Recombinant Bacteroides vulgatus ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Bacteroides vulgatus?

ATP synthase subunit c (atpE) in B. vulgatus is part of the membrane-embedded F₀ sector of the ATP synthase complex, forming a ring structure in the membrane. This c-ring works as a rotor that converts ion flow across the membrane into mechanical rotation, which is then used to drive ATP synthesis in the catalytic F₁ sector. In bacterial ATP synthases, the c subunit typically consists of two transmembrane helices connected by a short cytosolic loop, with each subunit harboring an ion binding site that is crucial for proton translocation . The number of c subunits in bacterial rings varies between species, ranging from 8 to 17, which affects the ion-to-ATP ratio during synthesis . The specific structure of B. vulgatus atpE would need to be experimentally determined, but based on other bacterial ATP synthases, it likely maintains the conserved features necessary for proton translocation and rotational coupling.

The c-ring rotates against a stationary subunit a, and this rotation is mechanically coupled to conformational changes in the F₁ sector through a central stalk, driving ATP synthesis in the catalytic sites formed by α and β subunits . The architecture of the membrane region shows how bacterial ATP synthases, despite their relative simplicity compared to mitochondrial complexes, perform the same core functions of energy conversion with remarkable efficiency .

How does the ATP synthase c subunit from Bacteroides vulgatus compare to those from other bacterial species?

Comparison of ATP synthase c subunits across bacterial species reveals both conservation of core functional elements and species-specific variations. While the search results don't provide specific information about B. vulgatus atpE, we can infer similarities and differences based on other bacterial ATP synthases. The basic hairpin structure with two transmembrane helices connected by a cytosolic loop is likely conserved, but variations may exist in the number of c subunits per ring, which directly affects the bioenergetic efficiency of the enzyme .

Bacterial ATP synthases like those from Bacillus PS3 show specific conformational states of catalytic β subunits (described as 'open', 'closed', and 'open'), which differ from those observed in E. coli and chloroplast ATP synthases . These structural differences reflect adaptations to specific energetic requirements and regulatory mechanisms. B. vulgatus, as an anaerobic gut bacterium, likely has adaptations in its ATP synthase to function optimally under the low-oxygen conditions of the intestinal environment.

The ion specificity (H⁺ vs Na⁺) of the binding site in subunit c is another point of variation among bacterial species. While many bacterial ATP synthases are proton-powered, some use sodium ions as the coupling ion, which affects their functional properties and environmental adaptability . Understanding these species-specific variations is crucial for interpreting experimental results and designing targeted modifications of recombinant atpE.

What techniques are available for expressing recombinant B. vulgatus atpE in heterologous systems?

Recombinant expression of B. vulgatus atpE can be accomplished using several heterologous expression systems, with E. coli being the most common for bacterial membrane proteins. Based on the successful expression of the Bacillus PS3 ATP synthase in E. coli reported in the search results, a similar approach could be applied to B. vulgatus atpE . This would typically involve PCR amplification of the atpE gene from B. vulgatus genomic DNA, followed by cloning into an appropriate expression vector with a suitable promoter and affinity tag for purification.

For optimal expression, codon optimization may be necessary to match the codon usage preferences of the host organism. Expression vectors containing inducible promoters (such as T7 or arabinose-inducible systems) allow controlled expression, which is particularly important for membrane proteins that can be toxic when overexpressed. The search results mention using "Q5 high-fidelity DNA polymerase from New England Biolabs" for PCR amplification and the "NEBuilder® HiFi DNA Assembly Cloning Kit" for DNA assembly, suggesting these as reliable tools for constructing expression plasmids .

Transformation into an expression host like E. coli β2155 can be performed via electroporation, as mentioned in the search results . Verification of successful transformation can be done through colony PCR and sequencing. It's important to optimize expression conditions (temperature, inducer concentration, expression time) to maximize yield while maintaining proper folding of the membrane protein. Alternative expression systems such as Lactococcus lactis or cell-free systems may be considered if E. coli expression proves challenging.

What are the best methods for purifying recombinant B. vulgatus ATP synthase subunit c while maintaining its native structure?

Purification of recombinant B. vulgatus ATP synthase subunit c requires specialized approaches due to its hydrophobic nature as a membrane protein. The most effective strategy typically begins with the optimal solubilization of membranes using detergents that maintain protein structure and function. A combination of detergent screening is recommended, testing mild detergents like n-dodecyl β-D-maltoside (DDM), digitonin, or lauryl maltose neopentyl glycol (LMNG) at varying concentrations to identify conditions that effectively extract atpE while preserving its native fold.

Affinity chromatography represents the initial purification step, utilizing engineered tags (His6, FLAG, or Strep-II) introduced during cloning. Following affinity purification, researchers should employ size exclusion chromatography to separate the c-ring from aggregates or dissociated subunits, yielding a more homogeneous preparation. Throughout the purification process, it's crucial to maintain a consistent buffer composition, temperature, and pH to prevent protein denaturation or dissociation of the c-ring complex .

For functional studies, reconstitution into liposomes may be necessary, as demonstrated with other ATP synthases in the search results. This process involves removing the detergent through methods like Bio-Beads addition or dialysis, allowing the protein to integrate into a lipid bilayer that better mimics its native environment. Quality assessment of the purified protein should include SDS-PAGE, Blue Native PAGE, and western blotting to confirm purity and integrity, while negative-stain electron microscopy and circular dichroism can provide insights into structural preservation .

How can one assess the functional integrity of recombinant B. vulgatus atpE after purification?

Assessing the functional integrity of recombinant B. vulgatus atpE requires multiple complementary approaches to confirm both structural and functional properties. Reconstitution into liposomes provides an essential first step toward functional assessment, creating a membrane environment where ion translocation can be measured. After reconstitution, researchers can employ fluorescent probes like ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor proton translocation activity, with quenching of fluorescence indicating successful proton pumping across the membrane in response to ATP hydrolysis.

For direct functional assessment, ATP synthesis assays can be performed by generating an artificial ion gradient across the liposome membrane and measuring ATP production using luciferase-based detection systems. As demonstrated with other ATP synthases, applying different driving forces (in the range of 90-150 mV) can help determine the threshold potential required for ATP synthesis, providing insights into the bioenergetic efficiency of the recombinant enzyme . Comparing these values with those obtained from other bacterial ATP synthases helps position B. vulgatus ATP synthase within the spectrum of bioenergetic adaptations.

Structural integrity can be assessed through techniques like limited proteolysis, which probes the accessibility of protease sites as an indicator of proper folding. More advanced structural analysis may include cryo-electron microscopy, which has successfully revealed the atomic structure of bacterial ATP synthases in different rotational states . For the c-ring specifically, mass spectrometry can determine the oligomeric state by analyzing intact complexes, providing crucial information about the stoichiometry of proton translocation to ATP synthesis.

What expression systems are most effective for producing functional B. vulgatus ATP synthase complexes?

When aiming to produce functional B. vulgatus ATP synthase complexes, researchers must consider several expression systems, each with distinct advantages for membrane protein production. E. coli remains the first-choice expression host for bacterial membrane proteins due to its rapid growth, genetic tractability, and availability of specialized strains. The C41(DE3) and C43(DE3) strains, derived from BL21(DE3), are particularly effective for membrane protein expression due to their ability to mitigate toxicity associated with overexpression. The search results indicate successful expression of the Bacillus PS3 ATP synthase in E. coli, suggesting this approach may be viable for B. vulgatus ATP synthase as well .

For expression of the complete ATP synthase complex, co-expression strategies utilizing multiple compatible plasmids may be necessary, as the complete complex comprises multiple subunits encoded by different genes. Alternatively, a single plasmid containing the entire ATP synthase operon could be constructed. Temperature optimization is crucial, with lower temperatures (16-25°C) often yielding better results for complex membrane proteins by slowing expression and allowing proper membrane insertion and folding.

Alternative expression systems worth considering include Lactococcus lactis, which provides a gram-positive membrane environment that may better accommodate certain membrane proteins, and cell-free systems that bypass issues related to toxicity and membrane insertion. For B. vulgatus ATP synthase specifically, expression in Bacteroides species (if genetic tools are available) might provide the most native-like environment. The search results mention that P. vulgatus is genetically accessible, suggesting that homologous expression might be feasible, potentially yielding more native-like complexes .

How does the ion binding site in B. vulgatus ATP synthase subunit c influence its bioenergetic efficiency?

The ion binding site in ATP synthase subunit c serves as the fundamental unit of energy conversion, directly influencing the bioenergetic efficiency of the entire complex. In B. vulgatus ATP synthase, the specific composition of this binding site would determine whether it translocates protons or sodium ions, which has significant implications for its function in the anaerobic gut environment. The search results indicate that the number of ion binding sites per c-ring varies among different species and is directly related to the number of ions required to synthesize one ATP molecule .

The ion-to-ATP ratio, determined by the number of c subunits in the ring divided by the three catalytic sites in the F₁ domain, establishes the thermodynamic threshold for ATP synthesis. According to the search results, this ratio can range from 2.66 to 5.6 ions per ATP across different species . A lower ratio (fewer ions per ATP) allows ATP synthesis at lower driving forces, which may be advantageous in energy-limited environments like the anaerobic human gut where B. vulgatus resides. The search results highlight that some ATP synthases with unusual c subunit arrangements can synthesize ATP at driving forces as low as 90-150 mV, which is physiologically relevant for anaerobic organisms .

Experimentally, this relationship can be investigated by reconstituting the purified ATP synthase into liposomes and measuring ATP synthesis rates at different artificially imposed membrane potentials. By determining the minimum driving force required for ATP synthesis, researchers can calculate the effective ion-to-ATP ratio and infer the number of c subunits in the ring. These experiments would provide valuable insights into how B. vulgatus has adapted its ATP synthase to function efficiently in its specific ecological niche.

What role might post-translational modifications play in regulating B. vulgatus ATP synthase subunit c function?

Post-translational modifications (PTMs) represent a sophisticated layer of regulation that can fine-tune the function of ATP synthase subunit c in response to changing environmental or metabolic conditions. Although the search results don't specifically address PTMs in B. vulgatus ATP synthase, research on other bacterial ATP synthases suggests several potential regulatory mechanisms. Phosphorylation of specific residues in subunit c could alter the proton binding affinity or the structural dynamics of the c-ring, potentially adjusting the enzyme's activity in response to energy demand.

In anaerobic bacteria like B. vulgatus, redox-sensitive modifications such as glutathionylation or sulfenylation might serve as mechanisms to coordinate ATP synthase activity with the redox state of the cell. Given the low oxygen tension in the gut environment, such redox-responsive regulation could help maintain energy homeostasis during fluctuations in nutrient availability. Proteomics approaches including mass spectrometry with electron-transfer dissociation (ETD) or higher-energy collisional dissociation (HCD) fragmentation can identify specific PTMs and their locations within the protein sequence.

The functional significance of identified PTMs can be investigated through site-directed mutagenesis, replacing modifiable residues with non-modifiable analogues (e.g., serine to alanine for phosphorylation sites) and assessing the impact on ATP synthase activity. In vitro modification systems can also be employed to enzymatically modify purified subunit c before reconstitution and functional assays. Understanding the PTM landscape of B. vulgatus ATP synthase would provide valuable insights into how this anaerobic gut bacterium regulates its energy metabolism in response to the dynamic intestinal environment.

How does the structure of the B. vulgatus c-ring compare to V-type and A-type ATP synthase c-rings?

The structural comparison between B. vulgatus c-ring and those from V-type and A-type ATP synthases represents a fascinating evolutionary question that bridges fundamental research with phylogenetic implications. According to the search results, traditional bacterial F-type ATP synthases typically have c subunits consisting of two transmembrane helices with one ion binding site per subunit . In contrast, V-type ATPases found in eukaryotes often contain duplicated c subunits with multiple transmembrane helices but a reduced number of ion binding sites per subunit mass.

Interestingly, some archaea possess ATP synthases with V-type or A-type features, including unusual c subunits with varied numbers of hairpins and reduced ion-translocating residues . The search results indicate that these enzymes with V-type c subunits were previously thought incapable of ATP synthesis, yet recent research has demonstrated their ability to synthesize ATP at physiologically relevant driving forces . This finding challenges previous assumptions about the evolutionary trajectory from a progenitor ATPase to modern variants.

For B. vulgatus, determining whether its ATP synthase c-ring more closely resembles typical bacterial F-type structures or incorporates features of A/V-type synthases would provide valuable insights into its evolutionary history and bioenergetic strategy. High-resolution structural determination through cryo-electron microscopy, as demonstrated for the Bacillus PS3 ATP synthase , would allow precise comparison of the B. vulgatus c-ring architecture with known structures. Mass spectrometry of the intact c-ring could reveal its stoichiometry, while hydrogen-deuterium exchange experiments could identify exposed regions and interfaces between subunits, collectively building a comprehensive structural model for comparative analysis.

What are common challenges in expressing recombinant B. vulgatus atpE and how can they be addressed?

Expression of recombinant B. vulgatus atpE presents several technical challenges that researchers commonly encounter. Toxicity represents a primary obstacle, as membrane protein overexpression can disrupt host cell membrane integrity, leading to growth inhibition and reduced yields. This can be mitigated by using specialized expression strains like C41(DE3) or C43(DE3) that are more tolerant to membrane protein expression, or by employing tightly regulated expression systems that minimize basal expression. The search results mention successful ATP synthase expression in E. coli, suggesting that with proper optimization, this host can support membrane protein production .

Protein misfolding constitutes another significant challenge, particularly for membrane proteins requiring specific lipid environments. Lowering the expression temperature (to 16-25°C) slows protein synthesis, potentially allowing more time for proper membrane insertion and folding. Addition of specific lipids to the growth medium may better mimic the native membrane environment of B. vulgatus. For particularly difficult proteins, fusion partners like Mistic or SUMO can improve membrane targeting and solubility.

Codon usage differences between B. vulgatus and expression hosts can limit translation efficiency. The search results indicate successful genetic work with P. vulgatus, suggesting that either native codons are compatible with common expression hosts, or codon optimization was employed . Commercial codon optimization services can design synthetic genes aligned with the codon preferences of the chosen expression host. Additionally, co-expression of rare tRNAs using plasmids like pRARE can alleviate codon-related bottlenecks. For complete ATP synthase complexes, ensuring balanced expression of all components through careful promoter selection and ribosome binding site engineering becomes essential for assembling functional units.

How can researchers distinguish between functional and non-functional recombinant ATP synthase preparations?

Distinguishing between functional and non-functional recombinant ATP synthase preparations requires a multi-faceted approach that combines structural and functional assays. The gold standard for functional assessment involves reconstituting the purified enzyme into liposomes and measuring ATP synthesis driven by an artificially imposed proton gradient. According to the search results, successful ATP synthesis has been demonstrated in reconstituted systems at driving forces ranging from 90 to 195 mV, providing a reference range for experimental design . The absence of ATP synthesis under these conditions would indicate non-functional preparations.

What approaches can be used to study the interaction between B. vulgatus ATP synthase subunit c and other subunits of the complex?

Studying the interactions between B. vulgatus ATP synthase subunit c and other components of the complex requires specialized techniques that can capture both stable and transient protein-protein interactions within membrane environments. Cross-linking mass spectrometry (XL-MS) represents a powerful approach, utilizing chemical cross-linkers of defined length to covalently connect interacting proteins, followed by enzymatic digestion and mass spectrometric identification of cross-linked peptides. This technique can map interaction interfaces between subunit c and adjacent proteins like subunits a, b, and the central stalk components.

Genetic approaches including suppressor mutation analysis can identify functional interactions between subunits. By introducing destabilizing mutations in subunit c and screening for compensatory mutations in other subunits that restore function, researchers can map networks of functionally important interactions. The search results demonstrate that P. vulgatus is genetically accessible, suggesting these approaches could be implemented directly in the native organism . Site-directed mutagenesis targeting predicted interface residues, followed by functional assays, can verify the importance of specific interactions identified through structural or computational approaches.

For visualizing dynamic interactions in intact complexes, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions of subunit c that become protected upon complex formation, indicating interaction surfaces. Fluorescence resonance energy transfer (FRET) using site-specifically labeled components can monitor conformational changes and subunit movements during catalysis. The search results describe ATP synthases as "marvelous machines that transform energy stored in the transmembrane electrochemical gradient of ions...first to rotational energy of protein masses, the c ring in the membrane" , highlighting the dynamic nature of these interactions that must be captured through complementary experimental approaches.

How might engineering B. vulgatus ATP synthase improve its application in bioenergetic studies or biotechnology?

Engineering B. vulgatus ATP synthase opens promising avenues for both fundamental bioenergetic research and innovative biotechnological applications. Strategic modification of the c-ring composition represents a particularly intriguing approach, as the number of c subunits directly determines the ion-to-ATP ratio and thus the thermodynamic efficiency of energy conversion. The search results indicate that this ratio varies naturally among different species and can range from 2.66 to 5.6 ions per ATP . By engineering c-rings with altered stoichiometry, researchers could develop ATP synthases with customized energy conversion properties tailored for specific applications or energy environments.

Site-directed mutagenesis of the ion binding sites in subunit c could alter ion specificity (H⁺ vs Na⁺) or binding affinity, potentially creating variants with enhanced performance under specific conditions. The search results mention that some ATP synthases with unusual c subunits can function at driving forces as low as 90-150 mV , suggesting that engineering efforts could potentially push these limits even further. For biotechnological applications like bio-electrochemical systems, engineering ATP synthases that operate efficiently at lower driving forces could significantly improve energy capture from dilute or fluctuating energy sources.

Chimeric ATP synthases, combining components from different species with B. vulgatus elements, might yield enzymes with novel properties. For instance, incorporating c subunits from extremophiles might enhance stability under harsh conditions, while regulatory elements from other species could provide new control mechanisms. The genetic accessibility of P. vulgatus demonstrated in the search results provides a foundation for implementing these engineering approaches, potentially leading to designer ATP synthases with optimized performance characteristics for both research and biotechnological applications.

What insights might B. vulgatus ATP synthase provide about bacterial adaptation to the human gut environment?

B. vulgatus ATP synthase likely represents a specialized adaptation to the unique bioenergetic challenges of the human gut environment, potentially offering profound insights into bacterial energetic strategies under nutritional fluctuation and low oxygen tension. As an abundant member of the gut microbiota, B. vulgatus must navigate an environment characterized by intermittent nutrient availability, competition with other microbes, and varying redox conditions. The configuration of its ATP synthase, particularly the c-ring stoichiometry and ion specificity, may reflect adaptations to maximize energy capture efficiency under these constraints.

The search results indicate that some ATP synthases can function at driving forces as low as 90-150 mV, which is particularly relevant for organisms living "near the thermodynamic limit of ATP synthesis" . This suggests that B. vulgatus may have evolved an ATP synthase capable of synthesizing ATP under the modest ion gradients achievable in the anaerobic gut environment. Comparative analysis of B. vulgatus ATP synthase with those from bacteria occupying different niches could reveal specific adaptations to the gut environment, potentially identifying signature features that confer a competitive advantage in this ecosystem.

Beyond basic energetics, the regulatory mechanisms of B. vulgatus ATP synthase may provide insights into how gut bacteria respond to environmental fluctuations. The search results mention that in some bacteria, ATP synthase inhibition is modulated by ATP concentration, allowing the enzyme to run in reverse (establishing a proton motive force by ATP hydrolysis) when ATP is abundant . Understanding whether B. vulgatus employs similar regulatory strategies could reveal how it balances energy production and consumption during feast-famine cycles characteristic of the gut environment, potentially informing broader questions about bacterial persistence and colonization in this important ecosystem.

How could structural studies of B. vulgatus ATP synthase contribute to our understanding of bacterial bioenergetics evolution?

Structural studies of B. vulgatus ATP synthase hold transformative potential for elucidating evolutionary trajectories in bacterial bioenergetics, particularly regarding the diversification of energy-converting enzyme complexes. The search results highlight the existence of ATP synthases with unusual c subunit arrangements, including those with characteristics typically associated with V-type ATPases rather than F-type ATP synthases . These findings challenge previous evolutionary models and suggest more complex evolutionary histories than previously recognized.

High-resolution structural determination of B. vulgatus ATP synthase through cryo-electron microscopy—similar to the approach used for Bacillus PS3 ATP synthase described in the search results —would allow precise comparison with other bacterial, archaeal, and eukaryotic ATP synthases. Such comparative analysis could identify conserved core elements that have remained invariant throughout evolution, as well as lineage-specific adaptations that reflect particular environmental pressures or metabolic strategies. The positioning of B. vulgatus ATP synthase within this structural landscape would provide valuable data points for reconstructing the evolutionary history of these essential energy-converting complexes.

Particularly significant would be structural insights into the c-ring, as variations in this component appear central to the functional diversification of ATP synthases and ATPases. The search results suggest an evolutionary scenario involving "duplication of the c subunit gene followed by loss of one ion-binding site" as a mechanism for transitioning between different functional modes . Structural evidence from B. vulgatus could support or refine such models, potentially revealing intermediate forms or unique adaptations that illuminate the evolutionary pathways connecting the diverse ATP synthase variants observed across the tree of life. These findings would contribute fundamentally to our understanding of how life has evolved diverse solutions to the universal challenge of energy conversion and storage.