Recombinant Acidovorax ebreus ATP synthase subunit c (atpE)

What is the structural organization of ATP synthase subunit c in bacteria like Acidovorax ebreus?

ATP synthase subunit c (atpE) forms a cylindrical oligomeric ring in the membrane domain (F0) of the ATP synthase complex. In bacteria, this c-ring typically consists of 10-15 identical c-subunits, though the exact stoichiometry varies between species. Each c-subunit contains two transmembrane α-helices with a critical conserved glutamic acid residue (equivalent to E56 in Bacillus PS3) that participates directly in proton translocation . This residue undergoes protonation and deprotonation cycles that drive rotation of the c-ring relative to the a-subunit. The c-ring functions as a rotor that mechanically couples proton flow across the membrane to ATP synthesis in the F1 domain.

The mature subunit c is highly hydrophobic and relatively small (approximately 8 kDa), making it challenging to work with in recombinant systems. The c-ring structure is critical for function as it determines the H+/ATP ratio during energy conversion, with each c-subunit translocating one proton per complete rotation.

What expression systems are most suitable for recombinant production of bacterial ATP synthase subunit c?

For bacterial ATP synthase subunit c production, Escherichia coli expression systems have proven most effective due to their compatibility with bacterial membrane proteins and scalability. Several specific approaches have demonstrated success:

• Fusion protein strategies: Expressing subunit c as a fusion with maltose-binding protein (MBP) significantly enhances solubility and facilitates purification through affinity chromatography . This approach allows for the recovery of functional protein after cleaving the fusion partner.

• Codon optimization: Optimizing the codon usage of the atpE gene according to the expression host improves translation efficiency and protein yield.

• Inducible expression systems: IPTG-inducible promoters (T7, lac, tac) allow controlled expression, which is essential as overexpression of membrane proteins can be toxic to host cells.

• Cell-free expression systems: For challenging membrane proteins, cell-free systems can circumvent toxicity issues and provide direct access to the synthesized protein.

The choice of expression system should be guided by the specific experimental requirements and downstream applications. For structural studies requiring high purity and yield, the MBP fusion approach in E. coli has proven particularly effective for chloroplast subunit c and can be adapted for bacterial orthologs .

What purification strategies overcome the hydrophobic nature of ATP synthase subunit c?

Purifying ATP synthase subunit c presents unique challenges due to its extreme hydrophobicity and tendency to form aggregates. Successful purification strategies include:

Table 1: Purification Strategies for Recombinant ATP Synthase Subunit c

The most effective approach combines multiple techniques: initial solubilization with detergents, affinity purification via fusion tags, and final polishing via size exclusion chromatography. For functional studies, it's critical to verify that the purified protein maintains its native alpha-helical secondary structure, which can be confirmed through circular dichroism spectroscopy .

How can researchers design experiments to assess the functional integrity of recombinant ATP synthase subunit c?

Assessing the functional integrity of recombinant ATP synthase subunit c requires experimental approaches that evaluate both its structural integration into the c-ring and its participation in proton translocation. Key experimental designs include:

• Reconstitution assays: Reconstitute purified subunit c into liposomes with other F0 components (particularly subunit a) to form a minimal functional unit capable of proton translocation. Measure proton pumping using pH-sensitive fluorescent dyes.

• Complementation studies: Express recombinant subunit c in ATP synthase-deficient bacterial strains and assess restoration of growth on non-fermentable carbon sources or ATP synthesis activity in membrane vesicles .

• Site-directed mutagenesis: Introduce mutations in the conserved glutamic acid residue (equivalent to E56 in Bacillus PS3) and assess the impact on proton translocation and ATP synthesis. The E to D mutation typically reduces but doesn't eliminate activity, while E to Q abolishes function .

• Single-molecule rotation assays: Attach fluorescent markers to the c-ring and observe rotation directly using total internal reflection fluorescence microscopy when ATP is added to the system.

• Proton transfer-coupled molecular dynamics simulations: Complement experimental approaches with computational modeling to understand the energetics and kinetics of proton transfer through the c-ring .

These approaches provide complementary data on different aspects of subunit c function and should be selected based on the specific research questions being addressed.

What are the common challenges in recombinant production of ATP synthase subunit c from Acidovorax ebreus and how can they be overcome?

Recombinant production of ATP synthase subunit c from bacteria like Acidovorax ebreus faces several significant challenges:

Table 2: Challenges and Solutions in Recombinant atpE Production

Implementing a Design of Transfections (DoT) workflow similar to that developed for neural progenitors can systematically identify optimal conditions for recombinant expression . By simultaneously varying key parameters (induction time, temperature, media composition, and host strain), researchers can mathematically model the relationship between these factors and protein yield to identify optimal conditions.

For Acidovorax ebreus atpE specifically, the use of specialized E. coli strains with enhanced membrane protein expression capabilities (such as C41/C43 or Lemo21) combined with a fusion tag strategy offers the most promising approach to overcome these challenges.

How can researchers accurately determine the stoichiometry of ATP synthase c-rings from Acidovorax ebreus?

Determining the precise stoichiometry of c-rings is crucial for understanding the bioenergetics of ATP synthesis, as it directly influences the H+/ATP ratio. Several complementary approaches can be employed:

• Cryo-electron microscopy (cryo-EM): High-resolution cryo-EM can directly visualize the c-ring structure and count the number of c-subunits. This technique requires purified c-rings reconstituted in detergent micelles or nanodiscs.

• Mass spectrometry of intact c-rings: Native mass spectrometry of purified c-rings can determine the oligomeric state based on the total molecular weight.

• Cross-linking and SDS-PAGE analysis: Chemical cross-linking of adjacent c-subunits followed by SDS-PAGE can create a ladder of cross-linked products that reveal the total number of subunits.

• Atomic force microscopy (AFM): AFM of membrane-embedded c-rings can resolve individual subunits and determine their number directly.

• X-ray crystallography: Although challenging, crystallographic analysis provides definitive structural data on c-ring composition.

The number of c-subunits per ring is known to range from c10 to c15 among organisms for which it has been determined, resulting in coupling ratios (ions transported:ATP generated) ranging from 3.3 to 5.0 . The c-ring stoichiometry directly affects the bioenergetic efficiency of ATP synthesis, with larger rings requiring more protons per ATP but potentially operating at lower proton motive force.

For accurate determination, multiple methods should be employed and compared, as each technique has specific limitations that could lead to artifacts in stoichiometry assessment.

What approaches are most effective for studying the functional impact of site-directed mutations in ATP synthase subunit c?

Site-directed mutagenesis of ATP synthase subunit c provides valuable insights into structure-function relationships, particularly regarding proton translocation. The most effective approaches include:

• Single-chain c-ring technology: Genetically fused c-subunits allow precise control over the position and number of mutations within the c-ring. This approach enables detailed studies of cooperation between c-subunits during rotation .

• Inverted membrane vesicles assays: Prepare inverted membrane vesicles from cells expressing mutated ATP synthase and measure ATP synthesis activity driven by artificial proton gradients .

• Proton pumping measurements: Assess ATP-driven proton pumping activity using pH-sensitive fluorescent dyes to quantify the impact of mutations on proton translocation.

• Molecular dynamics simulations: Complement experimental approaches with proton transfer-coupled molecular dynamics simulations to understand atomic-level changes in proton binding and transfer kinetics .

• Thermal stability assays: Investigate the impact of mutations on c-ring stability using thermal shift assays or differential scanning calorimetry.

Research on Bacillus PS3 ATP synthase has demonstrated that a single E56D mutation in one c-subunit reduces ATP synthesis activity to approximately 35.8% of wild-type, while double E56D mutations cause further activity reduction. Significantly, the activity decreases more as the distance between mutation sites increases, revealing functional cooperation among c-subunits .

These findings suggest that at least three c-subunits cooperate at the a/c interface during c-ring rotation, consistent with molecular dynamics simulations showing multiple deprotonated carboxyl residues facing the a-subunit simultaneously .

How does ATP synthase subunit c isoform diversity impact functional studies of recombinant proteins?

The diversity of ATP synthase subunit c isoforms presents both challenges and opportunities for functional studies:

Table 3: Impact of Subunit c Isoform Diversity on Functional Studies

In mammals, ATP synthase subunit c exists as three isoforms (P1, P2, P3) that differ only in their mitochondrial targeting peptides, with identical mature peptides . Surprisingly, these isoforms are not functionally redundant. Silencing any individual isoform results in ATP synthesis defects, indicating specialized functions conferred by the targeting peptides beyond protein import .

For recombinant expression studies of bacterial subunit c like that from Acidovorax ebreus, researchers should consider:

• Investigating whether multiple isoforms exist in the native organism

• Including any native targeting sequences when designing recombinant constructs

• Assessing the impact of heterologous expression on protein function

• Studying potential interactions with respiratory chain components

These considerations are particularly important when using recombinant subunit c for functional reconstitution experiments or when interpreting phenotypes of complementation studies.

How can researchers study cooperation among c-subunits in the ATP synthase c-ring?

Recent research has revealed functional cooperation among c-subunits during ATP synthase operation. Sophisticated approaches to study this cooperation include:

• Genetically fused single-chain c-rings: This innovative approach enables precise control over the position and number of mutations within the c-ring. By creating fused c10 polypeptides with specific mutations in defined positions, researchers can study how c-subunits interact functionally .

• Controlled introduction of mutations: Using the single-chain approach, researchers can introduce mutations (such as E56D in Bacillus PS3) at specific positions and assess their impact on ATP synthesis, ATP-driven proton pumping, and ATP hydrolysis activities .

• Analysis of spatial effects: By varying the distance between mutation sites, researchers can determine how spatial arrangement affects cooperation. Studies show that ATP synthesis activity decreases further as the distance between two E56D mutations increases, directly demonstrating cooperation .

• Proton transfer-coupled molecular simulations: Computational approaches can reveal how proton uptake times are shared between multiple c-subunits and how mutations affect these dynamics .

• Single-molecule rotation assays: Direct observation of c-ring rotation using fluorescence microscopy can reveal how mutations affect rotation speed and pausing.

Research on Bacillus PS3 ATP synthase suggests that at least three c-subunits cooperate at the a/c interface during rotation. Molecular dynamics simulations show that in wild-type ATP synthase, two or three c-subunits with deprotonated carboxyl residues face the a-subunit simultaneously, allowing sharing of proton uptake waiting times .

This cooperation explains why mutations at distant sites cause more severe functional defects than mutations at adjacent sites. The findings have profound implications for understanding the mechanism of rotary catalysis in ATP synthases across species.

What are the current limitations in our understanding of ATP synthase subunit c structure and function?

Despite significant advances, several important knowledge gaps remain in our understanding of ATP synthase subunit c:

• Species-specific c-ring stoichiometry: While c-ring stoichiometry is known to vary from c10 to c15 among organisms, the factors that determine this variation remain undefined . The evolutionary pressures and adaptive advantages of different stoichiometries are poorly understood.

• Assembly mechanisms: The process by which individual c-subunits assemble into functional c-rings in vivo remains unclear, particularly for bacteria like Acidovorax ebreus.

• Proton path details: The precise pathway of protons through the a/c interface and the atomic-level details of proton transfer require further elucidation.

• Rotational dynamics: The exact sequence and timing of events during c-ring rotation, including potential cooperativity between distant c-subunits, needs further investigation.

• Regulatory mechanisms: How ATP synthase activity is regulated at the level of c-ring function remains poorly understood, particularly in environmental bacteria that may face diverse bioenergetic challenges.

Addressing these knowledge gaps will require continued development of innovative experimental approaches, particularly those that can study the dynamics of the system under physiologically relevant conditions.

What future research directions are most promising for advancing our understanding of bacterial ATP synthase subunit c?

Future research on bacterial ATP synthase subunit c, including that from Acidovorax ebreus, should focus on several promising directions:

• Cryo-EM structural studies: High-resolution structures of intact ATP synthase complexes from diverse bacterial species will reveal species-specific features and common mechanisms.

• Single-molecule approaches: Real-time observation of c-ring rotation and proton translocation will provide insights into the dynamics of energy conversion.

• Systems biology integration: Understanding how ATP synthase function integrates with broader cellular metabolism in different environmental conditions.

• Comparative analyses: Systematic comparison of c-ring structures and functions across diverse bacterial species, including extremophiles, to understand evolutionary adaptations.

• Synthetic biology applications: Engineering c-rings with altered stoichiometry or proton binding properties to create ATP synthases with novel bioenergetic properties.

• Advanced computational models: Development of multiscale computational approaches that can bridge quantum mechanical aspects of proton transfer with macroscale rotation and ATP synthesis.