Recombinant Clostridium perfringens ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Clostridium perfringens and what is its function?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F-type ATP synthase in Clostridium perfringens. It forms part of the membrane-embedded Fo sector of ATP synthase, specifically contributing to the formation of the c-ring structure. This ring functions as a proton-translocating rotor that converts the energy from proton flow across the membrane into mechanical energy, ultimately driving ATP synthesis through the F1 sector of the enzyme complex .

The c-subunit is alternatively known as ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, or Lipid-binding protein. In C. perfringens, the full-length protein consists of 72 amino acids with the sequence: MDMKLLAAGIAVLAGIGAGIGIGIATAGALEATARQPEASDKIQSLFIMGAGLSEATAIYGLVVSIILLFVA .

How is the atpE gene organized within the ATP synthase operon of Clostridium species?

In Clostridium species, the ATP synthase genes are organized in an operon structure similar to that found in many other bacteria. Based on studies of the related organism C. pasteurianum, the ATP operon consists of nine genes arranged in the sequence: atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ɛ) .

The atpE gene, which encodes the c subunit, is positioned as the third gene in this operon. This organization allows for coordinated expression of all ATP synthase components. Transcription analysis using reverse transcription-PCR has confirmed the presence of transcripts for all nine genes in the operon .

What is the stoichiometry of c-subunits in ATP synthase complexes and why is it significant?

The stoichiometry of c-subunits in ATP synthase complexes varies among different organisms, with rings containing anywhere from 10 to 15 c-subunits reported in various species . This variation is significant because it directly affects the bioenergetics of ATP synthesis.

The number of c-subunits per ring (n) determines the H+/ATP coupling ratio, which ranges from 3.3 to 5.0 in characterized organisms. This ratio represents the number of protons that must be translocated to synthesize one ATP molecule. The relationship can be expressed as:

H+/ATP ratio = n/3

where n is the number of c-subunits and 3 represents the constant number of ATP molecules generated per complete rotation of the c-ring .

While the specific stoichiometry for C. perfringens has not been definitively established in the provided literature, understanding this parameter is crucial for characterizing the energetic efficiency of the organism's ATP synthase.

What expression systems are most effective for recombinant production of C. perfringens ATP synthase subunit c?

Based on approaches used for similar proteins, Escherichia coli expression systems have proven most effective for recombinant production of ATP synthase subunit c. Several vector systems can be employed, with the choice depending on specific research requirements:

• pMAL-c2x system (New England Biolabs): This system fuses the target protein to maltose-binding protein (MBP), which enhances solubility and provides an affinity tag for purification .

• pET-32a(+) system (Novagen): This system allows for high-level expression and includes options for adding thioredoxin fusion tags that can improve protein folding .

• pFLAG-MAC system (Sigma-Aldrich): This system incorporates a FLAG epitope tag for antibody detection and purification .

For optimal expression, parameters that should be optimized include:

• E. coli strain selection (BL21(DE3) or derivatives often preferred)

• Growth temperature (typically 37°C for initial growth, potentially lowered to 16-30°C after induction)

• IPTG concentration for induction (0.5-1.0 mM is standard)

• Post-induction incubation time (30 minutes to several hours)

What purification strategies work best for recombinant C. perfringens ATP synthase subunit c?

Purification of recombinant C. perfringens ATP synthase subunit c requires specialized approaches due to its hydrophobic nature as a membrane protein. Effective strategies include:

• Affinity chromatography: Using fusion tags such as His-tag, MBP, or FLAG tags to facilitate selective binding to affinity resins.

• Size exclusion chromatography: For further purification based on molecular size, TSK gel G3000SW or similar matrix can be used, employing fast protein liquid chromatography (FPLC) systems .

• Buffer optimization: The protein requires specific buffer conditions for stability:

  • Tris-based buffers (typically 20 mM Tris-HCl, pH 8.0)

  • High glycerol content (50%)

  • Protease inhibitor cocktails during extraction

• Storage conditions: Store at -20°C for short-term use, or -80°C for extended storage. Working aliquots can be maintained at 4°C for up to one week. Repeated freeze-thaw cycles should be avoided .

For membrane protein extraction, specialized lysis buffers containing detergents may be necessary to solubilize the protein from the membrane fraction.

How can researchers verify the correct folding and functionality of recombinant ATP synthase subunit c?

Verification of correct folding and functionality of recombinant ATP synthase subunit c can be accomplished through several complementary approaches:

• Secondary structure analysis: Circular dichroism (CD) spectroscopy can confirm the expected alpha-helical structure that characterizes correctly folded c-subunits .

• SDS-PAGE and immunoblotting: 12% polyacrylamide gels with subsequent immunoblotting using antibodies specific to c-subunit can verify expression and approximate molecular weight. Native ATP synthase from appropriate sources (e.g., spinach chloroplasts) can serve as positive controls .

• Functional reconstitution: The ultimate test of functionality involves reconstituting the c-subunit into proteoliposomes with other ATP synthase components and assessing ATP synthesis or hydrolysis activity. For ATP hydrolysis assays, the protein should display characteristic responses to activators and inhibitors similar to those observed in native ATP synthase complexes .

• Mass spectrometry: To confirm the exact molecular mass and identify any post-translational modifications.

How can recombinant C. perfringens ATP synthase subunit c be used to study antimicrobial targets?

Recombinant C. perfringens ATP synthase subunit c serves as a valuable tool for studying potential antimicrobial targets through several experimental approaches:

• Inhibitor screening assays: The purified recombinant protein can be used to screen for novel inhibitors that specifically target C. perfringens ATP synthase. This can be accomplished through:

  • Competitive binding assays with known inhibitors such as dicyclohexylcarbodiimide (DCCD)

  • ATP hydrolysis inhibition assays

  • In vitro reconstitution systems to measure proton translocation inhibition

• Structure-function studies: Site-directed mutagenesis of conserved residues in the recombinant protein can identify critical amino acids involved in function, providing insights for rational drug design .

• Comparative analysis: By comparing the properties of C. perfringens ATP synthase subunit c with those from other organisms, researchers can identify unique features that could be exploited for species-specific targeting .

• Immunological approaches: The recombinant protein can be used to raise antibodies for studying localization, expression levels, and potential immunotherapy approaches targeting this bacterial component.

Given that C. perfringens is a pathogenic organism causing gas gangrene and enterotoxaemia, developing inhibitors specific to its ATP synthase could lead to novel antimicrobial strategies with reduced effects on human ATP synthase .

What mutagenesis approaches are most informative for studying c-subunit structure-function relationships?

Several mutagenesis approaches provide valuable insights into the structure-function relationships of ATP synthase c-subunit:

• Alanine scanning mutagenesis: Systematic replacement of residues with alanine to identify amino acids essential for function, particularly those involved in proton binding and translocation. Key targets include conserved acidic residues like glutamate that are typically essential for proton binding .

• Conservative substitutions: Replacing amino acids with chemically similar residues (e.g., glutamate to aspartate, arginine to lysine) to determine the structural constraints on function. These substitutions can reveal subtle aspects of the protein's mechanism, as demonstrated in studies where replacement of key residues resulted in measurable changes in activity without complete loss of function .

• Cross-linking studies: Introduction of cysteine residues at specific positions allows for disulfide cross-linking experiments that can provide information about proximity relationships between residues and subunits in the assembled complex.

• Chimeric protein construction: Creating fusion proteins between c-subunits from different species can help identify species-specific domains that contribute to functional differences.

How can researchers study the assembly of c-subunits into the functional c-ring?

• In vitro reconstitution: Purified recombinant c-subunits can be reconstituted into liposomes under controlled conditions to study spontaneous assembly into c-rings. This approach allows for manipulation of lipid composition, pH, and ionic strength to determine factors influencing assembly .

• Cross-linking experiments: Chemical cross-linking of assembled complexes followed by mass spectrometry analysis can provide information about subunit arrangement and stoichiometry.

• Atomic force microscopy (AFM): This technique allows visualization of membrane protein complexes in near-native conditions and can provide information about c-ring diameter, which correlates with subunit stoichiometry.

• Native gel electrophoresis: Blue native PAGE can be used to analyze intact membrane protein complexes and estimate the size of assembled c-rings.

• Electron microscopy: Negative staining and cryo-EM approaches can provide structural information about assembled c-rings, including subunit arrangement and stoichiometry.

These methods complement each other and can collectively provide a comprehensive understanding of c-ring assembly dynamics and structural features .

How do regulatory factors affect the expression and assembly of ATP synthase c-subunits in C. perfringens?

The regulation of ATP synthase c-subunit expression and assembly in C. perfringens involves multiple factors that must be considered in research:

• Transcriptional regulation: As part of the ATP operon, expression of the atpE gene is likely coordinated with other ATP synthase components. Regulatory elements may include:

  • Promoter sequences upstream of the operon

  • Transcription factors responding to energy status

  • Environmental sensing mechanisms specific to anaerobic conditions

• Post-transcriptional regulation: mRNA stability and translation efficiency may be regulated by:

  • RNA secondary structures

  • Small RNAs

  • RNA-binding proteins

• Environmental factors influencing expression:

  • Oxygen levels (C. perfringens is an obligate anaerobe)

  • pH conditions

  • Nutrient availability

  • Growth phase

• Assembly factors: Specific proteins may assist in the correct folding and assembly of c-subunits into the c-ring structure. These factors remain largely uncharacterized in C. perfringens but are crucial targets for future research .

• Lipid environment effects: The composition of the membrane lipid environment significantly affects c-ring assembly and stability. Variations in lipid composition between different growth conditions may influence assembly efficiency and resulting ring stoichiometry .

Understanding these regulatory mechanisms could provide insights into how C. perfringens adapts its energy metabolism to different environmental conditions during infection and colonization .

How do the properties of C. perfringens ATP synthase c-subunit differ from those in other bacterial species?

Comparative analysis reveals several notable differences between C. perfringens ATP synthase c-subunit and those from other bacterial species:

These differences represent important areas for future research and may provide insights into the adaptation of energy metabolism to different ecological niches.

What are the current technical challenges in structural studies of ATP synthase c-rings and how can they be addressed?

Structural studies of ATP synthase c-rings face several technical challenges that researchers must address:

• Membrane protein crystallization barriers:

  • The hydrophobic nature of c-rings makes them difficult to crystallize for X-ray crystallography

  • Solution: Utilize lipidic cubic phase crystallization methods or develop fusion constructs with crystallization chaperones to enhance crystal formation

• Maintaining native structure during purification:

  • Detergent extraction can disrupt the native structure of membrane protein complexes

  • Solution: Employ gentle extraction using nanodisc technology or styrene maleic acid lipid particles (SMALPs) that preserve the native lipid environment

• Stoichiometry determination challenges:

  • Various analytical methods often yield conflicting results for c-ring stoichiometry

  • Solution: Combine multiple complementary techniques such as mass spectrometry, atomic force microscopy, and cryo-EM to build consensus data

• Expression and purification yield limitations:

  • Membrane proteins typically express at lower levels than soluble proteins

  • Solution: Optimize expression systems through fusion partners (such as MBP), codon optimization, and specialized E. coli strains designed for membrane protein expression

• Functional reconstitution complexity:

  • Demonstrating functionality of purified components requires complex reconstitution systems

  • Solution: Develop simplified in vitro assay systems that can monitor specific aspects of c-ring function, such as proton translocation or rotation

Addressing these challenges requires interdisciplinary approaches combining molecular biology, biochemistry, and advanced structural biology techniques .

How can researchers investigate the interactions between ATP synthase c-subunit and other components of the ATP synthase complex?

Investigating the interactions between ATP synthase c-subunit and other components of the complex requires specialized approaches:

• Co-immunoprecipitation studies:

  • Using antibodies against the c-subunit or other ATP synthase components to pull down interaction partners

  • Western blotting can identify co-precipitated proteins, confirming interactions

• Crosslinking coupled with mass spectrometry:

  • Chemical crosslinking agents can capture transient protein-protein interactions

  • Subsequent mass spectrometry analysis can identify crosslinked peptides, providing detailed information about interaction interfaces

• FRET (Förster Resonance Energy Transfer) analysis:

  • Fluorescently labeled components can reveal proximity and dynamics of interactions

  • This approach is particularly valuable for studying conformational changes during function

• Hybrid complex formation:

  • Mixing components from different species can reveal compatibility and critical interaction sites

• Site-directed mutagenesis of interface residues:

  • Targeted mutations at predicted interaction sites can disrupt specific protein-protein contacts

  • Functional assays following mutation can reveal the importance of specific residues

• Cryo-electron microscopy:

  • Recent advances in cryo-EM technology allow visualization of membrane protein complexes at near-atomic resolution

  • This can reveal the structural basis of c-subunit interactions with other ATP synthase components

These approaches collectively provide a comprehensive understanding of how the c-subunit integrates into the larger ATP synthase complex to enable energy conversion .

What are the most promising future research directions involving recombinant C. perfringens ATP synthase subunit c?

Several promising research directions involving recombinant C. perfringens ATP synthase subunit c warrant further investigation:

• Structural determination of the complete C. perfringens ATP synthase complex:

  • Using cryo-EM to resolve the structure at high resolution

  • Determining the c-ring stoichiometry and its implications for bioenergetics

  • Comparing structural features with ATP synthases from other organisms

• Development of specific inhibitors as potential antimicrobials:

  • Using the recombinant protein for high-throughput screening of inhibitor compounds

  • Structure-based drug design targeting unique features of C. perfringens ATP synthase

  • In vivo testing of candidate molecules in infection models

• Investigation of c-ring assembly mechanisms:

  • Factors influencing c-subunit oligomerization and c-ring formation

  • Role of lipid environment in determining ring stoichiometry

  • Identification of assembly chaperones or accessory proteins

• Adaptation of ATP synthase to anaerobic lifestyle:

  • Comparative studies between aerobic and anaerobic organisms' ATP synthases

  • Regulatory mechanisms specific to anaerobic energy conversion

  • Evolution of ATP synthase components in response to metabolic constraints

• Synthetic biology applications:

  • Engineering c-subunits with altered properties for biotechnological applications

  • Creating hybrid ATP synthases with novel functions

  • Developing energy-harvesting biohybrid systems based on ATP synthase components