Recombinant Clostridium novyi ATP synthase subunit c (atpE)
Description
Introduction to ATP Synthase and Subunit c
ATP synthase is a remarkable molecular machine found in the membranes of bacteria, mitochondria, and chloroplasts. This enzyme complex synthesizes adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi), utilizing the energy of a transmembrane proton gradient . The enzyme consists of two principal components: a membrane-intrinsic Fo sector and a membrane-extrinsic F1 sector .
In bacteria like Clostridium novyi, the ATP synthase complex is encoded by an operon containing multiple genes. While the specific operon structure in C. novyi is not directly documented in the available research, studies on the related organism Clostridium pasteurianum reveal an operon consisting of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ɛ) . This arrangement appears to be conserved across many bacterial species.
The functional mechanism of ATP synthase involves a rotary motor driven by proton translocation. When protons move through the interface between subunit a and the c-ring, they cause the rotor (which includes the c-ring and associated subunits) to turn . This rotation drives conformational changes in the F1 sector that catalyze ATP synthesis. Conversely, ATP hydrolysis can drive rotation in the opposite direction, resulting in proton pumping across the membrane .
Expression and Purification of Recombinant C. novyi atpE
The most common approach involves expressing the full-length C. novyi atpE protein with an N-terminal histidine (His) tag in Escherichia coli . This strategy leverages the well-established bacterial expression system while facilitating subsequent purification through affinity chromatography. Alternative expression platforms including yeast, baculovirus, and mammalian cell systems may also be employed depending on specific research requirements .
The following table summarizes the key characteristics of commercially available recombinant C. novyi atpE protein :
| Parameter | Specification |
|---|---|
| Species | Clostridium novyi |
| Source | E. coli |
| Tag | His |
| Protein Length | Full Length (1-84) |
| Form | Lyophilized powder |
| Amino Acid Sequence | MISSQAFVAGMCAIGAGLASIACIGGGIGTGNATAKAVEGVSRQPEASGKILSTMIIGSA LSEATAIYGFLIAILLVLKIGNIG |
| Purity | Greater than 90% as determined by SDS-PAGE |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Recommended Storage | -20°C/-80°C upon receipt; aliquoting necessary for multiple use |
| Reconstitution | In deionized sterile water to 0.1-1.0 mg/mL |
For optimal stability, the purified protein should be stored at -20°C or -80°C, with repeated freeze-thaw cycles avoided . Working aliquots may be maintained at 4°C for up to one week. The reconstitution of lyophilized protein typically involves dissolution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the potential addition of glycerol (5-50% final concentration) for long-term storage .
While not specifically documented for C. novyi atpE, research on c subunits from other organisms suggests that alternative strategies may also be effective. For instance, studies on chloroplast ATP synthase subunit c demonstrated successful expression as a fusion protein with maltose binding protein (MBP), followed by cleavage and purification using reversed phase chromatography . Similar approaches might be applicable to C. novyi atpE production when higher yields or specific structural studies are required.
Functional Properties and Role in ATP Synthesis
The c subunit of ATP synthase plays a fundamental role in the function of this enzymatic complex. Multiple copies of subunit c assemble to form a ring structure within the membrane-embedded Fo sector. This c-ring is directly involved in proton translocation across the membrane and couples this process to ATP synthesis.
The c-ring functions as a rotating component within the ATP synthase complex. Proton translocation at the interface between subunit a and the c-ring drives the rotation of the entire rotor assembly, which includes the c-ring and associated subunits . This rotation, in turn, induces conformational changes in the catalytic F1 sector that lead to ATP synthesis.
The efficiency of ATP synthesis is directly related to the H+:ATP ratio, which is determined by the number of c subunits in the ring and the number of catalytic sites in the F1 sector. With three catalytic sites in the F1 sector (a common feature across most ATP synthases), the synthesis of one ATP molecule typically requires the translocation of protons equal to the number of c subunits in the ring . This stoichiometric relationship has significant implications for the bioenergetics of the organism and may reflect adaptations to specific environmental conditions.
In addition to its role in ATP synthesis, the c subunit and the c-ring structure represent potential targets for regulation and inhibition. In various organisms, ATP synthase activity can be modulated through interactions with regulatory proteins or small molecules that affect the function of the c-ring or its interaction with other subunits of the complex.
Applications in Research and Potential Therapeutic Relevance
Recombinant Clostridium novyi ATP synthase subunit c (atpE) serves as a valuable tool for various research applications and may have potential therapeutic relevance.
Therapeutic and Biotechnological Applications
Recombinant C. novyi atpE has potential applications in therapeutic and biotechnological contexts. One notable application is in vaccine development, as indicated by its commercial availability for such purposes .
The ATP synthase complex and its components represent potential targets for antimicrobial interventions. Given the essential role of ATP synthase in bacterial metabolism and the structural differences between bacterial and human ATP synthases, selective targeting of bacterial ATP synthase components could provide a basis for novel antimicrobial strategies.
Of particular interest is the potential connection to cancer therapy. Certain strains of C. novyi, notably C. novyi-NT, have shown promise as anti-tumor agents . Although the direct relationship between atpE and these anti-tumor properties is not established in the available research, understanding the molecular components of C. novyi, including atpE, may contribute to the development and refinement of C. novyi-based therapeutic approaches.
Product Specs
Please note: We prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will strive to fulfill your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: cno:NT01CX_0535
STRING: 386415.NT01CX_0535
Q&A
What is the basic structure and function of ATP synthase subunit c (atpE) in Clostridium novyi?
ATP synthase subunit c (atpE) is a critical component of the F₀ portion of F₁F₀ ATP synthase, located in the cytoplasmic membrane of C. novyi. This multisubunit enzyme complex synthesizes ATP from ADP and inorganic phosphate (Pi) by utilizing the transmembrane chemiosmotic energy generated by proton or sodium gradients. The enzyme can also function in reverse, hydrolyzing ATP to generate chemiosmotic energy .
The F₀ component, which includes subunit c, is the membrane-intrinsic portion responsible for proton translocation across the membrane. Multiple c subunits form a ring structure that rotates during ATP synthesis/hydrolysis. The atpE gene in C. novyi encodes this subunit c protein, which plays a crucial role in energy conversion within the bacterial cell.
How does C. novyi ATP synthase organization compare to other bacterial species?
Based on comparative genomic analyses, the ATP synthase operon organization in Clostridium species follows the pattern seen in many bacteria. In C. pasteurianum, a related Clostridium species, the atp operon consists of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ɛ) . This organization is likely similar in C. novyi.
What distinguishes C. novyi is that unlike many other Clostridium species, C. novyi contains a homolog to the acid phosphatase of C. perfringens, suggesting potential unique regulatory mechanisms that may indirectly affect ATP synthase function . Additionally, while most ATP synthase subunits are highly conserved across species, variations in the c subunit can affect proton binding, ion specificity, and inhibitor sensitivity.
What expression systems are most effective for producing recombinant C. novyi atpE?
For heterologous expression of C. novyi atpE, several expression systems can be considered:
E. coli T7 Expression System: Similar to the approach used for C. perfringens proteins, recombinant atpE can be expressed using pET vectors in T7 E. coli expression strains . This system has been successful for expressing other clostridial proteins and would likely work for C. novyi atpE as well.
Optimization Considerations:
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Removal of N-terminal hydrophobic regions may improve soluble expression
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Addition of a C-terminal hexahistidine tag facilitates purification
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Temperature reduction during induction (16-20°C) may enhance proper folding
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Codon optimization for E. coli may be necessary due to the different codon usage between Clostridium and E. coli
The recombinant protein should be verified by DNA sequencing of the expression construct and Western blot analysis using anti-His antibodies or specific antibodies against the target protein.
What purification strategies are recommended for isolating recombinant C. novyi atpE?
Purification of recombinant C. novyi atpE requires specialized approaches due to its hydrophobic nature as a membrane protein:
Recommended Purification Protocol:
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Cell Lysis: Sonication or French press in buffer containing detergents (e.g., DDM, CHAPS)
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Initial Purification: Affinity chromatography using Ni-NTA for His-tagged protein
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Secondary Purification: Size exclusion chromatography to obtain highly purified protein
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Detergent Exchange: If functional studies are planned, exchange harsh detergents with milder ones
Buffer Considerations:
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Include glycerol (10-20%) to enhance stability
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Maintain pH around 6.0-7.5, as ATP synthase typically functions optimally in this range
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Add divalent cations (Mg²⁺) to stabilize the protein structure
The purified protein should be assessed for activity using ATPase assays with substrates such as para-nitrophenyl phosphate, similar to methods used for other ATP synthase studies .
What assays are available for measuring the activity of recombinant C. novyi atpE?
While isolated subunit c alone isn't typically used for activity assays (as it functions as part of the complete F₀F₁ complex), researchers can employ several approaches to assess ATP synthase function:
ATP Hydrolysis Activity:
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Measure release of inorganic phosphate from ATP using colorimetric methods
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Monitor para-nitrophenyl phosphate (pNPP) hydrolysis spectrophotometrically at 405nm
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Utilize 4-methylumbelliferyl phosphate (4MUP) for fluorescence-based detection
ATP Synthesis Assays:
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Reconstitute purified ATP synthase into liposomes
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Generate proton gradient using acid-base transition or ionophores
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Measure ATP production using luciferase-based luminescence assays
Data Analysis Parameters:
How do divalent cations influence C. novyi ATP synthase activity?
Divalent cations significantly affect ATP synthase function, with different effects depending on the specific ion:
Enhancing Cations:
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Mg²⁺ is typically required for optimal ATP synthase activity, forming complexes with ATP
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Mn²⁺ may substitute for Mg²⁺ with varying efficiency
Inhibitory Cations:
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High concentrations of Ca²⁺ can inhibit ATP synthase activity
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Heavy metal ions (Zn²⁺, Cu²⁺) may irreversibly inhibit the enzyme
Based on studies of related enzymes, activity assays should include 1-5 mM MgCl₂ for optimal function . The differential effects of various cations can provide insights into the catalytic mechanism and metal-binding sites within the enzyme complex.
What challenges exist in structural determination of C. novyi atpE?
The structural characterization of C. novyi atpE presents several significant challenges:
Membrane Protein Crystallization Barriers:
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Hydrophobic nature complicates protein handling and crystallization
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Detergent selection is critical for maintaining native structure
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Lipid environment requirements may necessitate lipidic cubic phase crystallization
Expression and Purification Hurdles:
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Low expression yields of membrane proteins
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Protein heterogeneity due to post-translational modifications
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Difficulty in obtaining sufficient quantities of pure, homogeneous protein
Methodological Approaches:
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Cryo-electron microscopy for structure determination without crystallization
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Homology modeling based on related ATP synthases (as demonstrated for M. tuberculosis AtpE )
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NMR spectroscopy for structural dynamics studies
Alternative Approaches: For initial structural insights, homology modeling using the Modeller9.16 platform followed by energy minimization and molecular dynamics simulation can provide a working structural model, as demonstrated for M. tuberculosis AtpE .
How can C. novyi atpE be explored as a potential drug target?
ATP synthase subunit c represents a promising target for antimicrobial development due to its essential role in bacterial energy metabolism:
Target Validation Approaches:
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Genetic studies to confirm essentiality (conditional knockdown)
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Structure-based drug design using homology models
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In silico screening against virtual libraries
Inhibitor Screening Methodology:
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Virtual screening against a 3D model structure using tools like RASPD and PyRx
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Selection of compounds with minimum binding energies
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Filtering candidates based on Lipinski's rule of five for physicochemical properties
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Molecular docking analysis to identify top candidates
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Experimental validation using enzymatic assays
Similar approaches for M. tuberculosis AtpE identified compounds with binding energies between -8.69 and -8.44 kcal/mol, which were more potent than ATP itself . These methodologies could be adapted for C. novyi atpE as a potential therapeutic target against clostridial infections.
What potential mechanisms of inhibition exist for C. novyi ATP synthase?
Several mechanisms can be exploited to inhibit ATP synthase function:
Direct c-Subunit Binding:
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Disruption of c-ring rotation
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Interference with proton binding sites
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Prevention of conformational changes
Catalytic Site Inhibition:
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Competitive inhibition at nucleotide binding sites
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Allosteric modulation affecting catalytic efficiency
Interface Disruption:
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Targeting the interface between F₁ and F₀ components
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Disrupting subunit interactions within the complex
Experimental Approaches to Study Inhibition:
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Site-directed mutagenesis to identify key residues
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Isothermal titration calorimetry for binding affinity determination
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Enzyme kinetics studies to elucidate inhibition mechanisms
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Computer modeling for rational drug design
How might C. novyi atpE structure inform understanding of antibiotic resistance mechanisms?
The structural features of ATP synthase subunit c can provide valuable insights into resistance mechanisms:
Mutation-Based Resistance Analysis:
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Identification of common mutation sites in resistant strains
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Structural mapping of resistance hotspots
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Computational prediction of resistance-conferring mutations
Comparative Studies:
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Analysis of atpE sequences from sensitive and resistant strains
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Structure-function correlations with resistance phenotypes
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Evolution of resistance mechanisms across bacterial species
Potential Applications:
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Design of novel antibiotics that overcome resistance
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Development of combination therapies targeting multiple sites
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Creation of diagnostic tools for rapid resistance detection
Understanding the structure-function relationship of C. novyi atpE could lead to the development of new antimicrobials that remain effective against resistant strains, similar to approaches being explored for M. tuberculosis AtpE as an alternative target to overcome isoniazid resistance .
What role might the unique features of C. novyi proteins play in membrane association?
Clostridium novyi possesses unique protein features that may influence membrane association and function:
LysM Domain Significance:
The putative acid phosphatase from C. novyi contains a LysM domain within its N-terminal region, making the protein 52 amino acids longer than similar enzymes in other Clostridium species . While this information pertains to another protein, it suggests C. novyi may employ unique mechanisms for protein anchoring that could potentially apply to other membrane-associated proteins like ATP synthase components.
Peptidoglycan Binding:
LysM domains are involved in peptidoglycan binding , suggesting a potentially unique form of surface anchoring. This may represent an adaptation specific to C. novyi's ecological niche or pathogenicity.
Research Implications:
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Investigation of potential unique membrane association mechanisms for ATP synthase
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Examination of protein-peptidoglycan interactions specific to C. novyi
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Exploration of how these unique features affect enzyme function and regulation
Experimental Approaches:
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Deletion studies of specific domains
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Fluorescence microscopy for localization studies
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Cross-linking experiments to identify interaction partners
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Comparative genomics across Clostridium species
