Recombinant Desulfovibrio vulgaris ATP synthase subunit c (atpE)

What is the structure of Desulfovibrio vulgaris ATP synthase subunit c (atpE)?

The ATP synthase subunit c (atpE) from Desulfovibrio vulgaris is a small, highly hydrophobic membrane protein consisting of 82 amino acids with the sequence: MDSSALGLTCLAAAIGMAIAAAGCGIGQGMGLKAACEGTARNPEAGGKIMVTLILGLAFVESLAIYALVVNLILLFANPFMG . This protein forms a critical component of the F₀ sector of ATP synthase, where it assembles into an oligomeric ring structure embedded in the membrane. The protein contains multiple transmembrane helices that create the proton-conducting pathway essential for ATP synthesis.

For structural studies, researchers typically use recombinant versions with affinity tags such as His-tags to facilitate purification . When working with this protein, consider these methodological approaches:

• Membrane protein crystallization techniques (detergent screening, lipidic cubic phase)

• Cryo-electron microscopy for visualization of assembled c-rings

• NMR spectroscopy with isotopically labeled protein for dynamic studies

• Molecular dynamics simulations to understand proton translocation mechanisms

The hydrophobic nature of this protein presents specific challenges for structural biology, requiring specialized handling techniques including appropriate detergent selection and reconstitution protocols to maintain native structure.

What is the primary function of ATP synthase subunit c in Desulfovibrio vulgaris?

The ATP synthase subunit c in Desulfovibrio vulgaris serves as a crucial component in cellular bioenergetics, functioning primarily in proton translocation across the membrane. Within the ATP synthase complex, the c-subunit forms a ring structure in the F₀ sector that rotates as protons move through the complex, driving conformational changes in the F₁ sector that catalyze ATP synthesis .

In sulfate-reducing bacteria like Desulfovibrio vulgaris, ATP synthase plays a particularly important role in energy conservation during anaerobic respiration. These organisms utilize lactate and hydrogen as electron donors and sulfate as the terminal electron acceptor, generating a proton gradient that drives ATP synthesis through the ATP synthase complex .

To study the function of ATP synthase experimentally, researchers typically employ:

• The ACMA (9-amino-6-chloro-2-methoxyacridine) assay - This method measures ATP synthase enzymatic activity by monitoring H⁺ ion sequestration in submitochondrial vesicles (SMVs) in response to ATP addition .

• Genetic approaches - Deletion mutants can reveal the physiological impact of altered ATP synthase function .

• Structural studies - These provide insights into the molecular mechanism of proton translocation and rotary catalysis.

Understanding the function of ATP synthase subunit c in Desulfovibrio vulgaris has implications beyond basic microbiology, including potential applications in bioremediation and biotechnology.

How does the atpE gene expression change under various growth conditions in Desulfovibrio vulgaris?

The expression of the atpE gene in Desulfovibrio vulgaris demonstrates significant plasticity in response to environmental conditions, reflecting the organism's metabolic adaptability. Expression patterns differ notably under various growth conditions:

• Standard growth conditions (lactate/sulfate medium): Under optimal anaerobic growth at 33°C, atpE expression maintains baseline levels necessary for energy production via ATP synthase .

• Nitrite stress conditions: When exposed to nitrite, Desulfovibrio vulgaris undergoes metabolic adjustments that affect energy production pathways, including alterations in ATP synthase gene expression .

• Substrate variation: Growth on different electron donors (besides lactate) can lead to differential expression of energy metabolism genes, including ATP synthase components.

• Stationary vs. exponential phase: Expression levels may vary according to growth phase, with potential implications for energy conservation strategies.

To investigate these expression patterns, researchers employ several methodological approaches:

• Quantitative RT-PCR to measure mRNA levels of atpE under different conditions

• Proteomic analyses to quantify protein levels and post-translational modifications

• Reporter gene assays using fusion constructs to monitor promoter activity

• Comparative growth studies of wild-type and mutant strains

These methodologies provide insights into how Desulfovibrio vulgaris regulates ATP synthase expression as part of its broader strategy for bioenergetic adaptation to environmental changes.

What experimental models are suitable for studying Desulfovibrio vulgaris ATP synthase?

Several experimental models offer distinct advantages for investigating different aspects of Desulfovibrio vulgaris ATP synthase:

• Whole-cell studies: Wild-type Desulfovibrio vulgaris Hildenborough grown anaerobically at 33°C in lactate/sulfate medium C provides a physiologically relevant system . This approach allows for the examination of ATP synthase function in its native cellular context, including interactions with other metabolic pathways.

• Deletion mutant strains: Genetic models with specific ATP synthase components removed enable comparative analyses of growth kinetics and enzymatic activities . These mutants reveal the functional significance of individual components within the ATP synthase complex.

• Recombinant protein systems: Expression of His-tagged atpE protein in E. coli allows for the production of purified protein components for biochemical and structural studies . This system is particularly valuable for obtaining significant quantities of protein for downstream applications.

• Submitochondrial vesicles (SMVs): These membrane preparations are enriched in ATP synthase and provide a system for functional assays such as the ACMA assay, which measures H⁺ translocation activity .

• In vitro reconstitution systems: Purified ATP synthase components can be reconstituted into liposomes to study assembly and function in a defined membrane environment.

When working with these models, several methodological considerations are crucial:

• Maintenance of strict anaerobic conditions during growth and enzyme preparation

• Appropriate buffer systems and stabilizing agents for protein stability

• Careful control of pH and ionic conditions that affect ATP synthase activity

• Proper handling of membrane fractions to maintain native protein organization

These experimental models collectively enable comprehensive investigation of Desulfovibrio vulgaris ATP synthase structure, function, and physiological significance in sulfate-reducing bacteria.

How does nitrite stress affect ATP synthase function in Desulfovibrio vulgaris?

Nitrite stress profoundly impacts ATP synthase function in Desulfovibrio vulgaris through multiple mechanisms, reflecting the organism's metabolic adaptations to this environmental stressor. Desulfovibrio vulgaris, while primarily a sulfate-reducing bacterium, can reduce nitrite as a defense mechanism, with significant energetic consequences .

The effects of nitrite stress on ATP synthase include:

• Altered proton motive force: Nitrite stress affects electron transport and proton translocation, altering the proton gradient that drives ATP synthase.

• Metabolic reprogramming: To counter nitrite toxicity, Desulfovibrio vulgaris shifts its metabolic pathways, prioritizing nitrite reduction over sulfate reduction, which changes the energetic landscape for ATP synthase operation .

• Protein expression changes: Transcriptional and translational regulation of ATP synthase components may occur in response to nitrite stress.

• Functional efficiency alterations: The catalytic efficiency of ATP synthase may be compromised under nitrite stress conditions.

To investigate these effects, researchers employ a multi-faceted experimental approach:

Understanding how nitrite stress affects ATP synthase function has implications for both fundamental microbial physiology and applied aspects such as bioremediation strategies using Desulfovibrio species in nitrite-contaminated environments.

What methodological approaches are effective for analyzing ATP synthase assembly in Desulfovibrio vulgaris?

Analyzing ATP synthase assembly in Desulfovibrio vulgaris requires specialized methodological approaches that address both the complexity of this multisubunit enzyme and the anaerobic nature of the organism:

• Native-PAGE electrophoresis with immunoblotting:

  • Separates intact ATP synthase complexes based on molecular weight and charge

  • Immunoblotting with antibodies against specific subunits (like atpE) identifies fully assembled complexes versus free subunits

  • Critical for distinguishing between assembled ATP synthase (monomer and dimer forms) and unassembled components

• Blue native polyacrylamide gel electrophoresis (BN-PAGE):

  • Preserves native protein-protein interactions during separation

  • Allows for second-dimension SDS-PAGE to identify individual components within complexes

• Sucrose gradient ultracentrifugation:

  • Separates macromolecular assemblies based on size and density

  • Fractions can be analyzed by Western blotting to identify co-migrating subunits

• Cross-linking mass spectrometry (CL-MS):

  • Chemical cross-linking captures transient protein-protein interactions

  • Mass spectrometry analysis identifies cross-linked peptides, revealing spatial relationships between subunits

• Fluorescence microscopy with tagged subunits:

  • Fluorescently labeled ATP synthase components can visualize assembly in vivo

  • FRET (Förster Resonance Energy Transfer) between differently labeled subunits confirms proximity

For anaerobic organisms like Desulfovibrio vulgaris, these techniques must be adapted to maintain anaerobic conditions throughout sample preparation and analysis. Rapid processing, oxygen-scavenging buffers, or anaerobic chambers are essential to preserve native protein states and assembly intermediates.

How does the c-subunit of ATP synthase contribute to energy conservation in sulfate-reducing bacteria?

The c-subunit of ATP synthase plays a pivotal role in energy conservation in sulfate-reducing bacteria like Desulfovibrio vulgaris, functioning as a critical component in the chemiosmotic mechanism that couples electron transport to ATP synthesis.

In Desulfovibrio species, the bioenergetic pathway involves several interconnected processes:

• Electron transport and proton translocation: During dissimilatory sulfate reduction, Desulfovibrio vulgaris oxidizes electron donors like lactate and hydrogen while using sulfate as the terminal electron acceptor . This process generates a proton gradient across the membrane.

• Proton conduction through the c-ring: The c-subunit forms an oligomeric ring in the membrane that contains essential residues for proton binding and release. As protons move through channels in the c-ring, they drive rotation of the entire ring structure.

• Mechanical energy transduction: The rotating c-ring is physically coupled to the central stalk of ATP synthase, which extends into the F₁ catalytic domain. This mechanical coupling converts the rotational motion into conformational changes in the catalytic sites that synthesize ATP.

• Energy yield optimization: The stoichiometry of c-subunits in the ring (the number of c-subunits per ring) determines the H⁺/ATP ratio, which is critical for energy conservation efficiency in low-energy environments.

The ATP synthase c-subunit is particularly important in sulfate-reducing bacteria due to the energetic constraints of their metabolic lifestyle:

• Sulfate reduction is a low-energy-yielding process compared to aerobic respiration

• The proton motive force generated during sulfate reduction is relatively small

• Efficient energy capture and conservation are essential for survival in anaerobic environments

To study the role of the c-subunit in energy conservation, researchers employ various approaches:

• Site-directed mutagenesis of key residues involved in proton translocation

• Kinetic measurements of ATP synthesis under varying proton motive force conditions

• Structural studies to determine c-ring stoichiometry and organization

• Comparative genomics to identify adaptations in c-subunit sequence across different sulfate-reducing bacteria

Understanding how the c-subunit contributes to energy conservation has implications for both basic science and biotechnological applications involving sulfate-reducing bacteria in bioremediation and bioelectrochemical systems.

What are the comparative differences between Desulfovibrio vulgaris ATP synthase and other bacterial ATP synthases?

Comparative analysis of Desulfovibrio vulgaris ATP synthase with those from other bacteria reveals important structural and functional adaptations that reflect its anaerobic, sulfate-reducing lifestyle:

Structural Differences:

• c-ring stoichiometry: The number of c-subunits in the ring affects the H⁺/ATP ratio and therefore the bioenergetic efficiency. While precise stoichiometry in Desulfovibrio vulgaris has not been definitively determined in the provided search results, anaerobic bacteria often have different c-ring compositions compared to aerobes.

• Amino acid sequence specialization: The atpE protein in Desulfovibrio vulgaris has a unique sequence (MDSSALGLTCLAAAIGMAIAAAGCGIGQGMGLKAACEGTARNPEAGGKIMVTLILGLAFVESLAIYALVVNLILLFANPFMG) with specific adaptations for function in low-energy, anaerobic environments.

• Membrane integration: Anaerobes like Desulfovibrio vulgaris have different membrane compositions than aerobes, requiring specialized adaptations in the membrane-embedded regions of ATP synthase.

Functional Differences:

• Energy coupling efficiency: Operating in low-energy environments, Desulfovibrio vulgaris ATP synthase likely has adaptations to maximize energy conservation from the relatively small proton motive force generated during sulfate reduction.

• Metabolic integration: ATP synthase function in Desulfovibrio vulgaris is tightly coupled to dissimilatory sulfate reduction pathways and lactate oxidation mechanisms , unlike in aerobes where it is linked to oxygen respiration.

• Regulatory mechanisms: The response of ATP synthase to environmental stressors differs between bacterial species. For example, Desulfovibrio vulgaris has specific adaptations for handling nitrite stress .

Methodological Approaches for Comparative Studies:

• Comparative genomics and phylogenetics: Analyzing ATP synthase gene sequences across bacterial species to identify conserved and divergent regions

• Heterologous expression: Expressing Desulfovibrio vulgaris ATP synthase components in other systems to assess functional differences

• Biochemical characterization: Comparing enzyme kinetics, inhibitor sensitivity, and pH optima

• Structural biology: Using techniques like cryo-EM to compare ATP synthase structures across species

These comparative studies provide insights into how ATP synthase has evolved to meet the unique bioenergetic challenges faced by different bacterial species in their respective ecological niches.

How can recombinant Desulfovibrio vulgaris ATP synthase subunit c be used in structural biology studies?

Recombinant Desulfovibrio vulgaris ATP synthase subunit c (atpE) serves as a valuable tool for structural biology studies, offering insights into this critical component of bacterial bioenergetics. Several methodological approaches utilize this recombinant protein:

• X-ray Crystallography:

  • Purified His-tagged atpE protein can be crystallized using specialized techniques for membrane proteins

  • Lipidic cubic phase or bicelle crystallization methods may improve crystal quality

  • Molecular replacement using known c-subunit structures can facilitate structure determination

  • The resulting structures reveal atomic details of proton-binding sites and oligomerization interfaces

• Cryo-Electron Microscopy (Cryo-EM):

  • Reconstituted c-rings or entire ATP synthase complexes can be visualized at near-atomic resolution

  • Single-particle analysis captures different conformational states relevant to the rotary mechanism

  • Subtomogram averaging can reveal the organization of ATP synthase in membrane environments

  • This approach is particularly valuable for understanding the c-ring in its native oligomeric state

• Solution and Solid-State NMR:

  • Isotopically labeled recombinant atpE produced in E. coli enables detailed NMR studies

  • Dynamic aspects of proton translocation can be probed using hydrogen/deuterium exchange experiments

  • Protein-detergent interactions can be characterized to understand membrane integration

  • Solid-state NMR is particularly suitable for studying the assembled c-ring structure

• Molecular Dynamics Simulations:

  • Atomistic models based on experimental structures can simulate proton movement through the c-ring

  • These computational approaches complement experimental data by providing dynamic information

  • Simulations can test hypotheses about proton-binding residues and conformational changes

Experimental Workflow for Structural Studies:

When working with recombinant Desulfovibrio vulgaris atpE, researchers must carefully control protein stability using appropriate buffers (typically Tris/PBS-based with trehalose at pH 8.0) and storage conditions (-20°C/-80°C with 5-50% glycerol) .

These structural biology approaches provide crucial insights into ATP synthase function, contributing to our understanding of energy conversion mechanisms in diverse bacterial systems including the anaerobic, sulfate-reducing Desulfovibrio vulgaris.

What experimental considerations are essential when working with recombinant ATP synthase components from anaerobic organisms?

Working with recombinant ATP synthase components from anaerobic organisms like Desulfovibrio vulgaris presents unique experimental challenges that require specific considerations throughout the research workflow:

Protein Handling and Storage:

• Oxygen Sensitivity Management:

  • Components may exhibit oxygen sensitivity requiring anaerobic handling

  • Use of anaerobic chambers, oxygen-scrubbing systems, or nitrogen-flushed containers

  • Rapid work protocols to minimize oxygen exposure time

• Optimal Storage Conditions:

  • Store lyophilized proteins at -20°C/-80°C upon receipt

  • Prepare small aliquots to avoid repeated freeze-thaw cycles

  • Addition of 5-50% glycerol (final concentration) for long-term storage

  • Use of oxygen-impermeable containers for storage

• Reconstitution Protocols:

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Briefly centrifuge vials before opening to collect material

  • Allow proper time for complete rehydration before use

Expression and Purification Strategies:

• Expression System Selection:

  • E. coli is commonly used for expressing Desulfovibrio vulgaris ATP synthase components

  • Consider codon optimization to improve expression efficiency

  • Evaluate expression under microaerobic conditions to improve folding

• Purification Approaches:

  • His-tagged proteins facilitate purification via affinity chromatography

  • Carefully select detergents compatible with membrane protein structure

  • Consider gradual detergent exchange during purification to improve stability

  • Implement quality control via SDS-PAGE (>90% purity standard)

Buffer Composition and Additives:

Functional Assay Considerations:

• Activity Measurements:

  • The ACMA assay measures proton translocation activity but requires careful preparation of submitochondrial vesicles

  • ATP hydrolysis assays should include controls for spontaneous ATP hydrolysis

  • Consider reconstitution into liposomes for more native-like functional studies

• Assembly Analysis:

  • Native-PAGE and BN-PAGE can distinguish between assembled complexes and free subunits

  • Cross-linking approaches can capture transient interaction states

  • Size-exclusion chromatography can separate oligomeric states

By carefully addressing these considerations, researchers can successfully work with recombinant ATP synthase components from anaerobic organisms like Desulfovibrio vulgaris, enabling detailed structural and functional studies that contribute to our understanding of bioenergetics in these specialized microorganisms.