Recombinant Escherichia coli O139:H28 ATP synthase subunit c (atpE)

What is the structural organization of ATP synthase subunit c in E. coli?

ATP synthase subunit c in E. coli, encoded by the atpE gene, forms a hairpin structure comprising two membrane-spanning α-helices connected by a hydrophilic loop at the cytoplasmic side of the membrane. The C-terminal α helix carries an essential ion carrier (typically Asp or Glu) that is critical for function. Subunit c molecules oligomerize to form a ring structure (C-ring) with a variable stoichiometry of 8-15 subunits depending on the species . This C-ring interacts with subunit a at its periphery, forming the a-c interface that is essential for proton translocation and rotational catalysis in ATP synthesis.

How does the function of atpE in E. coli O139:H28 compare to other bacterial species?

While the search results don't provide specific information about E. coli O139:H28 atpE function compared to other bacterial species, general principles can be inferred. ATP synthase subunit c is functionally conserved across bacterial species, though with some amino acid sequence variation. The essential glutamate residue (corresponding to Glu61 in M. tuberculosis and potentially at a similar position in E. coli) provides the main stabilizing interaction for H⁺/Na⁺ ions during proton translocation . Other conserved residues also play roles in ion binding and C-ring stability. The functional conservation despite sequence variation suggests evolutionary pressure to maintain core mechanisms while allowing species-specific adaptations.

What expression systems are commonly used for recombinant production of ATP synthase subunit c?

For recombinant production of ATP synthase subunits including subunit c, E. coli expression systems are commonly employed. Based on the search results, methodologies might include:

• PCR amplification of the target gene using specific primers (similar to atpBS and atpFAS primers mentioned for other ATP synthase components)

• Cloning into expression vectors compatible with E. coli (similar to pMOSBlue vector followed by subcloning)

• Transformation into appropriate E. coli strains for protein expression

For higher yield and purity, researchers may employ controlled induction systems and optimization of growth conditions, followed by appropriate purification strategies considering the hydrophobic nature of subunit c.

What are the typical yields for recombinant ATP synthase subunit c expression in E. coli?

Based on analogous recombinant protein expression systems, typical yields for membrane proteins like ATP synthase subunit c would be expected to vary based on expression conditions and purification methods. While specific yields for E. coli O139:H28 ATP synthase subunit c are not provided in the search results, recombinant expression platforms for other proteins have achieved yields of approximately 5 mg/L . Membrane proteins like subunit c often express at lower levels than soluble proteins due to potential toxicity and challenges with membrane insertion.

Optimizing expression parameters including temperature, induction timing, and media composition can significantly impact yield. Using specialized E. coli strains designed for membrane protein expression may further improve production levels.

What purification challenges are specific to ATP synthase subunit c?

Purification of ATP synthase subunit c presents several challenges due to its properties:

• Membrane integration: As a highly hydrophobic membrane protein, subunit c requires detergents or other membrane-mimicking environments for extraction and stability

• Oligomeric state: The tendency to form C-rings can complicate purification of individual subunits

• Small size: At approximately 8 kDa, subunit c can be difficult to separate from other small proteins

• Maintaining native conformation: Ensuring the purified protein retains its functional structure

Successful purification strategies typically employ affinity tags (His-tag, GST), detergent optimization, and specialized chromatography methods. Size exclusion chromatography may help separate individual subunits from oligomeric forms.

How do mutations in the atpE gene affect ATP synthase function and bacterial physiology?

Mutations in the atpE gene can significantly impact ATP synthase function and bacterial physiology. Studies on mycobacterial ATP synthase provide insight into how mutations might affect function. For example:

• Mutations at the proton-binding site (e.g., Glu61→Asp in M. tuberculosis) alter proton binding efficiency and can affect the protonation/deprotonation cycle critical for ATP synthesis

• Mutations in residues involved in subunit interactions (such as Asp28, Leu59, Ala63, and Ile66 in M. tuberculosis) can disrupt the structure of the C-ring or its interaction with other subunits

• Some mutations confer resistance to ATP synthase inhibitors like TMC207 (bedaquiline) by altering drug binding sites without completely abolishing enzymatic function

These mutations can lead to changes in membrane potential, energy metabolism, and growth rate. The specific physiological impact depends on the nature and location of the mutation within the protein structure.

Table 1: Examples of atpE Mutations and Their Effects Based on Mycobacterial Studies

What techniques are most effective for studying protein-protein interactions involving ATP synthase subunit c?

Several techniques are effective for studying protein-protein interactions involving ATP synthase subunit c:

• Cross-linking studies: Chemical cross-linking coupled with mass spectrometry can identify residues in close proximity between subunit c and other ATP synthase components

• Co-immunoprecipitation: Using recombinant antibody approaches similar to those described in the search results for other proteins

• FRET (Förster Resonance Energy Transfer): For analyzing dynamic interactions in reconstituted systems

• Cryo-electron microscopy: For structural determination of the complete ATP synthase complex, revealing the arrangement of subunit c in relation to other subunits

• Molecular dynamics simulations: Based on homology modeling, as mentioned for mycobacterial ATP synthase , to predict interactions and conformational changes

• Two-hybrid systems: Modified for membrane proteins to detect specific interaction partners

These approaches can be combined to provide complementary insights into how subunit c interacts with other components of the ATP synthase complex and potentially with regulatory proteins.

How can recombinant atpE be used to study the mechanism of proton translocation in ATP synthase?

Recombinant atpE can be employed in multiple experimental approaches to study proton translocation mechanisms:

• Site-directed mutagenesis: Systematically altering key residues (such as the conserved Glu corresponding to Glu61 in M. tuberculosis) to evaluate their role in proton binding and translocation

• Reconstitution studies: Incorporating purified recombinant subunit c into liposomes or nanodiscs with other ATP synthase components to measure proton pumping activity

• Structural studies: Using purified recombinant protein for X-ray crystallography or cryo-EM to resolve structural details at atomic resolution

• Isotope labeling: Incorporating specific isotopes to track proton movement using NMR or other spectroscopic methods

• Biophysical measurements: Employing techniques like surface plasmon resonance or microscale thermophoresis to measure binding affinities for protons under varying conditions

These approaches can illuminate how the C-ring facilitates proton movement across the membrane and couples this process to ATP synthesis.

What biosafety considerations should be addressed when working with recombinant E. coli expressing ATP synthase components?

When working with recombinant E. coli expressing ATP synthase components, several biosafety considerations should be addressed:

• Risk assessment: While ATP synthase itself is not typically associated with pathogenicity, the host strain characteristics (particularly for O139:H28) should be evaluated for potential virulence factors or toxin production

• Containment level: Work should be conducted under appropriate biosafety level conditions, typically BSL-1 for non-pathogenic E. coli strains, but potentially higher if the O139:H28 strain has pathogenic potential

• Handling of plasmids: Proper management of recombinant plasmids to prevent unintended horizontal gene transfer, particularly if they contain antibiotic resistance markers

• Waste decontamination: Proper sterilization and disposal protocols for all materials containing recombinant organisms

• Personnel training: Ensuring researchers are properly trained in biosafety procedures and good microbiological practices

The historic Asilomar conference established fundamental principles for biosafety in recombinant DNA research that remain relevant today, emphasizing the importance of appropriate containment measures proportional to perceived risks .

How do post-translational modifications affect ATP synthase subunit c function in different species?

While the search results don't directly address post-translational modifications (PTMs) of ATP synthase subunit c, this represents an important area of research. PTMs can regulate enzyme activity, protein-protein interactions, and protein stability. For ATP synthase subunit c:

• Phosphorylation: Potential phosphorylation sites on cytoplasmic loops could regulate assembly or activity

• Acetylation: N-terminal acetylation or lysine acetylation might influence stability or interaction with other subunits

• Methylation: Could affect proton binding or translocation efficiency

• Species-specific modifications: Different organisms may utilize distinct PTMs for specialized regulation

Comparative studies between E. coli and other species could reveal conserved or divergent regulatory mechanisms. Techniques such as mass spectrometry, phospho-specific antibodies, and site-directed mutagenesis of modification sites would be valuable for identifying and characterizing these modifications.

What are the optimal conditions for expressing and purifying recombinant E. coli ATP synthase subunit c?

Optimal conditions for expressing and purifying recombinant E. coli ATP synthase subunit c typically include:

Expression conditions:

• Host strain: C41(DE3) or C43(DE3) strains designed for membrane protein expression

• Growth temperature: Lower temperatures (16-25°C) often improve folding of membrane proteins

• Induction: Mild induction with lower IPTG concentrations (0.1-0.5 mM)

• Media: Rich media supplemented with glucose to prevent leaky expression

• Expression time: Extended expression periods (16-24 hours) at lower temperatures

Purification approach:

• Membrane isolation: Differential centrifugation to isolate bacterial membranes

• Solubilization: Mild detergents like DDM (n-dodecyl-β-D-maltoside) or LDAO (lauryldimethylamine oxide)

• Affinity chromatography: Utilizing His-tag or other affinity tags

• Size exclusion: To separate monomeric from oligomeric forms

• Detergent exchange: If necessary for downstream applications

These conditions may require optimization for the specific E. coli O139:H28 strain and expression construct used.

How can researchers verify the proper folding and oligomeric state of recombinant ATP synthase subunit c?

Researchers can employ several complementary techniques to verify proper folding and oligomeric state:

• Circular dichroism (CD) spectroscopy: To confirm secondary structure composition (predominantly α-helical for subunit c)

• Size exclusion chromatography (SEC): To evaluate oligomeric state and homogeneity

• Analytical ultracentrifugation: For precise determination of molecular weight and oligomeric state

• Native gel electrophoresis: To assess oligomer formation under non-denaturing conditions

• Functional assays: Reconstitution with other ATP synthase components to verify activity

• Limited proteolysis: Properly folded proteins often show characteristic proteolytic patterns

• Mass spectrometry: Native MS can determine the precise oligomeric state of membrane protein complexes

The C-ring of ATP synthase typically contains multiple c subunits (8-15 depending on species) , so verification of appropriate oligomerization is crucial for functional studies.

What are effective approaches for studying structure-function relationships in ATP synthase subunit c?

Effective approaches for studying structure-function relationships include:

• Site-directed mutagenesis: Systematic mutation of key residues followed by functional characterization, similar to the approach used in studying mycobacterial ATP synthase resistance to TMC207

• Chimeric proteins: Creating hybrid proteins with domains from different species to identify functionally important regions

• Truncation analysis: Evaluating the impact of terminal deletions on structure and function

• Cysteine scanning mutagenesis: Introducing cysteines at specific positions for subsequent labeling or cross-linking

• Homology modeling and molecular dynamics: Computational approaches to predict structural impacts of mutations, as demonstrated for mycobacterial ATP synthase

• High-resolution structural studies: X-ray crystallography, cryo-EM, or NMR to resolve atomic details

• In vitro reconstitution: Assembling purified components to measure specific activities

These approaches can illuminate the roles of specific residues in ion binding, C-ring formation, and interaction with other subunits.

How can researchers design experiments to evaluate the impact of mutations in the atpE gene?

Researchers can design comprehensive experiments to evaluate the impact of atpE mutations using these approaches:

• Complementation studies: Similar to the "isogenic complementation system" mentioned for M. smegmatis , where wild-type or mutant atpE genes are expressed in a strain lacking functional atpE

• Growth characterization: Measuring growth rates, final cell densities, and growth under different energy conditions (fermentable vs. non-fermentable carbon sources)

• ATP synthesis assays: Quantifying ATP production in membrane vesicles or reconstituted systems

• Membrane potential measurements: Using fluorescent dyes to assess the impact on proton gradient formation

• Proton pumping assays: Measuring pH changes in reconstituted liposomes containing wild-type or mutant proteins

• Structural studies: Analyzing how mutations affect C-ring formation and stability

• Drug binding studies: For mutations that affect inhibitor binding, as observed with TMC207 resistance in mycobacteria

Table 2: Experimental Design for Evaluating atpE Mutations

What recombinant antibody approaches can be used to study ATP synthase subunit c localization and interactions?

Recombinant antibody approaches offer significant advantages for studying ATP synthase subunit c:

• Phage display selection: As described in the search results , phage display can generate high-affinity recombinant antibodies (Fabs) that recognize specific epitopes on ATP synthase subunit c

• Automated platforms: High-throughput platforms can generate multiple antibodies against different epitopes on the same protein, allowing for comprehensive binding studies

• Fragment antibody libraries: Using diverse synthetic Fab scaffolds displayed on filamentous phage

• Specificity validation: Testing cross-reactivity against highly related antigens to ensure monospecificity

• Application in immunofluorescence: For cellular localization studies

• Proximity labeling: Antibody-based approaches can be combined with proximity labeling techniques to identify interaction partners

• Immunoprecipitation: For isolating intact ATP synthase complexes from cellular extracts

These approaches provide renewable reagents with consistent performance, addressing reproducibility concerns associated with traditional antibodies .

How does the structure of E. coli ATP synthase subunit c compare to that of other bacterial species?

While specific structural comparisons between E. coli O139:H28 and other bacterial species are not detailed in the search results, general principles can be inferred:

ATP synthase subunit c maintains a conserved hairpin structure across bacterial species, comprising two membrane-spanning α-helices connected by a hydrophilic loop . Despite variable amino acid composition across species, key functional residues are typically conserved, particularly the essential glutamate or aspartate residue in the C-terminal α-helix that serves as the proton carrier .

Based on crystal structures of ATP synthase C-rings from organisms like I. tartaricus and S. platensis, each ion binding site lies between two adjacent c subunits. The conserved glutamate residue (Glu65 in I. tartaricus, Glu62 in S. platensis, corresponding to similar positions in E. coli) provides the main stabilizing interaction for the H⁺/Na⁺ ion .

The number of c subunits in the C-ring varies between species (8-15), which affects the bioenergetics of ATP synthesis by changing the H⁺/ATP ratio .

What is known about the drug-binding properties of ATP synthase subunit c and potential applications?

The search results provide insights into drug-binding properties of ATP synthase subunit c, primarily from studies on mycobacterial ATP synthase:

TMC207 (bedaquiline) targets the C-ring of mycobacterial ATP synthase, binding to a cleft located between two adjacent c subunits that encompasses the proton-binding site (Glu61) . The drug appears to be anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28 .

Mutations in the atpE gene that confer resistance to TMC207 have been identified at positions 28 (Asp→Val/Pro), 61 (Glu→Asp), 63 (Ala→Pro), and 66 (Ile→Met) in M. tuberculosis . These mutations define the drug-binding pocket and highlight the importance of these residues in drug-target interactions.

Naturally resistant mycobacterial species like M. xenopi, M. novacastrense, and M. shimoidei display a Met at position 63 in subunit c in place of a conserved Ala in susceptible species .

This knowledge could inform the development of new ATP synthase inhibitors with applications in antimicrobial therapy, particularly against drug-resistant bacterial pathogens.

How do different experimental conditions affect the stability and activity of recombinant ATP synthase subunit c?

Various experimental conditions can significantly affect the stability and activity of recombinant ATP synthase subunit c:

pH effects:

• Protonation state of the key glutamate residue is pH-dependent

• Optimal pH range typically correlates with physiological conditions

• Extreme pH can destabilize the protein structure

Detergent selection:

• Detergent type significantly impacts stability and oligomerization

• Milder detergents (DDM, LMNG) generally better preserve function

• Detergent concentration affects protein-detergent ratio and stability

Lipid environment:

• Native-like lipid composition can enhance stability and function

• Specific lipids may be required for proper C-ring assembly

• Reconstitution in nanodiscs or liposomes can provide more physiological environment

Temperature sensitivity:

• Thermal stability likely varies between species

• Incubation at elevated temperatures may affect oligomeric state

• Thermal denaturation can provide insights into stability determinants

Ion concentrations:

• Specific ions may stabilize the structure and affect proton binding

• High salt can disrupt electrostatic interactions important for function

These factors should be systematically evaluated when establishing experimental protocols for functional or structural studies.

What computational approaches are most effective for modeling ATP synthase subunit c structure and dynamics?

Several computational approaches are effective for modeling ATP synthase subunit c:

• Homology modeling: Using known structures of ATP synthase C-rings (such as those from I. tartaricus and S. platensis) as templates to predict the structure of E. coli subunit c, similar to the approach described for mycobacterial ATP synthase

• Molecular dynamics simulations: To study conformational changes during proton translocation and interaction with other subunits

• Quantum mechanics/molecular mechanics (QM/MM): For detailed modeling of proton transfer events at the key glutamate residue

• Coarse-grained simulations: To model the assembly and stability of the complete C-ring in membrane environments over longer timescales

• Docking simulations: For predicting interactions with inhibitors or other binding partners, as demonstrated for TMC207 binding to mycobacterial ATP synthase

• Free energy calculations: To evaluate the energetics of proton binding and release during the catalytic cycle

• Network analysis: To identify communication pathways between the proton binding site and other functional regions

These approaches can provide insights into mechanistic details that are challenging to observe experimentally.

How can researchers integrate structural, functional, and genetic data to understand ATP synthase subunit c evolution?

Researchers can integrate multiple data types to understand ATP synthase subunit c evolution through these approaches:

• Comparative genomics: Analyzing atpE sequences across diverse species to identify conserved and variable regions

• Structural alignment: Comparing 3D structures or models across species to identify structurally conserved elements despite sequence divergence

• Functional conservation analysis: Evaluating the conservation of key functional residues (e.g., the proton-binding glutamate) across different lineages

• Ancestral sequence reconstruction: Predicting ancestral atpE sequences to understand evolutionary trajectories

• Selection pressure analysis: Identifying residues under positive or purifying selection

• Correlation with C-ring stoichiometry: Examining relationships between sequence features and the number of c subunits in different species

• Co-evolution with interacting partners: Analyzing how subunit c has co-evolved with other ATP synthase components, particularly subunit a

• Molecular clock analyses: Estimating the timing of key evolutionary changes in relation to major transitions in energy metabolism

This integrated approach can reveal how ATP synthase subunit c has evolved to maintain its critical role in cellular bioenergetics while adapting to different environmental conditions and organismal requirements.