Recombinant Bacillus megaterium ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its functional significance?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F₀ portion of ATP synthase. This membrane-embedded protein forms a multimeric ring structure that is essential for the mechanical coupling of proton translocation to ATP synthesis. The c-subunit ring rotates when protons flow along an electrochemical gradient across the membrane, driving the synthesis of ATP in the F₁ portion of the enzyme . The c-subunit ring is directly involved in energy conversion, transforming the electrochemical potential energy of the proton gradient into mechanical rotational energy that powers ATP production. Research has demonstrated that alterations in the c-subunit can lead to aberrant cellular metabolism, highlighting its crucial role in maintaining proper cellular energy homeostasis .

Why is Bacillus megaterium specifically used for recombinant atpE production?

Bacillus megaterium has emerged as an exceptional host for recombinant protein production, including ATP synthase components, for several research-significant reasons. This Gram-positive bacterium has been systematically optimized through the development of specialized plasmids with inducible promoter systems, compatible origins of replication, purification tags, and various protein secretion signals . These genetic tools, combined with optimized host strains and cultivation conditions, make B. megaterium particularly suitable for the expression of membrane proteins like atpE. The system allows for high protein yields (in the g/L scale) and has demonstrated success in the simultaneous co-production of up to 14 recombinant proteins, making it valuable for expressing multi-protein complexes like ATP synthase . Additionally, B. megaterium lacks certain proteases that can degrade heterologous proteins, offering enhanced stability for recombinant products.

How does the stoichiometry of c-subunits vary across species and what are the functional implications?

The c-subunit stoichiometry varies significantly across different organisms, with the number of c-subunits (n) in each ring ranging from c₁₀ to c₁₅ depending on the species . This variation directly affects the coupling ratio (protons translocated : ATP generated), which ranges from 3.3 to 5.0 among different organisms. The coupling ratio is entirely dependent on the number of c-subunits, since the number of ATP molecules generated per c₍ₙ₎ rotation is consistently three in all known ATP synthases .

While multiple hypotheses have been proposed, the evolutionary and physiological significance of this stoichiometric variation remains incompletely understood, making it an active area of research . Researchers speculate that the variation may reflect adaptations to different environmental conditions or energy demands across species.

What cloning strategies are most effective for recombinant B. megaterium atpE expression?

For effective cloning and expression of atpE from B. megaterium, a systematic approach utilizing optimized expression vectors is critical. Based on successful protein expression systems, a recommended workflow includes:

• Gene optimization: Codon optimization for B. megaterium expression, with consideration of mRNA secondary structure and removal of rare codons.

• Vector selection: Plasmids with inducible promoter systems (particularly xylose-inducible promoters) that have been specifically optimized for B. megaterium . These vectors should include:

  • Compatible origins of replication

  • Small purification tags (His₆ or Strep-tag II) for simplified purification

  • Signal sequences for appropriate protein localization

• Expression construct design: For membrane proteins like atpE, inclusion of a short linker between the tag and protein may improve folding and function.

The expression strategy should be designed with consideration of the unique properties of ATP synthase subunit c, particularly its hydrophobicity and tendency to form multimers. When cloning, researchers should verify sequence integrity through DNA sequencing to confirm the absence of mutations that might affect protein folding or function .

What purification protocols yield highest quality recombinant atpE protein?

Purification of recombinant atpE requires specialized approaches due to its hydrophobic nature and membrane localization. A comprehensive purification protocol includes:

• Cell lysis optimization: Gentle disruption using enzymatic methods (lysozyme treatment) followed by mechanical disruption (French press or sonication) in buffer containing stabilizing agents.

• Membrane fraction isolation: Differential centrifugation to isolate membrane fractions containing the c-subunit.

• Detergent solubilization: Careful selection of detergents (DDM, LDAO, or C₁₂E₈) at concentrations that efficiently solubilize the protein without causing denaturation.

• Affinity chromatography: Utilizing the engineered tag (typically His₆) for initial purification, with attention to detergent concentration in all buffers.

• Size exclusion chromatography: For separating properly folded monomers or assembled multimers from aggregates.

This multi-step approach has been successfully implemented for purifying recombinant c-subunits from various organisms, yielding milligram quantities of purified protein . Quality assessment through SDS-PAGE and western blotting is essential at each purification step to monitor protein integrity and purity.

How can researchers assess proper folding and functionality of recombinant atpE?

Assessing the proper folding and functionality of recombinant atpE requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy: To evaluate the secondary structure content, particularly the alpha-helical content that is characteristic of properly folded c-subunits.

• Reconstitution assays: Incorporation of purified atpE into liposomes or nanodiscs, followed by proton translocation assays using pH-sensitive fluorescent dyes.

• Assembly assessment: Analysis of c-subunit oligomerization through native gel electrophoresis, size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), or electron microscopy.

• Functional complementation: Introduction of recombinant atpE into atpE-deficient bacterial strains to assess functional rescue.

• Binding studies: Evaluation of interactions with other ATP synthase subunits, particularly the a-subunit, which forms the proton channel with the c-ring.

Each of these approaches provides distinct but complementary information about the structural integrity and functional capacity of the recombinant protein, allowing researchers to confirm that their purified protein is suitable for downstream applications .

How can recombinant atpE be used to study c-subunit ring assembly and stoichiometry determination?

Investigating c-subunit ring assembly and stoichiometry is critical for understanding ATP synthase function. Recombinant atpE provides a valuable tool for such studies through several advanced approaches:

• In vitro reconstitution: Purified recombinant c-subunits can be used for reconstitution experiments to study the factors that influence ring assembly. By controlling the experimental conditions (pH, lipid composition, ionic strength), researchers can investigate the parameters that affect ring formation and stability .

• Mass spectrometry: Native mass spectrometry of reconstituted c-rings can precisely determine the stoichiometry of the complex. This approach has been successfully used to identify the exact number of c-subunits in rings from various organisms.

• Cryo-electron microscopy: Reconstituted c-rings can be analyzed by cryo-EM to determine the structural arrangement of subunits and confirm stoichiometry through direct visualization.

• Hybrid approaches: Combining recombinant c-subunits with other ATP synthase components allows for the generation of chimeric complexes that can reveal the factors determining species-specific c-ring stoichiometries .

These methodologies are particularly valuable because they enable the systematic manipulation of the c-subunit structure through site-directed mutagenesis, allowing researchers to investigate how specific residues influence ring assembly and stoichiometry .

What is the relationship between atpE and regulatory subunits of ATP synthase?

The interaction between the c-subunit ring and regulatory subunits, particularly the ε subunit, represents a sophisticated regulatory mechanism in ATP synthase. Research has revealed that:

• Regulatory mechanisms: While the c-subunit ring is primarily involved in proton translocation, it functions in concert with regulatory subunits that modulate ATP synthase activity. The ε subunit is a key regulatory element in bacterial and plant F₁-ATPases .

• Distinct regulatory roles: In Bacillus subtilis, the ε subunit has been shown to relieve MgADP inhibition rather than inhibiting the enzyme, demonstrating a species-specific regulatory mechanism . This contrasts with the inhibitory role of the ε subunit in other organisms, highlighting the diverse regulatory strategies across bacterial species.

• Structural basis: The interaction between the c-ring and the ε subunit involves the C-terminal domain of ε, which can adopt different conformations that either permit or restrict rotation of the c-ring .

• Functional implications: The regulatory interactions affect the enzyme's ability to switch between ATP synthesis and hydrolysis modes, which is critical for maintaining cellular energy homeostasis under varying conditions.

Understanding these interactions is essential for developing a complete model of ATP synthase regulation and could potentially inform approaches to modulate ATP synthase activity in various biological contexts .

How do mutations in atpE affect proton translocation and ATP synthesis efficiency?

Mutations in the c-subunit can have profound effects on ATP synthase function due to the critical role of this subunit in proton translocation. Key research findings include:

• Proton-binding site mutations: Alterations to the conserved acidic residue (typically Asp or Glu) that serves as the proton-binding site can severely impair or completely abolish proton translocation, demonstrating the essential role of this residue in the proton transport mechanism.

• Interface mutations: Changes to residues at the interface between adjacent c-subunits can affect ring stability and assembly, potentially altering the stoichiometry and consequently the coupling ratio of the enzyme.

• Membrane interaction mutations: Modifications to residues that interact with the lipid bilayer can alter the positioning of the c-ring within the membrane, affecting its rotational dynamics and interaction with the a-subunit.

• Regulatory mutations: Some mutations can lead to a "leaky" c-subunit ring, resulting in proton leakage across the membrane without coupling to ATP synthesis. This has been observed in certain diseases, such as Fragile X syndrome, where ATP synthase c-subunit leak causes aberrant cellular metabolism .

These studies provide valuable insights into the structure-function relationship of the c-subunit and can inform the development of strategies to modulate ATP synthase activity in various biological contexts .

What are common challenges in recombinant atpE expression and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant atpE, each requiring specific troubleshooting approaches:

• Poor expression levels: The hydrophobic nature of atpE often leads to low expression levels due to protein aggregation or toxicity to the host cell.

  • Solution: Optimize expression conditions by testing different induction temperatures (typically lower temperatures of 18-25°C), inducer concentrations, and duration of induction. Using specialized B. megaterium strains that have been genetically improved for membrane protein expression can significantly enhance yields .

• Protein misfolding and inclusion body formation: The c-subunit may form inclusion bodies rather than properly integrating into membranes.

  • Solution: Employ fusion partners like thioredoxin or SUMO that enhance solubility, or develop refolding protocols from inclusion bodies using gradual detergent dialysis methods.

• Proteolytic degradation: The recombinant protein may be subject to degradation by host proteases.

  • Solution: Include protease inhibitors during purification and consider using B. megaterium strains with reduced protease activity .

• Difficulty in verifying expression: The small size and hydrophobic nature of atpE can make detection challenging.

  • Solution: Employ specialized gel systems for small hydrophobic proteins, such as Tricine-SDS-PAGE, and consider western blotting with antibodies against the fusion tag for enhanced sensitivity.

• Loss during purification: The protein may aggregate or bind non-specifically during purification steps.

  • Solution: Maintain an appropriate detergent concentration above the critical micelle concentration throughout all purification steps, and test different detergents for optimal extraction and stability.

These methodological refinements have been developed based on extensive troubleshooting across multiple studies of ATP synthase components and can significantly improve the success rate of recombinant atpE production .

How can researchers interpret contradictory results regarding c-subunit stoichiometry?

Contradictory findings regarding c-subunit stoichiometry are not uncommon in the literature and require careful analysis. Researchers should consider:

When interpreting contradictory results, researchers should carefully evaluate the experimental conditions, consider the limitations of each technique, and look for consensus across multiple independent approaches. The stoichiometric variation in c-rings across species is now well-established, ranging from c₁₀ to c₁₅, suggesting that some of the contradictions in the literature reflect genuine biological diversity rather than experimental artifacts .

What approaches can verify the functional integration of recombinant atpE into ATP synthase complexes?

Confirming that recombinant atpE properly integrates into functional ATP synthase complexes requires multiple verification approaches:

• Complementation studies: Transform atpE-deletion mutant strains with the recombinant atpE gene and assess restoration of ATP synthesis activity and growth phenotypes.

  • Analysis metrics: Growth rates in media requiring oxidative phosphorylation, ATP/ADP ratios, and membrane potential measurements.

• Co-purification analysis: Express tagged versions of other ATP synthase subunits alongside recombinant atpE and perform pull-down experiments to verify complex formation.

  • Analysis metrics: SDS-PAGE followed by western blotting or mass spectrometry to confirm the presence of atpE in the purified complex.

• Activity assays: Measure ATP synthesis or hydrolysis activities in reconstituted proteoliposomes containing the recombinant atpE.

  • Analysis metrics: ATP synthesis rates under an artificial proton gradient or ATP hydrolysis rates coupled to NADH oxidation, compared against native enzyme preparations.

• Structural verification: Employ electron microscopy or atomic force microscopy to visualize the integrated c-ring within the ATP synthase complex.

  • Analysis metrics: Structural parameters of the c-ring (diameter, symmetry) should match known values for the species being studied.

• Biophysical interaction studies: Use techniques like surface plasmon resonance (SPR) or microscale thermophoresis to quantify binding between recombinant atpE and other subunits, particularly subunit a.

  • Analysis metrics: Binding affinities between subunits should be in the expected range for stable complex formation.

These complementary approaches provide robust verification of functional integration and can help identify specific issues if the recombinant protein fails to properly incorporate into the complex .

How can atpE studies contribute to understanding bacterial energy metabolism in challenging environments?

Research on ATP synthase subunit c is increasingly important for understanding bacterial adaptation to extreme or changing environments:

• Adaptation mechanisms: Studies of atpE from extremophiles reveal adaptations in c-subunit structure that optimize ATP synthase function under challenging conditions (extreme pH, temperature, or pressure). Recombinant expression of these variants in B. megaterium provides a system for detailed characterization of these adaptations .

• Energy efficiency variations: The stoichiometric differences in c-rings (c₁₀-c₁₅) directly affect the energetic efficiency of ATP production. Research suggests that organisms in energy-limited environments may have evolved c-rings with fewer subunits to maximize ATP output per proton, while those in energy-rich environments may prioritize other factors .

• Regulatory responses: The interaction between atpE and regulatory subunits like the ε subunit reveals sophisticated mechanisms for modulating ATP synthase activity in response to changing environmental conditions, such as the relief of MgADP inhibition observed in Bacillus species .

• Antimicrobial targets: The essential nature of ATP synthase makes it a potential target for antimicrobials, with the c-subunit offering unique targeting opportunities due to its conserved structure but species-specific features. Research using recombinant atpE can help identify and validate such targets, particularly in pathogenic bacteria like Mycobacterium tuberculosis .

These studies not only advance our fundamental understanding of bioenergetics but also have potential applications in synthetic biology, where optimized ATP synthase variants could be engineered for specific applications in artificial systems or modified organisms .

What role might atpE play in developing new approaches to modulate bacterial metabolism?

ATP synthase subunit c represents a potential intervention point for controlling bacterial metabolism, with several promising research directions:

• Metabolic engineering: Manipulation of c-subunit stoichiometry through recombinant expression could potentially alter the energetic efficiency of ATP production, enabling fine-tuning of cellular metabolism for biotechnological applications .

• Synthetic biology applications: Engineered c-subunits with modified properties (proton affinity, ring stability, regulatory interactions) could serve as components in synthetic biological systems with customized energetic parameters.

• Antimicrobial development: The essential role of atpE in bacterial energy metabolism makes it a potential target for new antimicrobials. Research using recombinant B. megaterium systems can facilitate the screening of compounds that specifically target the c-subunit .

• Biotechnological applications: Understanding the mechanism of proton translocation through the c-ring could inform the development of biomimetic energy conversion systems or nano-scale rotary motors based on ATP synthase principles.

The ability to produce recombinant atpE and reconstitute functional c-rings provides researchers with powerful tools to investigate these possibilities, potentially leading to novel applications in medicine, biotechnology, and synthetic biology .