The recombinant atpE is utilized in:
ELISA Kits: Serves as an antigen for detecting anti-Shigella antibodies, aiding in serotyping or diagnostics .
Structural Biology: Provides material for studying F₀ sector dynamics, including proton channel formation and c-ring assembly .
Therapeutic Targeting: Potential use in developing inhibitors targeting ATP synthase to combat Shigella infections .
Genetic Context: The atpE gene resides in the Shigella boydii genome, part of the ATP synthase operon. Its expression is tightly regulated to maintain energy homeostasis .
Functional Redundancy: Subunit c (atpE) interacts with other F₀ components (e.g., subunits a, b) to form a functional proton channel .
Commercial Availability: Multiple vendors offer recombinant atpE, with variations in tag type (His-tag) and purity levels .
While this article focuses on atpE, contextualizing its role within the broader ATP synthase complex is essential. Below is a comparison of F₀F₁ ATP synthase subunits in Shigella boydii serotype 18:
KEGG: sbc:SbBS512_E4184
ATP synthase subunit c (atpE) from Shigella boydii serotype 18 (strain CDC 3083-94 / BS512) is a membrane-embedded protein component of the F0 sector of the bacterial ATP synthase complex. It consists of 79 amino acids with the sequence "MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA" . This protein is highly hydrophobic, allowing it to function within the lipid bilayer of bacterial membranes. The c-subunit is a critical component of the ATP synthase machinery that couples proton translocation to ATP synthesis, thereby playing an essential role in energy production for the bacterial cell.
The ATP synthase c-subunit forms a ring-like structure within the membrane-embedded F0 portion of the ATP synthase complex. Multiple c-subunits assemble into a circular arrangement that creates a central pore, which functions as a proton channel. This c-ring interacts with other membrane components of the F0 sector, particularly the a-subunit, to form the complete proton translocation pathway. The c-ring is coupled to the F1 sector of ATP synthase (containing the catalytic sites for ATP synthesis) via interactions with the central stalk components, primarily the gamma, delta, and epsilon subunits . This structural arrangement allows the rotational motion generated by proton flow through the F0 sector to drive conformational changes in the F1 sector that catalyze ATP synthesis.
Researchers have employed several experimental systems to study the function of ATP synthase c-subunit, including:
Reconstituted liposome systems: Purified c-subunit proteins incorporated into artificial membrane vesicles allow for controlled assessment of channel formation and ion conductance properties .
Submitochondrial vesicles (SMVs): These inside-out vesicles enriched in ATP synthase enable measurement of leak conductance sensitive to ATP/ADP and regulatory proteins like Bcl-xL .
Bacterial expression systems: Recombinant production in heterologous hosts like yeast provides sufficient quantities of the protein for structural and functional studies .
Electrophysiological techniques: Patch-clamp recordings and voltage-dependent conductance measurements have revealed channel-forming properties of the c-subunit ring .
Recent research has revealed that the c-subunit ring possesses functions beyond its role in ATP synthesis. Evidence indicates that the c-subunit can form a voltage-dependent channel when reconstituted into liposomes . This channel demonstrates sensitivity to adenine nucleotides, F1 beta-subunit protein, and anti-c-subunit antibodies . The channel formation capability suggests a potential role in membrane permeability regulation.
The c-subunit has been implicated as a component of the mitochondrial permeability transition pore (mPTP), which regulates inner membrane permeability in response to various cellular stresses . This leak conductance pathway may function as an uncoupling mechanism that dissipates the proton gradient without synthesizing ATP under specific conditions. The dual functionality of the c-subunit—participating in both energy coupling and potential uncoupling processes—represents an intriguing area of ongoing research with implications for understanding cellular metabolism regulation and pathological conditions.
While direct evidence linking ATP synthase c-subunit to Shigella pathogenesis is limited in the provided research materials, several connections can be inferred:
Energy requirement for virulence: Shigella pathogenesis involves numerous energy-dependent processes including invasion, intracellular multiplication, and secretion of virulence factors through the type three secretion system (T3SS) . Proper ATP synthase assembly and function are likely critical for generating the energy required for these virulence mechanisms.
Environmental adaptation: Shigella encounters diverse environmental conditions during infection, including variations in pH, temperature, oxygen availability, and osmolarity . These environmental cues trigger adaptive responses in bacterial gene expression and protein function, potentially including modifications to ATP synthase components.
Stress response coupling: The ATP synthase complex may serve as a sensor for environmental stress conditions, with changes in its assembly or activity feeding into regulatory networks that control virulence gene expression.
The c-subunit's central role in energy production positions it as a potential indirect contributor to pathogenesis, as energy metabolism and virulence are intrinsically linked in bacterial pathogens.
Post-translational modifications (PTMs) of the ATP synthase c-subunit can significantly impact its function, though this area remains underexplored specifically for Shigella boydii. Based on studies in related systems, several PTM-related functional consequences are possible:
Phosphorylation: Phosphorylation of specific amino acid residues may alter the conformational dynamics of the c-ring, affecting proton translocation efficiency or the rotation coupling mechanism.
Acetylation: Acetylation of lysine residues could modify the protein's interaction with lipids or other subunits of the ATP synthase complex.
Oxidative modifications: Under oxidative stress conditions, cysteine or methionine residues may undergo oxidation, potentially impacting protein stability or channel properties.
Proteolytic processing: Limited proteolysis could generate c-subunit fragments with altered functions, potentially contributing to membrane permeabilization under stress conditions.
These modifications may represent mechanisms for fine-tuning ATP synthase activity in response to changing environmental conditions, which would be particularly relevant for pathogenic bacteria like Shigella that must adapt to diverse host environments.
Based on the available data, the following handling and storage guidelines are recommended for recombinant Shigella boydii ATP synthase subunit c:
Parameter | Recommended Condition | Notes |
---|---|---|
Storage Temperature | -20°C for routine storage; -80°C for extended storage | Avoid repeated freeze-thaw cycles |
Working Storage | 4°C | Aliquots can be maintained for up to one week |
Buffer Composition | Tris-based buffer with 50% glycerol | Optimized for protein stability |
Reconstitution | Deionized sterile water to 0.1-1.0 mg/mL | Briefly centrifuge vial before opening |
Long-term Preservation | Add 5-50% glycerol (final concentration) | 50% is the default recommended concentration |
Shelf Life (Liquid) | 6 months at -20°C/-80°C | Dependent on storage conditions |
Shelf Life (Lyophilized) | 12 months at -20°C/-80°C | Preferred form for longest stability |
For optimal results, avoid repeated freezing and thawing cycles, as these can lead to protein denaturation and loss of activity . When preparing working aliquots, it is advisable to make small volumes sufficient for immediate experimental needs to minimize waste and preserve the integrity of the stock solution.
Several specialized techniques have been employed to investigate the channel-forming properties of ATP synthase subunit c:
Liposome Reconstitution Assays: Purified c-subunit can be incorporated into lipid vesicles to study its channel-forming capacity. This technique allows for controlled assessment of membrane permeability changes induced by the protein .
Electrophysiological Measurements: Patch-clamp techniques can be applied to c-subunit-containing membranes to directly measure ion conductance. These recordings have revealed that the c-subunit forms voltage-dependent channels sensitive to specific modulators .
Fluorescent Dye Release Assays: Liposomes loaded with fluorescent dyes can be used to monitor membrane permeabilization upon c-subunit incorporation, offering a high-throughput approach to assess channel activity.
Molecular Dynamics Simulations: Computational approaches can model the structural dynamics of the c-ring and predict ion conductance pathways, complementing experimental findings.
Site-Directed Mutagenesis: Introduction of specific amino acid substitutions can identify residues critical for channel formation versus ATP synthesis functions, helping to delineate structure-function relationships.
Crosslinking Studies: Chemical crosslinking combined with mass spectrometry can identify conformational changes associated with channel opening and closing, as well as interactions with regulatory proteins.
To investigate the role of ATP synthase subunit c in environmental stress adaptation, researchers can implement the following methodological approaches:
Gene Expression Analysis: qRT-PCR and RNA-seq can quantify changes in atpE transcript levels in response to various environmental stressors (pH changes, oxygen limitation, temperature shifts) . This approach can reveal transcriptional regulation patterns specific to stress conditions.
Protein Level Assessment: Western blotting with anti-c-subunit antibodies can track changes in protein abundance, while proteomic approaches can identify post-translational modifications induced by stress.
Genetic Manipulation: Construction of atpE mutants with altered expression levels or specific amino acid substitutions can help determine the protein's contribution to stress tolerance.
In Vivo Models: Animal infection models such as the rabbit ileal loop model or guinea pig model can be used to assess the impact of atpE modifications on bacterial survival and virulence under physiologically relevant conditions .
pH Adaptation Studies: Since Shigella encounters pH variations during infection, experiments examining ATP synthase function across pH gradients can reveal adaptations specific to the c-subunit's role in proton translocation .
Oxygen Tension Experiments: Modulating oxygen availability while monitoring ATP synthase assembly and function can illuminate the protein's role in adapting energy metabolism to hypoxic environments encountered during infection .
Proper analysis of free c-subunit versus assembled c-ring requires careful consideration of several factors:
Research has shown that increases in free c-subunit can correlate with altered cellular metabolism and increased membrane permeability, suggesting that the balance between free and assembled forms may serve as a regulatory mechanism for energy coupling efficiency .
Comparative analysis of ATP synthase c-subunit across bacterial species can yield valuable insights into evolutionary conservation, functional specialization, and potential therapeutic targets:
Sequence Conservation: Analysis of conserved versus variable regions can identify functionally critical amino acid residues versus those that may confer species-specific adaptations.
Structural Variations: Differences in c-subunit structure across species may correlate with variations in proton/sodium specificity, stoichiometry of the c-ring, or regulatory mechanisms.
Pathogen-Specific Features: Comparison between pathogenic and non-pathogenic bacteria may reveal adaptations in the c-subunit that contribute to survival in host environments.
Drug Target Potential: Regions of the c-subunit that differ significantly between bacterial and human ATP synthase could represent selective targets for antimicrobial development.
Horizontal Gene Transfer Assessment: Phylogenetic analysis can identify instances of horizontal gene transfer that might have contributed to virulence acquisition in pathogens like Shigella.
This comparative approach can be particularly valuable for understanding how Shigella boydii's ATP synthase has adapted to its pathogenic lifestyle and how these adaptations might contribute to its virulence mechanisms.
The ATP synthase c-subunit likely plays a multifaceted role in Shigella's adaptation to the host environment during infection:
pH Adaptation: Shigella encounters varying pH environments, from the acidic stomach to the more neutral intestine. The c-subunit's function in proton translocation may be modulated to maintain energy production across these pH gradients . The CpxA/CpxR two-component system, which responds to pH changes, influences virulence gene expression in Shigella and may indirectly affect ATP synthase function .
Oxygen Tension Response: As Shigella moves from oxygen-rich to oxygen-limited environments within the host, the efficiency of ATP synthase must be regulated accordingly. The c-subunit's structure or abundance might be adjusted to optimize energy production under different oxygen concentrations .
Nutrient Availability Adaptation: Changes in available carbon sources during infection may necessitate shifts in energy metabolism, with the c-subunit potentially serving as a regulatory point for adjusting ATP production efficiency.
Immune Response Evasion: The ATP synthase complex, including the c-subunit, might play a role in modulating host cell responses, potentially through effects on membrane potential or pH regulation that influence host signaling pathways.
Understanding these adaptation mechanisms could provide insights into Shigella pathogenesis and identify potential intervention strategies targeting the bacterium's energy metabolism during infection.