Recombinant Desulfococcus oleovorans ATP synthase subunit c (atpE)

What is the ATP synthase subunit c in Desulfococcus oleovorans?

The ATP synthase subunit c in Desulfococcus oleovorans is a critical component of the F1F0-ATP synthase complex encoded by the atpE gene. Like other c subunits, it assembles into a cylindrical oligomer (c-ring) within the membrane-embedded F0 portion of the ATP synthase. This subunit plays a fundamental role in the proton-pumping process during ATP synthesis, working in cooperation with subunit a (Atp6-equivalent) to convert the proton gradient generated by the respiratory chain into the mechanical energy needed for ATP production . The c-ring forms the rotor component of this molecular machine, and in Desulfococcus oleovorans, as in other sulfate-reducing bacteria, it has evolved specific adaptations to function in the organism's unique bioenergetic context.

How does the c-ring structure relate to ATP synthase function?

The c-ring structure is central to ATP synthase function as it determines the ion-to-ATP ratio, a fundamental parameter of cellular bioenergetics. Each c-subunit harbors a conserved ion-binding site, and as protons (or sodium ions in some organisms) bind to these sites, they drive the rotation of the c-ring. The stoichiometry of the c-ring—the number of c-subunits in the ring—directly affects how many ions must be transported to generate one ATP molecule . For example, if a c-ring contains 13 subunits, as has been observed in some alkaliphilic bacteria, then 13 protons would be required for a complete rotation, which leads to the synthesis of 3 ATP molecules (resulting in an H+/ATP ratio of approximately 4.3) . This stoichiometry is critically important for an organism's ability to generate sufficient ATP under different environmental conditions.

What experimental techniques are commonly used to study recombinant ATP synthase subunit c?

Multiple complementary techniques are essential for comprehensive characterization of recombinant ATP synthase subunit c. Researchers typically begin with molecular cloning of the atpE gene from Desulfococcus oleovorans genomic DNA, followed by heterologous expression in a suitable host such as E. coli. Purification often employs affinity chromatography using His-tagged constructs . Structural studies commonly utilize atomic force microscopy (AFM) and X-ray crystallography to determine c-ring stoichiometry and subunit arrangement . Functional characterization requires ATP hydrolysis or synthesis assays, proton-pumping measurements, and analysis of c-ring assembly using techniques such as blue native polyacrylamide gel electrophoresis. Cross-linking studies and mass spectrometry are valuable for investigating subunit interactions. Site-directed mutagenesis is crucial for examining the functional importance of specific residues in ion binding and c-ring assembly.

What are the optimal conditions for expressing recombinant D. oleovorans ATP synthase subunit c?

Optimal expression of recombinant D. oleovorans ATP synthase subunit c requires careful consideration of several parameters. Based on successful approaches with similar proteins from sulfate-reducing bacteria, expression in E. coli C43(DE3) or BL21(DE3) strains is recommended due to their tolerance for membrane protein expression . The atpE gene should be codon-optimized for E. coli and cloned into vectors with tightly controlled promoters such as pET or pBAD systems. Expression is best induced in mid-log phase (OD600 0.6-0.8) with low concentrations of inducer (0.1-0.5 mM IPTG for T7 promoter systems) at reduced temperatures (18-25°C) for 12-16 hours to minimize inclusion body formation and maximize proper membrane insertion. The growth medium should be supplemented with additional phosphate buffer and glucose to maintain pH and provide energy for protein folding. For membrane proteins like subunit c, addition of 0.5-1% glycerol can enhance membrane integrity and improve protein folding and stability during expression.

What purification strategy yields the highest purity and activity of the recombinant protein?

A multi-step purification strategy is necessary to obtain highly pure and functional recombinant ATP synthase subunit c. After cell lysis, membrane fractions are isolated by differential centrifugation and solubilized with appropriate detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin at concentrations just above their critical micelle concentration (CMC) . For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides initial purification. This should be followed by size exclusion chromatography to separate monomeric from oligomeric forms and remove aggregates. Ion exchange chromatography may be necessary for removing residual contaminants. Throughout purification, it's crucial to maintain a consistent detergent concentration above the CMC in all buffers to prevent protein aggregation. The purified protein should be assessed for purity using SDS-PAGE and Western blotting, with functionality verified through reconstitution in liposomes followed by proton translocation or ATP synthesis assays.

How can researchers confirm the correct folding and oligomerization of recombinant D. oleovorans ATP synthase subunit c?

Confirming correct folding and oligomerization of recombinant D. oleovorans ATP synthase subunit c requires a combination of structural and functional approaches. Circular dichroism spectroscopy can verify secondary structure content, which should show predominant α-helical characteristics typical of c-subunits . Blue native PAGE can determine the oligomeric state, with properly formed c-rings migrating at positions corresponding to their molecular weight as stable complexes. Mass spectrometry techniques, particularly native mass spectrometry, can precisely determine c-ring stoichiometry. Cross-linking experiments followed by mass spectrometry analysis can verify proper subunit-subunit contacts. For functional validation, reconstitution into liposomes or nanodiscs followed by proton translocation assays provides evidence of proper folding. A definitive approach involves reconstituting the c-subunit with other purified ATP synthase components to form a functional holoenzyme capable of ATP synthesis or hydrolysis . Microscopy techniques like AFM or cryo-EM provide direct visualization of ring formation and structural integrity.

How does the c-ring stoichiometry of D. oleovorans ATP synthase compare to other extremophiles?

The c-ring stoichiometry of D. oleovorans ATP synthase represents an important adaptation to its specific ecological niche as a sulfate-reducing bacterium. While the exact c-ring stoichiometry for D. oleovorans has not been definitively reported in the provided search results, comparative analysis with other extremophiles provides valuable insights. In the extreme alkaliphile Bacillus pseudofirmus OF4, the c-ring contains 13 subunits, which is adapted for optimal ATP synthesis at high pH environments (>10) . This contrasts with the c10 configuration commonly found in mesophilic bacteria and the c8 rings observed in some mammalian mitochondrial ATP synthases . The stoichiometry differences directly impact the ion-to-ATP ratio, with larger c-rings (higher c-subunit numbers) requiring more ions per ATP synthesized but generating greater torque at lower driving force. For D. oleovorans, which thrives in anoxic environments with limited energy availability, its c-ring stoichiometry has likely evolved to optimize ATP production under these constrained bioenergetic conditions.

What role does the ATP synthase c-subunit play in the bioenergetics of sulfate-reducing bacteria?

In sulfate-reducing bacteria like D. oleovorans, the ATP synthase c-subunit occupies a crucial position in cellular bioenergetics, particularly in the context of their unique energy metabolism. These organisms utilize sulfate as the terminal electron acceptor in a process that generates a relatively small proton gradient compared to oxygen respiration . The ATP synthase must therefore be optimized to function efficiently with this limited proton motive force. The c-subunit ring serves as the primary energy-converting element, transforming the electrochemical potential into rotational motion. The efficiency of this conversion depends on several factors: the c-ring stoichiometry, the binding affinity for protons, and the interaction with other components of the respiratory chain. In D. oleovorans and related sulfate reducers, the ATP synthase likely operates in close coordination with specialized respiratory complexes such as the Qmo (quinone-interacting membrane-bound oxidoreductase) complex, which has been demonstrated as essential for sulfate respiration in the related Desulfovibrio vulgaris . This coordination ensures that the limited energy available from sulfate reduction can be effectively harnessed for ATP synthesis.

How does the D. oleovorans ATP synthase subunit c differ from those in other sulfate-reducing bacteria?

The D. oleovorans ATP synthase subunit c shares key structural features with other sulfate-reducing bacteria (SRB), but exhibits unique adaptations reflecting its specific ecological niche. While the core functional regions, including the ion-binding site and transmembrane helices, maintain high conservation, variations in the amino acid sequence likely reflect adaptations to D. oleovorans' specific growth conditions. In comparison with Desulfovibrio species, another well-studied group of SRB, D. oleovorans belongs to a different phylogenetic lineage (Deltaproteobacteria, Desulfobacteraceae family) and has adaptations for growth on different carbon sources . One notable difference is that while Desulfovibrio vulgaris and related species have genes encoding the Qmo complex in close proximity to their ATP synthase genes, suggesting coordinated expression and function, the genomic arrangement in D. oleovorans shows some distinct features . The search results indicate that D. oleovorans lacks homologs to DVU0851 (a hypothetical protein found in Desulfovibrio strains), suggesting different regulatory mechanisms or protein interactions for its ATP synthase . These differences likely reflect the ecological specialization of D. oleovorans for degrading hydrocarbons while using sulfate as an electron acceptor.

What insights can be gained from site-directed mutagenesis studies of conserved motifs in ATP synthase subunit c?

Site-directed mutagenesis of conserved motifs in ATP synthase subunit c provides crucial insights into structure-function relationships that impact cellular bioenergetics. Studies in B. pseudofirmus OF4 demonstrated that changing the conserved AxAxAxA motif to GxGxGxG (the motif found in most other bacteria) altered the c-ring stoichiometry from c13 to c12, with significant consequences for growth at high pH . Similar mutagenesis approaches applied to D. oleovorans subunit c could reveal how specific amino acid positions influence:

• Ion binding affinity and specificity: Mutations in the ion-binding site can alter proton coordination, affecting the pKa and the energy required for protonation/deprotonation.

• C-ring assembly and stability: Changes to residues at subunit interfaces can affect oligomerization, potentially altering the c-ring stoichiometry and stability.

• Interactions with other ATP synthase subunits: Mutations at interfaces with subunits a or b can disrupt the rotational mechanism or proton path.

• Environmental adaptations: Specific residues may be crucial for function under the low-energy conditions typical of sulfate reduction.

These studies would not only enhance our understanding of D. oleovorans bioenergetics but could also provide transferable insights for biotechnological applications or understanding similar adaptations in other extremophiles.

Can the recombinant D. oleovorans ATP synthase subunit c be incorporated into functional hybrid ATP synthases?

Recombinant D. oleovorans ATP synthase subunit c can potentially be incorporated into functional hybrid ATP synthases, offering a powerful approach for understanding both fundamental principles and organism-specific adaptations. Based on successful hybrid ATP synthase experiments with other organisms, several approaches show particular promise:

• Heterologous expression systems: The entire D. oleovorans ATP synthase complex or its individual components can be expressed in model organisms like E. coli, as demonstrated by the successful production of a fully assembled A. aeolicus F1FO ATP synthase in E. coli (designated EAF1FO) . This system allowed researchers to study the properties of the thermophilic ATP synthase in a mesophilic host.

• Chimeric c-rings: Individual c-subunits from D. oleovorans could be combined with those from other organisms to create chimeric c-rings with novel properties. This approach can reveal which regions of the protein are responsible for specific functional characteristics.

• Complete hybrid complexes: The D. oleovorans c-subunit could be incorporated into ATP synthase complexes where other components come from different organisms. This strategy can identify compatibility factors and essential interaction surfaces.

The research with A. aeolicus ATP synthase provides a methodological blueprint, showing that hybrid ATP synthases can maintain enzymatic activity comparable to the native complex. In that study, the EAF1FO complex catalyzed ATP hydrolysis at the same rate as the native AAF1FO complex, and structural analysis confirmed identical organization .

What are the major challenges in working with recombinant ATP synthase subunit c and how can they be overcome?

Working with recombinant ATP synthase subunit c presents several significant challenges that require specialized methodological solutions:

• Membrane protein expression: The hydrophobic nature of subunit c often leads to toxicity, aggregation, or inclusion body formation during expression. This can be addressed by using specialized E. coli strains (C43(DE3), Lemo21(DE3)) designed for membrane protein expression, employing lower temperatures (16-25°C), and fine-tuning inducer concentrations. Adding specific membrane-stabilizing compounds like glycerol (0.5-1%) to the growth medium can also improve yields of properly folded protein.

• Maintaining native oligomeric structure: The c-ring can dissociate during purification, losing its native stoichiometry and functional properties. Selecting appropriate detergents is crucial—mild detergents like digitonin, DDM, or GDN are preferred over harsh detergents like SDS. Crosslinking methods using optimized glutaraldehyde concentrations or specific chemical crosslinkers prior to purification can help maintain the native oligomeric state.

• Functional assessment: Verifying that the recombinant protein retains native function is challenging. Reconstitution into proteoliposomes followed by proton translocation assays provides functional validation. Co-reconstitution with other ATP synthase components to form a functional complex capable of ATP synthesis offers definitive proof of proper folding and assembly.

• Low protein yields: ATP synthase subunit c often expresses at low levels. Optimization strategies include using strong promoters with tight regulation, codon optimization for the expression host, and adding fusion partners that enhance expression and solubility (while ensuring these can be removed without affecting the native structure).

• Protein stability during purification: Maintaining stability throughout multiple purification steps is difficult. Incorporating stabilizing additives (glycerol, specific lipids, sucrose) in all buffers and minimizing exposure to room temperature improves stability. Performing purification steps as quickly as possible and keeping samples cold (4°C or on ice) helps preserve native structure.

What techniques can be used to determine the c-ring stoichiometry of D. oleovorans ATP synthase?

Determining the precise c-ring stoichiometry of D. oleovorans ATP synthase requires a combination of complementary techniques, each with specific advantages:

• Atomic Force Microscopy (AFM): AFM can directly visualize individual c-rings embedded in lipid bilayers or on surfaces, allowing direct counting of subunits. This technique was successfully employed to determine that the c-ring of B. pseudofirmus OF4 contains 13 subunits, and that specific mutations reduced this number to 12 . AFM provides high-resolution topographical information that can distinguish individual subunits within the assembled ring.

• X-ray Crystallography: When high-quality crystals can be obtained, X-ray crystallography provides atomic-resolution structures clearly showing the number of subunits in the c-ring. This approach was used alongside AFM to confirm the c-ring stoichiometry changes in B. pseudofirmus OF4 mutants .

• Mass Spectrometry: Native mass spectrometry of intact c-rings can accurately determine the oligomeric state based on the precise molecular mass of the complex. This approach requires careful optimization of ionization conditions to maintain the intact complex during analysis.

• Crosslinking coupled with SDS-PAGE: Chemical crosslinking can "freeze" the c-ring in its native oligomeric state, and the resulting complex can be analyzed by SDS-PAGE to estimate stoichiometry based on apparent molecular weight.

• Cryo-Electron Microscopy (cryo-EM): High-resolution cryo-EM can resolve individual subunits within the c-ring structure and has become increasingly powerful for membrane protein complexes.

• Quantitative Amino Acid Analysis: By comparing the ratio of unique amino acids from different subunits in a purified ATP synthase complex, researchers can calculate the stoichiometry of different components, including the number of c-subunits per complex.

A combination of these approaches provides the most reliable determination of c-ring stoichiometry, with AFM and X-ray crystallography being particularly valuable for direct visualization of the ring structure.

How can researchers investigate the interaction between recombinant ATP synthase subunit c and other components of the ATP synthase complex?

Investigating interactions between recombinant ATP synthase subunit c and other components of the ATP synthase complex requires sophisticated biochemical, biophysical, and structural approaches:

The combination of these complementary approaches can provide a comprehensive understanding of how subunit c interacts with other ATP synthase components to form a functional complex.