromatography using the fusion tag for purification

• Additional purification steps such as size exclusion or ion exchange chromatography if needed

• Confirmation of purity by SDS-PAGE (>90% purity is typically achievable)

The purified protein is often supplied as a lyophilized powder and requires proper reconstitution before use in experimental applications .

What are the optimal storage conditions for recombinant C. botulinum atpE protein?

Based on product specifications, the following storage guidelines are recommended for maintaining recombinant C. botulinum atpE protein stability and functionality:

These storage conditions are critical for maintaining protein integrity and bioactivity for research applications.

What protocols are recommended for reconstituting lyophilized recombinant C. botulinum atpE protein?

Proper reconstitution of lyophilized recombinant C. botulinum atpE protein is crucial for downstream applications. The following step-by-step protocol is recommended:

• Briefly centrifuge the vial containing lyophilized protein to ensure the contents are at the bottom

• Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Gently mix the solution to ensure complete dissolution

• For long-term storage of reconstituted protein, add glycerol to a final concentration of 5-50% (50% is commonly recommended)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store aliquots at -20°C/-80°C for long-term storage

This methodology helps maintain protein stability and functionality after reconstitution, which is essential for obtaining reliable experimental results.

How can researchers incorporate recombinant C. botulinum atpE into functional studies of ATP synthase?

To incorporate recombinant C. botulinum atpE into functional studies, researchers can employ several methodological approaches:

• Liposome reconstitution: The purified atpE can be reconstituted into liposomes with appropriate lipid composition to study its proton channel activity. This requires:

  • Preparation of liposomes with defined lipid composition

  • Incorporation of purified atpE protein using detergent removal methods

  • Verification of successful incorporation by proteoliposome characterization

  • Measurement of proton flux using pH-sensitive dyes or electrodes

• In vitro ATP synthase assembly: Researchers can attempt to reconstitute the complete ATP synthase complex by combining purified atpE with other ATP synthase subunits. The c-ring formed by atpE subunits interacts with the a-subunit through half-channels, which is critical for proton translocation during ATP synthesis .

• Structure-function studies: Site-directed mutagenesis of key residues in the atpE sequence can help elucidate the functional importance of specific amino acids in proton translocation and c-ring assembly.

These approaches provide valuable insights into the functional characteristics of C. botulinum atpE within the context of ATP synthesis mechanisms.

How does the expression of atpE in C. botulinum change under different stress conditions?

Research on gene expression in C. botulinum under stress conditions has revealed important insights about atpE regulation. During heat shock stress (temperature shift from 37°C to 45°C), several genes involved in energy production and conversion, including ATP synthase components, show altered expression patterns.

Specifically, CBO0155 (atpG, ATP synthase subunit gamma) was downregulated under heat shock stress . While the search results don't directly mention atpE regulation under heat shock, the downregulation of atpG suggests that ATP synthesis as a whole may be reduced during heat stress, potentially as an energy conservation mechanism.

This downregulation of ATP synthase components aligns with the broader observation that heat shock stress leads to reduced energy-requiring processes in C. botulinum ATCC 3502, which may represent a survival strategy during stress conditions . Researchers studying atpE regulation should consider these patterns when designing experiments involving stress conditions.

What expression systems yield the most functional recombinant C. botulinum atpE protein?

Several expression systems can be used for producing recombinant C. botulinum atpE, each with advantages for different research applications:

E. coli is the most commonly used system for expressing recombinant C. botulinum atpE . When expressing membrane proteins like atpE, specific E. coli strains designed for membrane protein expression (such as C41(DE3) or C43(DE3)) may improve yield and functionality. The addition of fusion tags, particularly His-tags, facilitates purification while typically maintaining protein function.

How does the c-ring stoichiometry in C. botulinum ATP synthase compare to other bacterial species?

The c-ring stoichiometry (number of c-subunits per ring) is a critical parameter that determines the H⁺/ATP ratio in ATP synthase. According to the half-channel model, protons from the periplasmic space enter through one half-channel of the a-subunit and are transferred to a c-subunit in the c-ring. After one complete revolution of the c-ring, the proton is released into the cytoplasmic solution via the opposite half-channel .

The stoichiometry of H⁺/turn is determined by the number of c-subunits in the c-ring. While the exact stoichiometry for C. botulinum has not been explicitly stated in the provided search results, this is an active area of research for ATP synthases across species.

Comparative data on c-ring stoichiometry across bacterial species:

The engineering of ATP synthase to enhance the proton-to-ATP ratio demonstrates the importance of understanding c-ring stoichiometry for bioenergetic studies . Researchers interested in C. botulinum ATP synthase function would need to experimentally determine this parameter, potentially using cryo-electron microscopy or other structural biology techniques.

What structural features of C. botulinum atpE contribute to its function in ATP synthase?

The C. botulinum atpE protein contains several structural features that are critical for its function in ATP synthase:

• Transmembrane helices: The amino acid sequence (MDPKAFVSGMAALGAGIAALACIGAGIGTGNATGKAVEGVSRQPEASGKIMSTLVIGSAFSEATAIYGLIIALFLIFKI) suggests a highly hydrophobic protein typical of membrane-spanning segments . These transmembrane regions allow the protein to embed in the membrane and form the c-ring structure.

• Proton-binding site: While not explicitly described in the search results for C. botulinum, c-subunits typically contain a conserved carboxylate residue (aspartate or glutamate) that is essential for proton binding and translocation. This site undergoes protonation/deprotonation cycles during ATP synthesis.

• Oligomerization interfaces: The c-subunits assemble into a ring structure through specific protein-protein interactions. The interfaces between adjacent subunits are critical for proper assembly and function.

• Interaction surfaces with other subunits: The c-ring interacts with both the a-subunit (for proton translocation) and the γ and ε subunits (for mechanical coupling to the F1 sector). These interaction surfaces are essential for the coordinated function of the entire ATP synthase complex.

Understanding these structural features is crucial for researchers investigating the mechanism of ATP synthesis and for potentially developing targeted approaches that could affect C. botulinum bioenergetics.

What methodologies are most effective for studying interactions between atpE and other ATP synthase subunits?

Several advanced methodologies can be employed to study the interactions between atpE and other ATP synthase subunits:

• Crosslinking studies: Chemical crosslinking combined with mass spectrometry can identify specific interaction sites between atpE and other subunits, particularly the a-subunit and the γ/ε subunits.

• Cryo-electron microscopy: This technique has revolutionized the structural biology of membrane protein complexes and can provide high-resolution structures of the entire ATP synthase complex, revealing the detailed interactions between subunits.

• FRET (Förster Resonance Energy Transfer): By labeling different subunits with appropriate fluorophores, researchers can monitor subunit interactions and conformational changes in real-time.

• Molecular dynamics simulations: Computational approaches can model the dynamic interactions between atpE and other subunits, providing insights into the mechanism of proton translocation and rotary catalysis.

• Reconstitution studies: Purified recombinant subunits can be combined in defined ratios to reconstitute partial or complete ATP synthase complexes, allowing the study of specific interactions and their functional consequences.

• Site-directed mutagenesis combined with functional assays: Specific residues at presumed interaction surfaces can be mutated, and the effects on ATP synthase assembly and function can be assessed through various assays.

These methodologies provide complementary information about subunit interactions and can be combined to develop a comprehensive understanding of ATP synthase function in C. botulinum.

How can research on C. botulinum atpE contribute to understanding bacterial bioenergetics?

Research on C. botulinum atpE can provide valuable insights into bacterial bioenergetics for several reasons:

• Comparative bioenergetics: Studying ATP synthase from different bacterial species, including pathogens like C. botulinum, allows researchers to identify common principles and species-specific adaptations in energy conservation mechanisms.

• Environmental adaptation: Understanding how C. botulinum regulates ATP synthase expression and activity under different environmental conditions (like heat stress) provides insights into bacterial adaptation strategies . The downregulation of ATP synthase components under heat stress suggests energy conservation as a survival mechanism.

• Structure-function relationships: Detailed structural and functional studies of C. botulinum atpE can contribute to our understanding of the fundamental mechanisms of rotary catalysis and proton translocation in ATP synthases.

• Evolution of bioenergetic systems: Comparative analysis of ATP synthase components across bacterial species can illuminate the evolutionary history of these essential molecular machines.

These contributions extend beyond C. botulinum biology and can inform broader questions in bacterial physiology, evolution, and potentially antimicrobial development.

What are the challenges in expressing and purifying functional C. botulinum atpE for structural studies?

Researchers face several challenges when expressing and purifying C. botulinum atpE for structural studies:

• Membrane protein expression: As a hydrophobic membrane protein, atpE can be difficult to express in soluble form. Aggregation and inclusion body formation are common issues that require optimization of expression conditions.

• Maintaining native conformation: Extracting atpE from membranes while preserving its native conformation requires careful selection of detergents and solubilization conditions.

• Oligomeric state preservation: The c-subunits naturally form a ring structure in ATP synthase. Preserving this oligomeric state during purification is challenging but essential for functional and structural studies.

• Reconstitution for functional studies: After purification, reconstituting atpE into lipid environments that support its native function requires optimization of lipid composition and reconstitution procedures.

• Stability during structural analysis: Maintaining protein stability during the timeframes required for structural studies (crystallography, cryo-EM) is particularly challenging for membrane proteins.

To address these challenges, researchers can employ specialized expression systems, fusion partners that enhance solubility, and careful optimization of purification and reconstitution protocols specific to membrane proteins.

How might understanding atpE function contribute to potential biotechnological applications?

Research on C. botulinum atpE could lead to several biotechnological applications:

• Bioenergetic engineering: Understanding the relationship between c-ring stoichiometry and ATP synthase efficiency could inform efforts to engineer ATP synthases with enhanced proton-to-ATP ratios for biotechnological applications .

• Membrane protein expression platforms: Methods developed for expressing and purifying functional atpE could be applied to other challenging membrane proteins of biotechnological interest.

• Biosensors: ATP synthase components, including atpE, could potentially be incorporated into biosensors for detecting compounds that affect membrane potential or proton gradients.

• Model systems for drug development: While not a direct target for antibiotics against C. botulinum, understanding ATP synthase function provides insights into bacterial energy metabolism that could inform novel antimicrobial strategies.

• Synthetic biology applications: Engineered ATP synthases with modified properties could be incorporated into synthetic biological systems designed for specific energy conversion tasks.

These potential applications highlight the broader significance of fundamental research on ATP synthase components like atpE beyond their specific role in C. botulinum biology.

How does the genomic context of the atpE gene in C. botulinum compare across different strains?

Whole genome sequencing analysis of different C. botulinum strains provides insights into genomic variation and conservation. While the search results don't specifically address the genomic context of atpE across strains, the methodology for such comparative analysis is well-established through studies of other genes in C. botulinum .

For comprehensive analysis of atpE across C. botulinum strains, researchers would typically:

• Identify the atpE gene location in reference genomes (such as C. botulinum ATCC 3502)

• Examine synteny (conservation of gene order) in the ATP synthase operon across strains

• Calculate nucleotide and amino acid sequence conservation using tools like average nucleotide identity (ANI) analysis

• Identify single nucleotide polymorphisms (SNPs) that might affect protein function

• Construct phylogenetic trees based on atpE sequences to understand evolutionary relationships

The high-quality SNP (hqSNP) analysis approach demonstrated for botulinum neurotoxin genes could be applied to atpE to identify strain-specific variations that might affect ATP synthase function or regulation.

How does recombinant C. botulinum atpE protein differ from native atpE in structure and function?

When comparing recombinant and native C. botulinum atpE, several differences may arise that researchers should consider:

To address these differences, researchers often employ strategies such as:

• Removing fusion tags after purification when possible

• Reconstituting in lipid compositions that mimic native membranes

• Co-expressing with partner subunits to facilitate proper assembly

• Validating results with native protein when feasible

Understanding these differences is crucial for interpreting results from studies using recombinant atpE and extrapolating to the native protein's behavior in C. botulinum.

What are common challenges in reconstituting active recombinant C. botulinum atpE and how can they be addressed?

Researchers working with recombinant C. botulinum atpE may encounter several technical challenges during reconstitution and activity assessment:

• Protein aggregation after reconstitution:

  • Solution: Optimize buffer conditions (pH, salt concentration), add stabilizing agents like glycerol (5-50%) , and ensure slow, controlled rehydration of lyophilized protein.

• Loss of activity during freeze-thaw cycles:

  • Solution: Avoid repeated freeze-thaw cycles by preparing single-use aliquots and storing at appropriate temperatures (-20°C/-80°C for long-term, 4°C for working aliquots up to one week) .

• Difficulty in verifying functional reconstitution:

  • Solution: Develop and apply functional assays specific to atpE, such as proton translocation assays in reconstituted liposomes or monitoring assembly into c-ring structures by native PAGE.

• Incomplete solubilization:

  • Solution: Centrifuge briefly before opening the vial to bring all contents to the bottom, and ensure complete dissolution in reconstitution buffer before use .

• Protein instability in experimental conditions:

  • Solution: Add stabilizers appropriate for the specific experimental conditions, monitor protein stability throughout experiments, and optimize buffer composition.

These technical considerations are critical for ensuring that experiments with recombinant C. botulinum atpE yield reliable and reproducible results.

How can researchers verify the structural integrity of purified recombinant C. botulinum atpE?

Multiple analytical techniques can be employed to verify the structural integrity of purified recombinant C. botulinum atpE:

• SDS-PAGE analysis: Confirms protein purity (>90% as specified in product information) and approximate molecular weight.

• Circular dichroism (CD) spectroscopy: Assesses secondary structure content (primarily alpha-helical for atpE) and can detect major conformational changes.

• Size exclusion chromatography (SEC): Evaluates oligomeric state and detects aggregation.

• Mass spectrometry: Confirms exact molecular weight and can identify post-translational modifications or degradation products.

• Limited proteolysis: Properly folded proteins often show characteristic proteolytic patterns different from misfolded versions.

• Thermal shift assays: Measure protein stability and can be used to optimize buffer conditions.

• Functional assays: While technically challenging for membrane proteins like atpE, assays that measure specific functional properties provide the most relevant validation of structural integrity.

For membrane proteins like atpE, additional techniques may include:

• Native PAGE in the presence of mild detergents: Can assess c-ring assembly

• Electron microscopy: Can visualize c-ring formation with negative staining or cryo-EM

Combining multiple orthogonal techniques provides the most comprehensive assessment of structural integrity for recombinant C. botulinum atpE.

How might systems biology approaches enhance our understanding of C. botulinum atpE in cellular energetics?

Systems biology approaches offer powerful tools for understanding C. botulinum atpE within the broader context of cellular energetics:

These approaches could help address fundamental questions about how C. botulinum coordinates energy production with other cellular processes, particularly under stress conditions or during different growth phases.

What are the implications of C. botulinum atpE research for understanding other pathogenic clostridia?

Research on C. botulinum atpE has broader implications for understanding energy metabolism in other pathogenic clostridia:

• Comparative bioenergetics: Insights from C. botulinum atpE structure and function can inform studies of ATP synthase in related pathogens like C. difficile, C. tetani, and C. perfringens.

• Stress response mechanisms: The observed downregulation of ATP synthase components during heat stress in C. botulinum suggests a conserved energy conservation strategy that may be present in other clostridia.

• Evolutionary adaptations: Comparing atpE sequences and ATP synthase organization across clostridia can reveal how these organisms have adapted their energy metabolism to different ecological niches.

• Metabolic regulation during pathogenesis: Understanding how ATP synthase activity is regulated could provide insights into energy metabolism during infection and toxin production in various clostridial pathogens.

The genus Clostridium contains approximately 100 species, including both free-living bacteria and important pathogens . Comparative studies of ATP synthase across these species could reveal both conserved features essential to all clostridia and specialized adaptations in pathogenic species.