Recombinant Pseudomonas putida ATP synthase subunit c (atpE)

Basic Research Questions

• What is the structure and function of ATP synthase subunit c (atpE) in Pseudomonas putida?

  ATP synthase subunit c (atpE) in P. putida is a small hydrophobic protein consisting of 85 amino acids that forms a ring-like structure in the F0 sector of ATP synthase embedded in the cell membrane. The protein has a predominantly alpha-helical secondary structure and contains the sequence: METVVGLTAIAVALLIGLGALGTAIGFGLLGGKFLEGAARQPEMVPMLQVKMFIVAGLLDAVTMIGVGIALFFTFANPFVGQIAG .

  Functionally, the c-subunit ring plays a crucial role in the rotational mechanism of ATP synthesis. The rotation of this ring is driven by proton translocation across the membrane along an electrochemical gradient. This mechanical energy is then coupled to the synthesis of ATP in the F1 sector of the enzyme complex . Each c-subunit can bind and transport one proton across the membrane as the ring makes a complete rotation, which ultimately drives the synthesis of 3 ATP molecules per rotation .

• How does the c-subunit stoichiometry affect ATP synthesis efficiency in different organisms?

  The stoichiometry of c-subunits in the ring varies among organisms, ranging from 8 to 15 subunits per ring . This variation directly impacts the energetic efficiency of ATP synthesis.

  The coupling ratio of ATP synthesis (protons translocated:ATP generated) ranges from 3.3 to 5.0 among different organisms, depending on the number of c-subunits per ring (n) . Since each c-subunit binds and transports one proton, and a complete rotation of the ring results in the synthesis of 3 ATP molecules regardless of ring size, the energetic cost of ATP synthesis is higher in organisms with larger c-rings.

  Organism	c-ring size	Coupling ratio (H+:ATP)
  Various bacteria	10-15	3.3-5.0

  This variability may represent evolutionary adaptations to different environmental conditions and energy availability, though the exact purpose of stoichiometric variation remains under investigation .

• What are the key sequence similarities and differences between P. putida atpE and atpE from other organisms?

  P. putida atpE shares structural and functional homology with ATP synthase c-subunits from other organisms while maintaining species-specific characteristics. All c-subunits contain:

  • Predominantly hydrophobic amino acids forming transmembrane helices

  • A conserved acidic residue (typically aspartate or glutamate) essential for proton binding

  • Alpha-helical secondary structure

  What distinguishes P. putida atpE is its specific sequence adaptations that may contribute to the organism's remarkable stress tolerance and metabolic versatility. These adaptations could influence membrane integration and stability under various environmental conditions, including exposure to organic solvents and oxidative stress, which P. putida is known to withstand .

  Comparative structural analysis of atpE from diverse organisms provides insights into how evolutionary pressure shapes this essential component of energy metabolism across species.

Advanced Research Questions

• What expression systems are optimal for recombinant P. putida atpE production and what challenges must be overcome?

  The optimal expression system for recombinant P. putida atpE production is E. coli, but several challenges must be addressed:

  Challenges and Solutions:

  1. Hydrophobicity: The high hydrophobicity of atpE makes it difficult to express in soluble form.

     • Solution: Expression as a fusion protein with a soluble partner (MBP, His-tag) improves solubility .

  2. Toxicity to host cells: Membrane protein overexpression can disrupt host membrane integrity.

     • Solution: Tightly regulated inducible promoters (like IPTG-inducible systems) allow controlled expression .

  3. Codon bias: Differences in codon usage between P. putida and E. coli.

     • Solution: Codon optimization of the atpE gene for E. coli expression has been successfully implemented .

  4. Proper folding: Ensuring correct folding of the recombinant protein.

     • Solution: Fine-tuning induction conditions (temperature, inducer concentration) and co-expression with chaperones .

  For optimal expression, a strategy involving expression as an MBP-fusion protein (MBP-c1) under a controlled induction system with moderate temperature (30°C) has shown success in producing properly folded, functional atpE that can be purified in milligram quantities .

• How can researchers study the assembly and stoichiometry of the c-ring in recombinantly produced P. putida ATP synthase?

  Studying c-ring assembly and stoichiometry requires a multi-technique approach:

  1. Reconstitution experiments: Purified recombinant c-subunits can be reconstituted into liposomes to form c-rings under controlled conditions. This allows observation of the natural assembly process and determination of the native oligomeric state .

  2. Cryo-electron microscopy (cryo-EM): This technique can directly visualize the c-ring structure and count the number of subunits. Sample preparation involves purification of intact c-rings or reconstituted c-rings in nanodiscs or liposomes.

  3. Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify interaction interfaces between c-subunits and help understand the assembly pattern.

  4. Mass spectrometry of intact complexes: Native mass spectrometry can determine the exact molecular weight of the intact c-ring, allowing calculation of the subunit stoichiometry.

  5. Molecular dynamics simulations: Computational approaches can predict stable conformations of c-rings with different stoichiometries based on the P. putida atpE sequence.

  6. Mutational analysis: Strategic mutations at the interfaces between c-subunits can probe factors determining stoichiometry, potentially revealing how the number of subunits is controlled during assembly .

  These approaches together provide complementary information on both the process of assembly and the final stoichiometry of the c-ring.

• What role does the ATP synthase play in P. putida's remarkable tolerance to environmental stresses?

  ATP synthase, including the atpE subunit, plays several critical roles in P. putida's stress tolerance:

  1. Energy supply during stress: P. putida rapidly modulates ATP production through ATP synthase to maintain energy homeostasis during stress. During glucose starvation, P. putida can quickly access cellular reserves (PHA, amino acids, glycogen) to obtain ATP through respiration, replenishing reduced ATP levels and adenylate energy charge .

  2. Membrane adaptation: The c-ring, composed of atpE, is embedded in the cell membrane and must maintain functionality despite membrane modifications that occur during stress responses. P. putida modifies its membrane composition under stress, including shifts in the conformation of unsaturated fatty acids from cis to trans .

  3. Integration with stress response pathways: ATP synthase activity appears to be coordinated with stringent response pathways mediated by the alarmone ppGpp. Proteomic analyses have shown that toluene exposure in P. putida affects proteins involved in sugar transport and stress responses, including those required for ATP generation .

  4. Role in redox balance: P. putida's central metabolism, including ATP generation via ATP synthase, contributes to its high redox capacity. The organism can adjust NADPH formation at the expense of ATP, which is a key factor in oxidative stress endurance .

  These adaptations allow P. putida to maintain energy production even under challenging environmental conditions, contributing to its robustness as a platform for biotechnological applications.

Methodological Questions

• What purification strategies work best for recombinant P. putida atpE?

  Purification of recombinant P. putida atpE requires specialized approaches due to its hydrophobicity. A successful multi-step purification strategy includes:

  1. Expression as a fusion protein: Expression as a fusion with a soluble partner (e.g., MBP or His-tag) improves solubility and facilitates initial purification .

  2. Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin allows specific capture of the fusion protein. For MBP fusions, amylose resin affinity chromatography provides high selectivity .

  3. Detergent solubilization: Addition of appropriate detergents (e.g., DDM, LDAO) during or after cell lysis helps solubilize the membrane protein component.

  4. Protease cleavage: The fusion tag can be removed using specific proteases (TEV, Factor Xa) in the presence of detergent to prevent aggregation of the released atpE .

  5. Reversed-phase chromatography: Final purification can be achieved using reversed-phase chromatography with ethanol as an eluent, which has been successfully used for c-subunit purification .

  6. Size exclusion chromatography: This can be used as a final polishing step to separate monomeric from oligomeric forms and remove any remaining contaminants.

  This multi-step approach has been successful in obtaining highly purified (>90%) recombinant atpE in milligram quantities suitable for structural and functional studies .

• How can researchers verify the proper folding and structure of recombinant P. putida atpE?

  Verifying proper folding of recombinant P. putida atpE is crucial for functional studies. Several complementary techniques can be employed:

  1. Circular Dichroism (CD) Spectroscopy: This technique can confirm the alpha-helical secondary structure characteristic of properly folded atpE. The CD spectrum of properly folded atpE should show typical alpha-helical signatures with negative peaks at 208 and 222 nm .

  2. Thermal Stability Assays: Monitoring the unfolding of the protein with increasing temperature using CD or differential scanning calorimetry (DSC) can provide information about the stability of the folded structure.

  3. Functional Reconstitution: Incorporation of purified atpE into liposomes followed by proton translocation assays can verify functional integrity .

  4. Limited Proteolysis: Properly folded proteins typically show resistance to proteolytic digestion compared to misfolded variants. The digestion pattern can be analyzed by SDS-PAGE or mass spectrometry.

  5. Structural Studies: For definitive structural verification, techniques like NMR spectroscopy (for the monomer) or cryo-EM (for assembled rings) can be employed.

  6. Binding Assays: The ability to bind specific ligands or inhibitors (such as oligomycin) that target ATP synthase can provide evidence of proper folding.

  These methods collectively provide robust evidence for the correct folding and structural integrity of recombinant P. putida atpE .

• What techniques are most effective for studying the interaction of atpE with other ATP synthase subunits?

  Several techniques are particularly effective for investigating interactions between atpE and other ATP synthase subunits:

  1. Co-immunoprecipitation (Co-IP): Using antibodies against one subunit to pull down interacting partners, followed by mass spectrometry identification.

  2. Chemical Cross-linking coupled with Mass Spectrometry (XL-MS): This approach can identify specific residues involved in subunit interactions by creating covalent bonds between closely associated proteins, which are then identified by mass spectrometry .

  3. Surface Plasmon Resonance (SPR): Quantitative measurement of binding affinities between purified atpE and other subunits.

  4. Förster Resonance Energy Transfer (FRET): Labeling different subunits with fluorophore pairs to detect proximity and interactions in reconstituted systems.

  5. Two-hybrid Systems: Modified bacterial or yeast two-hybrid systems can detect interactions between membrane proteins like atpE and other subunits.

  6. Cryo-EM and X-ray Crystallography: These methods can provide structural information about the assembled complex, revealing the interfaces between atpE and other subunits at the atomic level.

  7. Molecular Dynamics Simulations: Computational approaches can predict interaction interfaces and binding energies between atpE and other ATP synthase components.

  8. Site-directed Mutagenesis: Strategic mutations at predicted interaction sites can validate the importance of specific residues in subunit assembly.

  These techniques provide complementary information about both the static structure of subunit interfaces and the dynamic nature of their interactions during ATP synthase function.

Research Application Questions

• How can recombinant P. putida atpE be used to study bacterial bioenergetics and metabolism?

  Recombinant P. putida atpE serves as a powerful tool for studying bacterial bioenergetics through several research applications:

  1. Investigation of proton-coupling mechanisms: By introducing specific mutations in recombinant atpE and studying their effects on proton translocation, researchers can elucidate the precise mechanism of proton binding, release, and coupling to ATP synthesis .

  2. Comparison of energetic efficiency across species: Recombinant expression allows researchers to create chimeric ATP synthase complexes with components from different species to investigate how variations in c-ring stoichiometry affect energetic efficiency .

  3. Study of metabolic adaptation under stress: P. putida modifies its energy metabolism under stress conditions. Recombinant atpE can be used to investigate how ATP synthase activity is regulated during stress responses, including solvent exposure and nutrient limitation .

  4. Analysis of membrane integration and protein folding: The process by which hydrophobic proteins like atpE fold and integrate into membranes is of fundamental importance. Recombinant atpE provides a model system for these studies .

  5. Investigation of ATP synthase assembly: By expressing recombinant atpE alongside other ATP synthase components, researchers can study the assembly process and identify factors that influence the formation of functional complexes .

  These applications collectively advance our understanding of bacterial energy metabolism and adaptation mechanisms, with potential implications for biotechnological applications of P. putida.

• How might structural studies of atpE inform the design of antimicrobial agents targeting bacterial ATP synthase?

  Structural studies of P. putida atpE can provide valuable insights for antimicrobial agent design through several mechanisms:

  1. Identification of unique structural features: Detailed structural analysis of P. putida atpE can reveal bacterial-specific features absent in human ATP synthase. These differences can be exploited for selective targeting by antimicrobial compounds.

  2. Understanding binding sites: Structural studies can identify potential binding pockets in the c-ring that could accommodate small molecules designed to disrupt ATP synthesis. The c-subunit is the target of several natural antibiotics like oligomycin, and structural information can guide the design of new inhibitors.

  3. Protein-protein interaction interfaces: The interfaces between atpE and other ATP synthase subunits represent potential targets for compounds designed to disrupt complex assembly.

  4. Species-specific variations: Comparative structural analysis of atpE from P. putida and pathogenic bacteria can reveal conservation patterns and unique features that might allow for species-selective targeting.

  5. Rational drug design: High-resolution structural data enables structure-based virtual screening and molecular docking approaches to identify candidate compounds that could selectively inhibit bacterial ATP synthases.

  6. Mechanism-based inhibitor design: Understanding the mechanistic details of proton translocation through the c-ring can inform the design of compounds that specifically interfere with this process.

  This research could ultimately lead to the development of novel antimicrobials with mechanisms distinct from current antibiotics, potentially addressing the growing challenge of antimicrobial resistance .

• What insights can studies of P. putida atpE provide about bacterial adaptation to extreme environments?

  P. putida is known for its remarkable adaptability to harsh conditions, and studies of its atpE can provide several insights into bacterial adaptation mechanisms:

  1. Energy conservation strategies: Analysis of P. putida atpE structure and function can reveal how ATP synthesis is maintained under stress conditions. The bacterium demonstrates the ability to rapidly modulate ATP production during stress, such as glucose starvation .

  2. Membrane integrity under stress: P. putida modifies its membrane composition under stress, including shifts in unsaturated fatty acid conformation. Since atpE is a membrane protein, its structure likely co-evolved with these membrane adaptations to maintain functionality under various conditions .

  3. Coordination with stress response pathways: The activity of ATP synthase in P. putida appears to be coordinated with stress response pathways, particularly the stringent response mediated by ppGpp . Understanding how atpE function is integrated with these pathways provides insights into cellular stress adaptation.

  4. Solvent tolerance mechanisms: P. putida is known for its tolerance to organic solvents, which typically disrupt membrane integrity. Studies of atpE can reveal adaptations that maintain ATP synthase function despite membrane perturbations caused by solvents .

  5. Evolutionary adaptations in energy metabolism: The specific sequence adaptations in P. putida atpE compared to other bacteria may reflect evolutionary responses to the ecological niches this organism inhabits, including contaminated soils and water sources.

  These insights contribute to our understanding of bacterial adaptation mechanisms and have implications for biotechnological applications of P. putida, such as bioremediation and metabolic engineering for production of valuable compounds under challenging conditions .