Recombinant Exiguobacterium sibiricum ATP synthase subunit c (atpE)

What is the structural role of subunit c (atpE) in ATP synthase complexes?

The c subunit (atpE) forms the rotor element in the F0 domain of ATP synthase. As shown in structural studies, it exists as a homo-oligomeric ring composed of hairpin-like c-subunits. The number of c-subunits per rotor varies from 10-15 depending on the organism, with each subunit containing ion-binding sites crucial for the rotary mechanism . The c-ring interacts with the a-subunit at a specific interface where ion translocation occurs, driving rotation of the entire c-ring and the attached central stalk (γ and ε subunits), which ultimately powers ATP synthesis in the F1 domain .

The structure of atpE is typically a 2-helix hairpin that spans the membrane, though some variations exist. For example, in Acetobacterium woodii, the c-ring contains both standard 2-helix subunits and a larger 4-transmembrane helix subunit similar to those found in V-type ATPases .

How does the ion specificity of atpE affect ATP synthase function?

ATP synthases can be coupled to either H+ (protons) or Na+ (sodium ions) depending on the organism and its environmental adaptations. The ion specificity is determined by specific amino acid residues in the c-subunit binding sites. In E. sibiricum, as with most bacterial species, these binding sites contain a conserved carboxylate group (usually from an aspartate or glutamate residue) that binds the coupling ion .

During operation, ions from the periplasmic (P) side of the membrane are channeled through the a-subunit to the c-subunits of the rotor. After rotation, the ions are released through an exit pathway into the cytoplasm (N-side), completing ion translocation across the membrane . The specific residues lining the ion-binding pocket determine whether H+ or Na+ is preferentially bound and transported.

What expression systems are most effective for producing recombinant atpE?

For recombinant expression of membrane proteins like atpE, E. coli remains the most commonly used host system due to its ease of use and high yield potential. Based on similar recombinant proteins, such as ATP synthase subunit delta from E. sibiricum, expression in E. coli can produce proteins with >85% purity as determined by SDS-PAGE .

For optimal expression of membrane proteins like atpE, consider:

• Using specialized E. coli strains designed for membrane protein expression (C41, C43)

• Employing low-temperature induction protocols (16-20°C)

• Testing various fusion tags (His, MBP, SUMO) to improve solubility

• Using controlled expression systems (like IPTG-inducible promoters with tight regulation)

What are the recommended storage conditions for recombinant atpE protein?

Based on storage recommendations for similar ATP synthase subunits from E. sibiricum, recombinant atpE should be stored at -20°C, with extended storage at -20°C or -80°C . To maintain protein stability:

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50% is recommended)

• Aliquot into small volumes to avoid repeated freeze-thaw cycles

• For working solutions, store aliquots at 4°C for up to one week

The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms maintain stability for approximately 12 months .

How can recombinant E. sibiricum atpE be used in drug discovery research?

ATP synthase has emerged as an important drug target, particularly for antimicrobial development. Drawing from research on M. tuberculosis AtpE, similar approaches can be applied to E. sibiricum atpE:

• Structure-based virtual screening can identify potential inhibitors that bind to atpE with high affinity. For example, in studies with M. tuberculosis AtpE, compounds with binding energies ranging between -8.69 and -8.44 kcal/mol were identified as potential inhibitors .

• Molecular docking analysis can be used to screen compounds for binding to specific sites on atpE. The compounds should ideally have binding energies lower than ATP itself to effectively compete for binding .

• Promising compounds should be further evaluated for ADME (absorption, distribution, metabolism, excretion) and toxicity properties. Studies on M. tuberculosis AtpE identified three compounds (ZINC14732869, ZINC14742188, and ZINC12205447) that demonstrated suitable properties for further development .

• Molecular dynamics simulations can assess the stability of ligand-protein complexes and predict binding energetics using approaches like MM-GBSA (Molecular Mechanics Generalized Born and Surface Area) .

What insights can comparative studies of E. sibiricum atpE provide about ATP synthase adaptation to extreme environments?

Exiguobacterium sibiricum is a psychrophilic bacterium isolated from Siberian permafrost, making its ATP synthase particularly interesting for studying cold adaptation. Comparative studies of E. sibiricum atpE with those from mesophilic and thermophilic organisms can reveal:

• Amino acid substitutions that contribute to cold stability and flexibility

• Altered ion-binding properties that may optimize function at low temperatures

• Modifications in subunit-subunit interactions that maintain rotor integrity in cold conditions

Similar comparative approaches with alkaliphilic bacteria have revealed specific adaptations in ATP synthase components that enable function at high pH . For example, alkaliphiles maintain ATP synthesis despite adverse pH gradients through specialized adaptations in both ATP synthase structure and the arrangement of respiratory chain components .

How do the stoichiometry and composition of the c-ring affect the bioenergetics of ATP synthesis?

The number of c-subunits in the rotor ring directly impacts the bioenergetic efficiency of ATP synthesis. Each c-subunit typically binds one coupling ion (H+ or Na+), and the complete rotation of the c-ring produces three ATP molecules (one at each catalytic site in the F1 domain) .

Therefore, the c-subunit stoichiometry determines the ion-to-ATP ratio. A c-ring with more subunits requires more ions to complete a full rotation, resulting in a higher ion-to-ATP ratio. For example:

• A 10-subunit c-ring: 10 ions translocated per 3 ATP = 3.33 ions/ATP

• A 15-subunit c-ring: 15 ions translocated per 3 ATP = 5 ions/ATP

This stoichiometry is significant for understanding how E. sibiricum balances energy efficiency with the thermodynamic constraints of its environment. Research in other species has shown that the c-ring stoichiometry can vary from 10 in yeast to 14 in spinach chloroplasts , with specialized adaptations in extremophiles.

What methods are most effective for purifying recombinant E. sibiricum atpE?

Purification of membrane proteins like atpE requires specialized approaches:

• Membrane extraction: Use mild detergents (DDM, LDAO, or Triton X-100) to solubilize membranes without denaturing the protein.

• Affinity chromatography: If expressing with a tag (His, FLAG, etc.), use appropriate affinity resins. For His-tagged proteins, immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co-NTA resins is effective.

• Size exclusion chromatography: Apply as a final polishing step to separate monomeric atpE from aggregates and other impurities.

• Quality assessment: Confirm purity by SDS-PAGE (target >85% purity as achieved with other E. sibiricum ATP synthase subunits) .

For reconstitution into functional assays, the purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of glycerol as a stabilizer .

How can researchers assess the assembly and function of recombinant atpE in c-rings?

Several complementary approaches can evaluate the assembly and function of atpE in c-rings:

• Blue Native PAGE: To analyze intact c-ring assemblies and determine approximate molecular weights.

• Cross-linking mass spectrometry: To identify interaction interfaces between adjacent c-subunits.

• Atomic Force Microscopy (AFM): To visualize assembled c-rings and measure their dimensions.

• Reconstitution assays: Incorporate purified c-subunits into liposomes with purified a-subunit to measure ion translocation using pH-sensitive or ion-sensitive fluorescent dyes.

• Single-molecule FRET: To detect conformational changes and rotary motion when assembled with other ATP synthase components.

For functional assessment, researchers can measure ion translocation activity by reconstituting the c-ring with other components of the F0 domain in proteoliposomes and monitoring ion flux across the membrane using ion-selective electrodes or fluorescent probes.

What site-directed mutagenesis targets are most informative for studying E. sibiricum atpE function?

Based on studies of ATP synthase c-subunits from other organisms, several key residues can be targeted for site-directed mutagenesis to elucidate function:

• Ion-binding site residues: Mutate the conserved carboxylate residue (Asp or Glu) that forms the ion-binding site to assess its role in ion specificity and translocation .

• a-subunit interface residues: Modify residues that interact with the essential arginine in the a-subunit TMH4 region to understand the mechanism of ion release and uptake .

• Rotor-stator interaction sites: Mutate residues at the interface with the γ and ε subunits to investigate torque generation and transmission.

• Subunit-subunit interaction sites: Alter residues at the interface between adjacent c-subunits to study c-ring assembly and stability.

A systematic alanine-scanning mutagenesis approach can be particularly informative for identifying critical functional residues throughout the protein.

What techniques provide the most detailed structural information about E. sibiricum atpE?

Several complementary approaches can provide structural insights:

• X-ray crystallography: While challenging for membrane proteins, this can provide atomic-resolution structures of the c-ring, particularly if crystallized in lipidic cubic phases.

• Cryo-electron microscopy: Increasingly the method of choice for membrane protein complexes, providing near-atomic resolution without crystallization.

• Solid-state NMR: Particularly useful for membrane proteins like atpE, providing information about dynamics and structural changes during function.

• Homology modeling: When experimental structures are unavailable, computational models based on homologous proteins can be constructed. For example, the 3D model of M. tuberculosis AtpE was built using homology modeling with Modeller9.16, followed by energy minimization and molecular dynamics simulation for refinement .

• Molecular dynamics simulations: To investigate dynamic properties and conformational changes under various conditions, as was done with ligand-bound AtpE complexes from M. tuberculosis .

How does the ion specificity of E. sibiricum atpE compare to other bacterial species?

ATP synthases can be coupled to either H+ or Na+ ions, depending on the organism and its environmental adaptations. Based on studies of other bacteria, several patterns emerge:

• Most aerobic bacteria, including those from the Bacillus genus (related to Exiguobacterium), utilize H+-coupled ATP synthases .

• Some anaerobic bacteria, particularly those from alkaline environments, use Na+-coupled ATP synthases to overcome bioenergetic challenges .

• The ion specificity is determined by specific residues in the c-subunit ion-binding site.

A comparative table of ATP synthase coupling ion specificity in various bacteria shows this pattern:

Based on this pattern and its taxonomic relationship to Bacillus species, E. sibiricum likely utilizes a H+-coupled ATP synthase .

What are common challenges in expressing and purifying recombinant E. sibiricum atpE?

Membrane proteins like atpE present several challenges during recombinant expression and purification:

• Toxicity to host cells: Overexpression of membrane proteins can disrupt host cell membranes. Solution: Use tightly regulated expression systems and consider specialized host strains like C41(DE3) designed for membrane protein expression.

• Protein aggregation: Hydrophobic membrane proteins can aggregate during expression. Solution: Lower expression temperature (16-20°C), add membrane-stabilizing agents like glycerol to growth media, or use fusion partners known to enhance solubility.

• Detergent selection: Finding the optimal detergent for extraction without denaturation is critical. Solution: Screen multiple detergents (DDM, LDAO, Fos-Choline, etc.) at various concentrations to identify conditions that maintain native structure.

• Protein stability: Maintaining stability during purification can be difficult. Solution: Include stabilizers in all buffers (glycerol at 5-50%, as recommended for other E. sibiricum ATP synthase subunits) , work at 4°C throughout purification, and minimize exposure to air.

How can researchers troubleshoot reconstitution of functional atpE into proteoliposomes?

Reconstitution of membrane proteins into proteoliposomes for functional studies presents several challenges:

• Proper orientation: atpE must insert with the correct orientation in liposomes. Solution: Use rapid dilution or detergent removal methods (Bio-Beads, dialysis) that favor proper insertion.

• Lipid composition: Lipid environment strongly affects function. Solution: Screen various lipid compositions, including E. coli polar lipids, POPC/POPE mixtures, and lipids matching E. sibiricum's native membrane composition.

• Protein-to-lipid ratio: Suboptimal ratios can lead to aggregation or insufficient incorporation. Solution: Test multiple protein-to-lipid ratios (typically 1:50 to 1:1000 w/w) to find optimal conditions.

• Functional assessment: Confirming activity can be challenging. Solution: Employ multiple complementary assays (ion flux, ATP synthesis when combined with F1) to verify function from different angles.

What strategies can overcome protein degradation during purification and storage?

Protein degradation is a common challenge with membrane proteins like atpE:

• Protease inhibitors: Include a comprehensive protease inhibitor cocktail during all purification steps.

• Temperature control: Maintain samples at 4°C during purification and store at -20°C or -80°C for long-term storage .

• Buffer optimization: Include stabilizers such as glycerol (5-50%) and consider adding specific lipids that may stabilize the native structure.

• Aliquoting: Divide purified protein into small aliquots to avoid repeated freeze-thaw cycles, which significantly contribute to degradation. For working solutions, store at 4°C for no more than one week .

• Lyophilization: For very long-term storage, lyophilization can extend shelf life to approximately 12 months at -20°C/-80°C, compared to 6 months for liquid preparations .