Recombinant Pelobacter carbinolicus ATP synthase subunit c 2 (atpE2)

What is the structure and function of P. carbinolicus ATP synthase subunit c 2?

ATP synthase subunit c 2 (atpE2) in Pelobacter carbinolicus is a small, highly hydrophobic 88-amino acid peptide with the sequence: MDFFSWVMITAGFGMAIGSLGTGIGQGLAVKSALEGVARNPGASGKILTTMMIGLAMIES LAIYVFVVAMIILFANPFQDVVLELLAK . This protein forms part of the F0 sector of ATP synthase, specifically as a component of the c-ring embedded in the membrane. Under physiological conditions, the protein folds into an α-helical hairpin structure .

The c-ring functions as the main constituent of the rotor in ATP synthase, facilitating the translocation of protons across the membrane along an electrochemical gradient, which drives the rotation of the ring and mechanically couples this rotation to ATP synthesis . The number of c subunits in each ATP synthase complex varies depending on the species, typically ranging from 8-16 units, which affects the ratio of protons translocated to ATP synthesized .

How does gene duplication affect P. carbinolicus ATP synthase compared to other bacteria?

P. carbinolicus exhibits species-specific duplication (SSD) of ATP synthase subunits, including the atpE2 gene. According to evolutionary analysis, P. carbinolicus contains both standard ATP synthase and a N-ATPase variant . This duplication pattern is summarized in the following table adapted from the comprehensive analysis of ATP synthase subunit duplications:

This duplication pattern, particularly the presence of both standard ATP synthase and N-ATPase variants, suggests evolutionary adaptation possibly related to the bacterium's diverse metabolic capabilities, including fermentation and syntrophic growth .

What expression systems are most effective for producing recombinant P. carbinolicus atpE2?

Based on protocols developed for similar proteins, Escherichia coli expression systems are most commonly used for recombinant production of ATP synthase subunit c proteins . The key methodological considerations include:

• Vector selection: Expression vectors containing strong promoters such as T7 are recommended. For example, pMAL-c2x has been successfully used for expression of similar ATP synthase c subunits .

• Fusion tags: Due to the highly hydrophobic nature of atpE2, expression as a fusion protein with solubility-enhancing tags is advisable. His-tags for purification or maltose-binding protein (MBP) fusions have shown success with similar ATP synthase c subunits .

• Host strain selection: T7 Express lysY/Iq E. coli strains have proven effective for expression of similar membrane proteins .

• Co-expression with chaperones: Co-transformation with plasmids expressing chaperone proteins DnaK, DnaJ, and GrpE (such as pOFXT7KJE3) can substantially increase production yields, particularly important since membrane proteins often cause toxicity or expression difficulties .

• Storage conditions: The recombinant protein should be stored at -20°C/-80°C, with 50% glycerol recommended for long-term storage. Working aliquots can be kept at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided .

What purification methods yield the highest purity of recombinant P. carbinolicus atpE2?

For optimal purification of recombinant P. carbinolicus atpE2, a multi-step approach should be employed:

• Initial extraction: Due to its hydrophobic nature, extraction should utilize a Tris-based buffer containing glycerol, which helps maintain protein stability .

• Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) is effective. For MBP fusion proteins, amylose resin chromatography provides high specificity .

• Tag removal: If required for downstream applications, fusion tags can be removed using specific proteases like TEV or Factor Xa, depending on the constructed cleavage site .

• Secondary purification: Size exclusion chromatography or ion exchange chromatography can be employed to remove tag-cleaved protein or other contaminants.

• Quality control: SDS-PAGE analysis should confirm >90% purity, and mass spectrometry can verify the identity and integrity of the purified protein .

• Storage formulation: Final storage in a buffer optimized for the protein (typically Tris/PBS-based with 6% trehalose or 50% glycerol, pH 8.0) helps maintain stability .

Notably, for reconstitution studies, the protein can be reconstituted in deionized water to a concentration of 0.1-1.0 mg/mL with addition of 5-50% glycerol for long-term storage .

How does P. carbinolicus atpE2 contribute to the organism's unique metabolic capabilities?

P. carbinolicus has surprising metabolic capabilities, including fermentation, syntrophic hydrogen/formate transfer, and electron transfer to sulfur from alcohols, hydrogen, or formate . The ATP synthase system, including atpE2, plays a crucial role in these processes:

• Energy conservation: In P. carbinolicus, ATP synthase with its multiple isozymes (including atpE2) has been differentiated and assigned roles according to structural properties and genomic contexts . The presence of multiple isozymes likely allows the bacterium to optimize ATP production under different growth conditions.

• Fermentation pathways: Unlike its Geobacter relatives, P. carbinolicus utilizes fermentation via simpler pathways of cytoplasmic enzymes. During fermentation, NAD+ is regenerated either by ethanol production or by proton reduction to hydrogen (requiring a syntrophic partner), and ATP is generated by substrate-level phosphorylation through acetate kinase .

• Adaptation to ecological niches: The specialized ATP synthase configuration helps P. carbinolicus adapt to fermentative and syntrophic niches, differentiating it from its respiratory ancestors in the Geobacteraceae family .

The distinct configuration of ATP synthase in P. carbinolicus, including the role of atpE2, thus contributes to its metabolic versatility, allowing it to thrive in environments where its Geobacter relatives cannot.

What methods can be used to assess the oligomeric state of recombinant P. carbinolicus atpE2?

To evaluate the oligomeric state of recombinant P. carbinolicus atpE2, researchers can employ several complementary techniques:

• Blue Native PAGE (BN-PAGE): This non-denaturing electrophoresis technique preserves native protein complexes and can resolve different oligomeric states of membrane proteins like atpE2.

• Size Exclusion Chromatography (SEC): SEC can separate oligomers based on their hydrodynamic radius, with calibration against protein standards of known molecular weight providing size estimation.

• Analytical Ultracentrifugation (AUC): Both sedimentation velocity and equilibrium experiments can determine molecular weight and oligomerization state in solution.

• Crosslinking Studies: Chemical crosslinking followed by SDS-PAGE and mass spectrometry can identify oligomeric associations.

• Atomic Force Microscopy (AFM): As demonstrated with similar c subunits, AFM can visualize oligomeric assemblies and fibril formation, particularly in the presence of calcium .

• Thioflavin T (ThT) Binding Assays: For investigating potential amyloidogenic properties, ThT fluorescence can monitor the formation of cross-β aggregates, as has been shown with other c subunits .

Research has shown that c subunits can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a calcium-dependent manner . When studying P. carbinolicus atpE2, it's important to consider these potential conformational changes and their functional implications.

How can researchers distinguish between the functional roles of different ATP synthase isozymes in P. carbinolicus?

P. carbinolicus contains multiple isozymes of ATP synthase, including the atpE2 protein. To distinguish their functional roles, researchers should implement the following methodological approaches:

• Genomic context analysis: Examining the genomic environment of each isozyme can provide clues about functional associations. In P. carbinolicus, genomic context analysis has already been used to differentiate and assign roles to various ATP synthase isozymes .

• Gene expression profiling: RNA-Seq or qRT-PCR analysis under different growth conditions (fermentative vs. syntrophic) can reveal condition-specific expression patterns of different isozymes.

• Gene knockout/knockdown studies: Selective deletion or suppression of individual isozyme genes followed by phenotypic characterization can reveal their specific contributions.

• Protein-protein interaction studies: Co-immunoprecipitation, bacterial two-hybrid systems, or crosslinking coupled with mass spectrometry can identify differential interaction partners of each isozyme.

• Heterologous expression and complementation: Expression of individual P. carbinolicus ATP synthase isozymes in model organisms with deleted endogenous ATP synthase can demonstrate functional capabilities.

• Biochemical characterization: Purification of individual isozymes followed by activity assays under varying conditions (pH, ion concentrations, temperature) can reveal specialized functions.

Research has indicated that the multiple isozymes may be involved in adaptation to different energy conservation strategies, including standard ATP synthesis and specialized roles in the bacterium's diverse metabolic capabilities .

What role does the c subunit play in calcium-dependent membrane permeabilization, and how might this apply to P. carbinolicus atpE2?

Recent research has revealed that ATP synthase c subunits may play a critical role in calcium-induced membrane permeabilization beyond their canonical function in ATP synthesis. For recombinant P. carbinolicus atpE2, this raises important considerations:

• Amyloidogenic properties: C subunits can function as amyloidogenic calcium-activated proteins, capable of spontaneously folding into β-sheets and self-assembling into fibrils and oligomers in a calcium-dependent manner .

• Conformational transitions: Studies have shown that purified c subunits can undergo conformational transitions from their native α-helical structure to β-sheet formation under certain conditions .

• Membrane permeabilization mechanisms: In the presence of elevated calcium levels, c subunit oligomers might form ion channels in lipid bilayers, potentially similar to mechanisms observed with amyloidogenic peptides like Aβ and α-synuclein .

• Experimental approaches: To investigate these properties in P. carbinolicus atpE2, researchers can:

  • Monitor calcium-dependent structural changes using circular dichroism spectroscopy

  • Assess membrane permeabilization using liposome leakage assays

  • Conduct ThT fluorescence measurements to detect amyloid formation

  • Use AFM to visualize oligomer formation in the presence/absence of calcium

  • Perform black lipid membrane experiments to measure potential ion channel activity

These findings suggest that under stress conditions or elevated calcium levels, P. carbinolicus atpE2 might exhibit functions beyond energy production, potentially related to stress responses or adaptation to environmental conditions.

How can researchers assess the potential impact of CRISPR spacers on P. carbinolicus atpE2 expression?

P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) that can produce RNA molecules interfering with genes containing identical sequences . To investigate potential CRISPR-mediated regulation of atpE2, researchers should:

• CRISPR spacer sequence analysis: Search the P. carbinolicus genome for CRISPR spacers with sequence homology to the atpE2 gene, similar to the analysis that identified spacer #1's match to the hisS gene .

• Expression verification of identified spacers: Use RT-PCR or RNA-Seq to confirm expression of any spacers with homology to atpE2.

• Genetic complementation studies: Design experiments similar to those used for hisS, where a chimeric CRISPR containing the spacer of interest is expressed in a heterologous host (e.g., G. sulfurreducens) containing the P. carbinolicus atpE2 gene .

• Plasmid construction: For heterologous expression, construct IPTG-inducible expression vectors containing the chimeric CRISPR with spacers between repeats typical of the CRISPR locus of the host organism .

• Growth inhibition assays: Monitor growth of transgenic strains to assess if spacer expression inhibits growth, which would suggest interference with atpE2 expression .

• Protein expression analysis: Use Western blotting to directly measure atpE2 protein levels in the presence/absence of spacer expression.

Methodology based on previous research with P. carbinolicus can be adapted, where plasmid vectors like pMA36 were constructed for IPTG-inducible expression of chimeric CRISPRs, and the impact on gene expression and cell growth was assessed .

What are the key considerations for reconstituting functional c-rings using recombinant P. carbinolicus atpE2?

Reconstituting functional c-rings from recombinant P. carbinolicus atpE2 requires careful consideration of several factors:

• Purification quality: Highly purified atpE2 with preserved native structure is essential. Avoid protein aggregation and ensure removal of detergents that might interfere with proper assembly .

• Lipid environment: The choice of lipids is critical for successful reconstitution. Consider using lipid compositions that mimic the native P. carbinolicus membrane environment or established lipid mixtures known to support c-ring assembly.

• Buffer conditions: Optimal pH, ionic strength, and presence of specific ions (particularly Na+ or H+, depending on the coupling ion of the ATP synthase) are crucial for proper folding and assembly.

• Temperature control: Since P. carbinolicus is a mesophilic bacterium, reconstitution should typically be performed at moderate temperatures (20-30°C).

• Assembly verification methods:

  • Negative-stain electron microscopy to visualize ring structures

  • AFM to examine topography and dimensions of reconstituted rings

  • Functional assays such as proton/sodium translocation in proteoliposomes

  • Size exclusion chromatography to assess complex formation

• Stoichiometry determination: The number of c subunits in the reconstituted ring can be determined by:

  • Mass determination of intact rings by native mass spectrometry

  • Structural analysis through cryo-electron microscopy

  • Crosslinking followed by SDS-PAGE analysis

• Functional testing: Reconstituted c-rings can be tested for functionality by incorporation into proteoliposomes and assessment of ion translocation activity or, if combined with other ATP synthase components, ATP synthesis/hydrolysis activity.

The successful reconstitution of functional c-rings would enable detailed structural and functional studies, including investigation of the factors that influence stoichiometric variation of the intact ring .