Recombinant Mycoplasma gallisepticum ATP synthase subunit c (atpE)

What is the basic structure of Mycoplasma gallisepticum ATP synthase subunit c (atpE)?

Mycoplasma gallisepticum ATP synthase subunit c (atpE) is a relatively small protein consisting of 96 amino acids with the sequence: MNIFLVIHELINQADQVNVTLTNHVGAYIGAGMAMTAAAGVGVGQGFASGLCATALARNPELLPKIQLFWIVGSAIAESSAIYGLIIAFILIFVAR . Structural analyses suggest two possible conformations: a hairpin-like structure with two hydrophobic domains transversing the membrane . The protein is highly hydrophobic, which is characteristic of F0 sector components that are embedded in the membrane. As part of the ATP synthase complex, it forms the c-ring structure within the F0 sector that is crucial for proton translocation across the membrane during ATP synthesis.

What are the alternative designations for the atpE protein in scientific literature?

The Mycoplasma gallisepticum ATP synthase subunit c is known by several alternative designations in scientific literature, including:

• ATP synthase F(0) sector subunit c

• F-type ATPase subunit c (shortened to F-ATPase subunit c)

• Lipid-binding protein

These alternative names reflect different aspects of the protein's function and localization within the ATP synthase complex. When conducting literature searches, researchers should use all these terms to ensure comprehensive coverage of relevant publications.

What are the optimal conditions for handling recombinant M. gallisepticum atpE protein in laboratory settings?

For optimal handling of recombinant M. gallisepticum ATP synthase subunit c (atpE) in laboratory settings, researchers should follow these methodological guidelines:

• Storage conditions: Store the protein at -20°C for regular use, or at -80°C for extended storage to maintain stability. The protein is typically supplied in a Tris-based buffer with 50% glycerol, which has been optimized for this specific protein .

• Working with the protein: Create working aliquots that can be stored at 4°C for up to one week to minimize freeze-thaw cycles. Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of activity .

• Buffer considerations: When designing experiments, consider that the protein's native environment is lipid-rich membrane, so buffer systems that maintain proper folding of hydrophobic proteins are essential.

• Sample preparation: For structural or functional studies, gentle detergents may be required to maintain the protein in solution while preserving its native conformation.

What experimental approaches are most effective for studying the function of atpE in ATP synthesis?

Several experimental approaches are particularly effective for studying the function of atpE in ATP synthesis:

• Reconstitution studies: Incorporating the recombinant protein into liposomes or nanodiscs to create a minimal system for studying proton translocation and ATP synthesis.

• Site-directed mutagenesis: Creating targeted mutations in the protein to identify key residues involved in proton binding, oligomerization, or interaction with other ATP synthase subunits.

• Cross-linking experiments: Using chemical cross-linkers to identify interaction partners and conformational changes during the catalytic cycle.

• Spectroscopic methods: Techniques such as circular dichroism (CD) and nuclear magnetic resonance (NMR) can provide insights into the protein's secondary structure and dynamics in different environments.

• Inhibitor studies: Using specific inhibitors of the c-subunit, such as oligomycin derivatives, to probe the mechanism of action and potential binding sites.

These approaches can be combined to provide a comprehensive understanding of how atpE contributes to ATP synthesis in M. gallisepticum.

What are the challenges in expressing and purifying recombinant M. gallisepticum atpE?

Researchers face several significant challenges when expressing and purifying recombinant M. gallisepticum atpE:

• Membrane protein solubility: As a highly hydrophobic membrane protein, atpE tends to aggregate during expression and purification, requiring careful optimization of detergent conditions.

• Expression systems: Selection of an appropriate expression system is critical. While E. coli is commonly used, the differences in membrane composition between E. coli and Mycoplasma may affect proper folding.

• Codon optimization: The different codon usage between M. gallisepticum and expression hosts necessitates codon optimization for efficient expression .

• Purification strategy: Affinity tags must be carefully selected and positioned to avoid interfering with protein function or structure. The tag type will typically be determined during the production process to optimize for yield and functionality .

• Functional verification: Confirming that the recombinant protein retains its native conformation and function is essential, particularly when studying structure-function relationships.

How does M. gallisepticum atpE differ structurally and functionally from ATP synthase c subunits in other bacteria?

M. gallisepticum atpE exhibits several notable differences compared to ATP synthase c subunits in other bacteria:

• Size and sequence variation: At 96 amino acids, the M. gallisepticum atpE is relatively compact compared to some other bacterial homologs. Sequence analysis reveals specific adaptations that may reflect M. gallisepticum's minimal genome and parasitic lifestyle .

• Membrane integration: The hairpin-like structure with two hydrophobic domains that transverse the membrane represents a common motif in F-type ATP synthases, but the specific arrangement and interactions may differ in M. gallisepticum due to its unique membrane composition.

• Oligomerization properties: The c-subunit forms a ring structure whose stoichiometry can vary between species. While the exact number of c-subunits in the M. gallisepticum c-ring is not specified in the available data, this parameter significantly impacts the bioenergetic efficiency of ATP synthesis.

• Ion specificity: Most bacterial ATP synthases use protons (H⁺) for coupling, but some use sodium ions (Na⁺). The specific residues in M. gallisepticum atpE suggest it functions as a proton-coupled ATP synthase.

These structural and functional adaptations reflect M. gallisepticum's evolutionary history and ecological niche as a minimal, host-dependent pathogen.

Does atpE play a role in M. gallisepticum pathogenesis or virulence?

While ATP synthase is primarily involved in energy metabolism rather than direct virulence, several lines of evidence suggest atpE may contribute to M. gallisepticum pathogenesis:

• Energy provision for virulence: ATP synthase provides the energy necessary for various virulence mechanisms, including the sophisticated immune evasion tactics employed by M. gallisepticum .

• Persistence in host: M. gallisepticum's ability to persist in the host through immune evasion results in long-term chronic infection . The energy provided by ATP synthase is essential for maintaining metabolic activities during this persistent state.

• Adaptation to host environment: The specific properties of M. gallisepticum ATP synthase may reflect adaptations to the energy constraints of its parasitic lifestyle within the respiratory tract of avian hosts.

• Potential interactions with host factors: Though not directly documented in the available data, bacterial ATP synthases can sometimes interact with host factors, potentially contributing to pathogenesis through non-canonical functions.

Understanding the role of atpE in M. gallisepticum pathogenesis requires considering both its primary role in energy metabolism and potential secondary contributions to virulence mechanisms.

Can M. gallisepticum atpE be targeted for vaccine development?

While atpE itself is not prominently mentioned as a vaccine target in the available data, there are several considerations for its potential in vaccine development:

• Conservation and essentiality: As an essential component of energy metabolism, atpE is likely to be conserved across M. gallisepticum strains, potentially providing broad protection against different isolates.

• Accessibility concerns: As a membrane-embedded protein, many epitopes of atpE may not be accessible to antibodies, potentially limiting its effectiveness as a traditional vaccine antigen.

• Multi-epitope approaches: Recent research has focused on developing multi-epitope peptide vaccines (MEPV) against M. gallisepticum using immunogenic segments from various proteins . A similar approach could potentially incorporate conserved, accessible epitopes from atpE.

• Plant-based expression systems: The successful expression of M. gallisepticum antigens in plant systems, as demonstrated for certain cytoadherence proteins , provides a potential platform for producing atpE-derived antigens for vaccine development.

• Immune response metrics: When evaluating potential vaccine candidates, researchers measure immunoglobulin Y (IgY) antibody titers as a key indicator of immune response in avian hosts .

Research on plant-derived vaccines against M. gallisepticum has shown promising results in triggering immune responses in chickens, suggesting that carefully designed vaccines incorporating essential proteins could be effective against chronic respiratory disease .

How does the c-ring stoichiometry of M. gallisepticum ATP synthase affect its bioenergetic efficiency?

The c-ring stoichiometry (number of c-subunits forming the ring) is a critical parameter that directly determines the H⁺/ATP ratio and therefore the bioenergetic efficiency of ATP synthesis. For M. gallisepticum, this represents a particularly interesting question:

• Theoretical considerations: The number of c-subunits determines how many protons must pass through the ATP synthase to generate one ATP molecule. A smaller c-ring requires fewer protons per ATP, increasing energetic efficiency.

• Methodological approaches: Determining c-ring stoichiometry typically requires advanced structural techniques such as:

  • Atomic force microscopy (AFM) of isolated c-rings

  • Cryo-electron microscopy (cryo-EM) of the intact ATP synthase complex

  • Mass spectrometry of chemically cross-linked c-rings

• Evolutionary considerations: As one of the smallest self-replicating organisms , M. gallisepticum may have evolved specific adaptations in its ATP synthase to optimize energy conversion under the constraints of its minimal genome and parasitic lifestyle.

• Comparative analysis: Comparing the c-ring stoichiometry of M. gallisepticum to that of other minimal organisms and related mycoplasmas could provide insights into the evolutionary pressures shaping ATP synthase structure and function.

Understanding the c-ring stoichiometry would provide fundamental insights into M. gallisepticum's bioenergetic strategy and could inform broader questions about energy limitations in minimal cellular systems.

What is the role of specific amino acid residues in the function of M. gallisepticum atpE?

Several key amino acid residues in M. gallisepticum atpE likely play critical roles in its function, based on what is known about c-subunits in other organisms:

• Proton-binding site: Typically, a conserved carboxylic acid residue (Asp or Glu) in the middle of one of the transmembrane helices serves as the proton-binding site. In the M. gallisepticum atpE sequence (MNIFLVIHELINQADQVNVTLTNHVGAYIGAGMAMTAAAGVGVGQGFASGLCATALARNPELLPKIQLFWIVGSAIAESSAIYGLIIAFILIFVAR) , potential candidates include the glutamic acid (E) residues.

• Helix-helix interactions: Residues involved in the packing of helices within and between c-subunits are crucial for the stability of the c-ring. These typically include glycine-rich motifs that allow close packing of helices.

• Interactions with other subunits: Specific residues mediate interactions with other components of the ATP synthase, particularly the a-subunit, which is essential for proton translocation.

• Lipid interactions: As a lipid-binding protein , certain residues likely mediate interactions with the membrane lipids, potentially affecting the protein's stability and function.

Methodological approaches to study these residues include site-directed mutagenesis, molecular dynamics simulations, and structural studies using NMR or X-ray crystallography.

How can structural data on M. gallisepticum atpE inform the development of specific inhibitors?

Structural information on M. gallisepticum atpE can guide the development of specific inhibitors through several approaches:

• Binding site identification: Detailed structural data can reveal unique pockets or interfaces that could serve as targets for small-molecule inhibitors. The hairpin-like structure with two hydrophobic domains may present unique binding opportunities.

• Structure-based drug design: Once binding sites are identified, computational methods such as molecular docking can be used to screen virtual libraries for compounds predicted to bind these sites.

• Selectivity considerations: Comparing the structure of M. gallisepticum atpE with that of the host (avian) ATP synthase can identify differences that could be exploited to develop inhibitors that selectively target the bacterial protein.

• Rational design strategies: Understanding the mechanism of proton translocation and c-ring rotation can inform the design of molecules that interfere with these processes. For example, compounds that bind at the interface between the c-ring and a-subunit could block proton transfer.

• In vitro validation systems: Developing reliable assays to test candidate inhibitors requires reconstituted systems where atpE function can be measured, such as proteoliposomes capable of generating ATP in response to a proton gradient.

The development of specific inhibitors against M. gallisepticum ATP synthase could provide new avenues for treating chronic respiratory disease in poultry, addressing the challenges posed by antibiotic resistance and antibiotic residues in meat and eggs .

What are the most appropriate controls when working with recombinant M. gallisepticum atpE in functional studies?

When designing functional studies with recombinant M. gallisepticum atpE, researchers should include these essential controls:

• Denatured protein control: Heat-inactivated or chemically denatured atpE protein to distinguish specific from non-specific effects.

• Related but distinct c-subunit: Using the c-subunit from a related Mycoplasma species or another bacterium to assess the specificity of observed effects.

• Site-directed mutants: Variants with mutations in key functional residues (e.g., proton-binding site) serve as negative controls for activity.

• Buffer controls: Samples containing all components except the protein to control for buffer effects, especially important given the specialized buffers needed for membrane proteins.

• Reconstitution controls: When performing reconstitution experiments, liposomes or nanodiscs without the protein should be tested to control for effects of the membrane environment.

• Intact ATP synthase complex: Where possible, comparing the behavior of isolated atpE to that of the complete ATP synthase complex can provide important context for interpreting results.

These controls help ensure that observed effects are specifically attributable to the functional properties of atpE rather than experimental artifacts.

How can researchers distinguish between effects on ATP synthesis versus ATP hydrolysis when studying atpE?

Distinguishing between effects on ATP synthesis versus ATP hydrolysis requires careful experimental design:

• Directional reconstitution: When reconstituting atpE into liposomes, ensuring a defined orientation allows researchers to distinguish inward versus outward pumping of protons.

• Separate assays for each direction:

  • ATP synthesis: Establishing a proton gradient (typically using acid-base transition or valinomycin with a K⁺ gradient) and measuring ATP production

  • ATP hydrolysis: Providing ATP and measuring either proton pumping (using pH-sensitive dyes) or phosphate release

• Inhibitor studies: Some inhibitors preferentially affect ATP synthesis versus hydrolysis, providing tools to distinguish these activities.

• Real-time monitoring: Using continuous assays rather than endpoint measurements allows detection of rate changes and transition effects.

• Coupled enzyme systems: For ATP synthesis measurements, luciferase-based detection systems provide sensitive, real-time monitoring.

These approaches help researchers determine whether experimental manipulations or potential therapeutic compounds target the synthetic or hydrolytic activities of the ATP synthase complex, which is crucial for understanding both basic biology and potential applications.

What are common pitfalls in interpreting data from studies on M. gallisepticum atpE, and how can they be avoided?

Researchers studying M. gallisepticum atpE should be aware of these common pitfalls in data interpretation:

• Aggregation artifacts: The hydrophobic nature of atpE can lead to aggregation, which may be mistaken for specific oligomerization. Solution: Use analytical ultracentrifugation or size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to distinguish specific oligomers from non-specific aggregates.

• Detergent effects: Detergents used to solubilize the protein can affect its structure and function. Solution: Test multiple detergent conditions and verify results using complementary techniques such as functional reconstitution into liposomes.

• Tag interference: Affinity tags may interfere with structure or function. Solution: Compare tagged and tag-cleaved versions of the protein, or use tags at different positions to confirm that observed properties are intrinsic to atpE.

• Expression system artifacts: Recombinant expression may result in proteins with non-native conformations. Solution: Validate key findings using protein isolated from M. gallisepticum when feasible.

• Oversimplification of the ATP synthase system: Studying atpE in isolation ignores its interactions with other ATP synthase subunits. Solution: Complement studies of isolated atpE with experiments on the intact ATP synthase complex when possible.

What statistical approaches are most appropriate for analyzing data from atpE functional studies?

When analyzing data from functional studies of M. gallisepticum atpE, researchers should consider these statistical approaches:

• Enzyme kinetics analysis: For functional assays measuring ATP synthesis or hydrolysis:

  • Michaelis-Menten kinetics to determine Km and Vmax

  • Hill equation analysis when cooperative behavior is observed

  • Competitive versus non-competitive inhibition models for inhibitor studies

• Time-series analysis: For measurements of proton translocation or conformational changes:

  • Exponential fitting to determine rate constants

  • Comparison of initial rates across experimental conditions

• Dose-response analysis: For inhibitor studies or activation by different substrates:

  • Determination of IC50 or EC50 values

  • Comparison of dose-response curves using extra sum-of-squares F test

• Comparative statistical methods: When comparing wild-type versus mutant proteins or different experimental conditions:

  • Analysis of variance (ANOVA) followed by appropriate post-hoc tests

  • Two-way ANOVA for experiments with multiple variables

• Sample size considerations: Power analysis to determine appropriate sample sizes, particularly important for in vivo studies such as those measuring immune responses to atpE-containing vaccines .

What emerging technologies could advance our understanding of M. gallisepticum atpE structure and function?

Several cutting-edge technologies show promise for advancing research on M. gallisepticum atpE:

• Cryo-electron microscopy (cryo-EM): Recent advances in resolution now permit visualization of membrane proteins at near-atomic resolution, potentially allowing determination of the complete structure of the M. gallisepticum ATP synthase complex.

• Single-molecule techniques: Methods such as single-molecule FRET (Förster Resonance Energy Transfer) can provide insights into the conformational dynamics of atpE during the catalytic cycle.

• Nanodiscs and native mass spectrometry: These approaches allow study of membrane proteins in more native-like environments while maintaining compatibility with analytical techniques.

• Integrative structural biology: Combining multiple techniques (X-ray crystallography, NMR, cryo-EM, computational modeling) to build comprehensive structural models.

• High-throughput mutagenesis coupled with deep sequencing: Systematic analysis of the effects of mutations throughout the protein to identify key functional residues.

• Advanced computational approaches: Molecular dynamics simulations with specialized force fields for membrane proteins can provide insights into atpE dynamics and interactions that are difficult to access experimentally.

These technologies could help resolve key questions about M. gallisepticum atpE, including its precise structure, dynamic behavior during ATP synthesis, and interactions with other ATP synthase components.

What are the most promising directions for translational research on M. gallisepticum atpE?

Translational research on M. gallisepticum atpE shows promise in several directions:

• Vaccine development: The essential nature of ATP synthase makes it a potentially valuable target for vaccines against M. gallisepticum. Plant-based expression systems have already demonstrated promise for producing M. gallisepticum antigens that elicit immune responses in chickens .

• Specific inhibitors: Structure-based design of inhibitors targeting unique features of M. gallisepticum atpE could lead to new antimicrobials with minimal cross-reactivity with host ATP synthases.

• Diagnostic applications: Antibodies against distinctive epitopes of M. gallisepticum atpE could be used in diagnostic tests for detecting the pathogen in poultry.

• Synthetic biology applications: Understanding the minimal functionality of ATP synthase in one of the simplest self-replicating organisms could inform efforts to design synthetic biological systems with optimized energy metabolism.

• Cross-species protection strategies: Identifying conserved features of atpE across multiple Mycoplasma species could lead to broader-spectrum approaches for controlling mycoplasmosis in various hosts.