Recombinant Pseudomonas entomophila ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its fundamental role?

ATP synthase subunit c (atpE), also known as proteolipid or subunit 9 in F-ATPases, is the main transmembrane component of the F0 complex in ATP synthases. This subunit forms an oligomeric structure called the c-ring that constitutes a critical part of the F0/V0/A0 rotor in the membrane-embedded portion of ATP synthases . The primary function of subunit c is to facilitate proton translocation across the membrane, which drives the rotary mechanism essential for ATP synthesis. In ATP synthases, the sequential protonation and deprotonation of conserved residues (typically Asp61) in subunit c is coupled to the rotational movement that drives ATP production .

The significance of this subunit cannot be overstated, as it directly couples the proton gradient generated by the respiratory chain to the mechanical energy needed for ATP synthesis. Research has demonstrated that ATP synthase subunit c catalyzes the production of ATP from ADP in the presence of a sodium or proton gradient, making it an essential enzyme in cellular energy metabolism .

How is the c-ring assembled and what determines its stoichiometry?

The c-ring in ATP synthases is composed of multiple copies of the subunit c protein arranged in a circular formation. The exact number of subunit c proteins in the c-ring (stoichiometry) varies significantly between species and even between different ATP synthase types. This variation has important functional implications for the bioenergetics of the cell.

Researchers studying c-ring assembly should consider these methodological approaches:

• Cryo-electron microscopy (Cryo-EM): This technique has become the gold standard for determining c-ring stoichiometry and structure. Sample preparation involves purification of intact ATP synthase complexes or isolated c-rings followed by vitrification and imaging.

• Cross-linking experiments: Chemical cross-linking followed by mass spectrometry can identify interactions between adjacent subunit c proteins and help elucidate the assembly process.

• Molecular dynamics simulations: Based on homology models (as described for AtpE ), simulations can predict stable c-ring configurations and the energetics of assembly.

The number of c subunits in the ring is thought to be determined by specific protein-protein interactions and the lipid environment of the membrane. This stoichiometry directly affects the ATP/H+ ratio and thus the bioenergetic efficiency of the organism.

What are the evolutionary implications of ATP synthase subunit c conservation?

Despite variations in the mitochondrial targeting peptides (as seen in mammalian isoforms), the mature sequence of ATP synthase subunit c is highly conserved across species, reflecting its fundamental role in bioenergetics . This conservation extends across bacteria, archaea, and eukaryotes, making it an excellent subject for evolutionary studies.

When analyzing sequence conservation in ATP synthase subunit c, researchers should:

• Employ multiple sequence alignment tools to identify conserved regions across diverse species

• Pay particular attention to the ion-binding residues (such as Asp61) that are critical for function

• Consider how differences in the c-ring stoichiometry might reflect adaptation to different environmental conditions or energy requirements

The high conservation of functional regions contrasted with the diversity in targeting peptides suggests differential selective pressures on different domains of the protein, with implications for the evolution of bioenergetic systems.

What are the optimal approaches for expressing recombinant Pseudomonas entomophila ATP synthase subunit c?

Successful expression of recombinant ATP synthase subunit c from Pseudomonas entomophila requires careful consideration of expression systems and conditions. Based on research practices with similar proteins, consider the following methodological approaches:

Expression System Selection:

• E. coli-based systems: BL21(DE3) strains typically provide good expression levels for bacterial membrane proteins. The inclusion of chaperones may improve proper folding.

• Cell-free expression systems: These can be advantageous for membrane proteins like subunit c, allowing direct incorporation into supplied lipid environments.

Expression Optimization Parameters:

• Temperature: Lower temperatures (16-25°C) often improve proper folding of membrane proteins.

• Induction conditions: IPTG concentration should be optimized (typically 0.1-0.5 mM) for membrane protein expression.

• Media supplements: Addition of specific lipids may improve membrane protein folding and stability.

• Fusion tags: N-terminal tags such as His6, MBP, or SUMO can improve expression and subsequent purification.

When designing expression experiments for ATP synthase subunit c, it's crucial to include controls with known expression patterns (ideally other membrane proteins successfully expressed in your system) and to verify the integrity of the expressed protein through techniques such as Western blotting with antibodies against the subunit c or any included tag .

What purification strategies work best for recombinant ATP synthase subunit c?

Purification of ATP synthase subunit c presents challenges due to its hydrophobic nature and membrane association. Based on established protocols for similar proteins, consider this strategic purification workflow:

• Membrane isolation: After cell lysis, isolate membrane fractions through differential centrifugation.

• Detergent solubilization: Test multiple detergents (DDM, LMNG, CHAPS) at various concentrations to optimize solubilization without denaturing the protein.

• Affinity chromatography: If using tagged constructs, employ appropriate affinity resins (Ni-NTA for His-tagged proteins).

• Size exclusion chromatography: For separating different oligomeric states and removing aggregates.

• Ion exchange chromatography: Can provide additional purification based on the protein's charge properties.

Critical considerations:

• Maintain detergent concentration above CMC throughout purification

• Consider using lipid additives to stabilize the protein

• Monitor protein quality after each purification step using SDS-PAGE and potentially mass spectrometry

• Assess protein functionality through proton transport assays or ATP synthesis activity measurements

For experimental validation, always include quality control analysis of the purified protein using techniques such as circular dichroism to confirm secondary structure integrity and dynamic light scattering to assess homogeneity .

How can ATP synthase activity be accurately measured in recombinant systems?

Measuring ATP synthase activity requires assessing both ATP synthesis and hydrolysis functions. For recombinant ATP synthase containing subunit c from Pseudomonas entomophila, consider these methodological approaches:

ATP Synthesis Activity:

• Reconstituted liposome assays: Reconstitute purified ATP synthase into liposomes, establish a proton gradient (using pH jump or valinomycin/K+), and measure ATP production over time using luciferase-based detection systems.

• Inside-out membrane vesicle assays: Prepare inside-out vesicles from cells expressing the recombinant protein, establish a proton gradient, and measure ATP production.

ATP Hydrolysis Activity:

• Spectrophotometric coupled enzyme assays: Measure ADP production by coupling to NADH oxidation through pyruvate kinase and lactate dehydrogenase.

• Malachite green phosphate release assay: Directly quantify released phosphate from ATP hydrolysis.

• Luciferin/luciferase assay: Measure ATP consumption in real-time.

Control Experiments and Validation:

• Include inhibitors such as oligomycin or DCCD to confirm specificity

• Compare with native ATP synthase activity as a reference point

• Establish the pH and temperature optima for the enzyme

• Measure activity in the presence of various ions to determine specificity

For example, a study examining ATP dynamics across different growth phases in various carbon sources utilized a genetically encoded ATP biosensor (iATPsnFR1.1) containing a circularly permuted super-folder green fluorescent protein integrated within the ATP-binding epsilon subunit of the F0-F1 ATP synthase . This biosensor showed that ATP binding induces conformational changes leading to enhanced green fluorescence with a response time within 10 milliseconds, providing real-time information about cellular ATP levels .

What techniques are available for studying the proton translocation function of ATP synthase subunit c?

Proton translocation through the c-ring is fundamental to ATP synthase function. These methodological approaches can help researchers investigate this process:

Direct Proton Flux Measurements:

• pH-sensitive fluorescent probes: Incorporate probes like ACMA or pyranine into proteoliposomes containing reconstituted ATP synthase to monitor pH changes.

• SSM-based electrophysiology: Use solid-supported membrane electrophysiology to measure transient currents associated with proton translocation.

• Patch-clamp techniques: For measuring proton currents in larger membrane systems.

Indirect Assessment Methods:

• Membrane potential measurements: Use voltage-sensitive dyes to monitor membrane potential changes associated with proton translocation.

• Proton-motive force determination: Calculate from measured ΔpH and membrane potential.

Structural Insights:

• Site-directed mutagenesis: Modify key residues (particularly the conserved Asp61) to assess their role in proton translocation.

• Hydrogen/deuterium exchange mass spectrometry: To identify regions exposed to the aqueous environment during the proton translocation cycle.

The mechanism of proton translocation involves sequential protonation and deprotonation of key residues in subunit c (typically Asp61) coupled to the rotational movement of the c-ring . This process is directly linked to ATP synthesis and represents a critical step in cellular energy metabolism.

How do mutations in ATP synthase subunit c affect enzyme function and bacterial viability?

Mutations in ATP synthase subunit c can have profound effects on enzyme function and organism viability due to this protein's central role in energy metabolism. Research approaches to study these effects include:

Mutation Analysis Strategies:

• Site-directed mutagenesis: Target conserved residues (particularly ion-binding sites like Asp61) and assess functional consequences.

• Random mutagenesis: Generate libraries of mutations and screen for functional phenotypes.

• Naturally occurring mutations: Study naturally occurring variants or resistance mutations (e.g., those conferring resistance to certain antibiotics targeting ATP synthase).

Functional Assessment Methods:

• Growth phenotyping: Measure growth rates and yields under various energy sources and conditions.

• ATP synthesis and hydrolysis assays: Quantify the impact on enzymatic activities.

• Proton leakage measurements: Determine if mutations affect the coupling between proton translocation and ATP synthesis.

Research has shown that silencing individual subunit c isoforms in mammalian systems results in ATP synthesis defects, indicating non-redundant functions even between highly similar isoforms . In bacteria, mutations in the ion-binding site can confer resistance to certain antibiotics while potentially compromising ATP synthesis efficiency. Complete loss of functional ATP synthase is often lethal in obligate aerobes but may be tolerated in facultative anaerobes under fermentative conditions.

What insights have structural studies provided about ATP synthase subunit c function?

Structural studies of ATP synthase subunit c have been instrumental in understanding its function. For researchers investigating Pseudomonas entomophila ATP synthase, these methodological approaches are valuable:

Structural Determination Methods:

• Homology modeling: When direct structural data is unavailable, homology modeling based on related proteins can provide valuable insights. For example, the 3D model structure of AtpE was constructed using Modeller9.16, followed by energy minimization and refinement using molecular dynamic simulation .

• Cryo-electron microscopy: Has revolutionized our understanding of the entire ATP synthase complex, including the c-ring arrangement.

• X-ray crystallography: Has provided high-resolution structures of isolated c-rings from various organisms.

• NMR spectroscopy: Particularly useful for studying dynamics and conformational changes.

Key Structural Insights:

• c-ring organization: The c-ring consists of multiple identical subunits arranged in a circle, with each subunit contributing to proton binding sites.

• Proton-binding site: The conserved acidic residue (typically Asp61) forms the proton-binding site, which undergoes protonation/deprotonation during rotation.

• Interface with other subunits: Structural studies have revealed how the c-ring interfaces with the a-subunit to form the proton channel and with the γ-subunit to couple rotation to ATP synthesis.

The 3D structure determination process typically involves template selection based on sequence identity, sequence alignment, model building, energy minimization, and validation. For example, AtpE structure modeling identified six protein templates (4V1F, 4MJN, 3ZK1, 3V3C, 2WIE, and 2W5J) with 4V1F selected due to high resolution (90.1% sequence identity) .

How can ATP synthase subunit c be targeted for antimicrobial development?

ATP synthase subunit c represents a promising target for antimicrobial development due to its essential role in bacterial energy metabolism. Researchers exploring this avenue should consider these methodological approaches:

Target Validation Methods:

• Genetic approaches: Conditional knockdown systems to confirm essentiality under various conditions.

• Chemical validation: Use of known ATP synthase inhibitors to establish proof-of-concept.

Inhibitor Discovery Strategies:

• Structure-based drug design: Using homology models or crystal structures to design compounds that interact with critical residues.

• High-throughput screening: Assays based on ATP synthesis inhibition to identify novel inhibitors.

• Molecular docking studies: Computational methods to identify potential binding sites and predict compound binding. For example, AtpE structure has been used for virtual screening against Zinc and PubChem databases to identify compounds with minimum binding energy using RASPD and PyRx tools .

Inhibitor Optimization Process:

• Structure-activity relationship studies: Systematic modification of lead compounds to improve potency and selectivity.

• Binding mode analysis: Techniques like X-ray crystallography or cryo-EM to confirm binding modes.

• Resistance mechanism studies: Investigation of potential resistance mechanisms to guide inhibitor design.

In one study, compounds with minimum binding energies ranging between -8.69 and -8.44 kcal/mol (less than the free binding energy of ATP) were selected as potential inhibitors of AtpE . The molecular docking process involved converting both AtpE and ligands to PDBQT files, calculating gasteiger charges, and determining free binding energy using Lamarckian genetic algorithms .

How can ATP synthase subunit c be leveraged for biotechnological applications?

ATP synthase and specifically its subunit c have potential applications in biotechnology beyond their natural role. These methodological approaches can help researchers explore such applications:

Bioenergy Applications:

• Engineered ATP synthases: Modify the c-ring stoichiometry to alter the H+/ATP ratio for optimized energy conversion in specific applications.

• Artificial photosynthesis systems: Incorporate ATP synthase into artificial membranes coupled to light-harvesting systems.

Biosensing Applications:

• ATP biosensors: Develop sensors based on conformational changes in ATP synthase components. For example, the iATPsnFR1.1 biosensor uses a circularly permuted super-folder green fluorescent protein integrated within the ATP-binding epsilon subunit of the F0-F1 ATP synthase, with ATP binding causing enhanced green fluorescence .

• Proton gradient sensors: Utilize subunit c's sensitivity to proton gradients for developing novel pH sensors.

Bioproduction Enhancement:

• Metabolic engineering: Modify ATP synthase to enhance ATP availability for biosynthetic pathways. Research has shown that ATP dynamics significantly impact bioproduction in microbial strains, with transient ATP accumulations coinciding with fatty acid and polyhydroxyalkanoate production .

• Carbon source optimization: Different carbon sources significantly impact ATP levels within each species. For example, during exponential growth under aerobic conditions, E. coli exhibits the highest ATP levels when cultivated with acetate .

What are common challenges in ATP synthase subunit c research and how can they be overcome?

Research on ATP synthase subunit c presents several technical challenges. Here are methodological solutions to common problems:

Expression and Purification Challenges:

• Low expression levels:

  • Solution: Optimize codon usage for the expression host

  • Alternative: Test different promoter systems or expression hosts

  • Validation: Compare expression levels using Western blotting

• Protein aggregation:

  • Solution: Adjust detergent type and concentration during solubilization

  • Alternative: Express as fusion with solubility-enhancing partners

  • Validation: Assess aggregation state using size exclusion chromatography

Functional Analysis Challenges:

• Low activity in reconstituted systems:

  • Solution: Optimize lipid composition to match native environment

  • Alternative: Verify complete assembly of the ATP synthase complex

  • Validation: Compare with native membrane activity as benchmark

• Inconsistent activity measurements:

  • Solution: Standardize preparation methods and assay conditions

  • Alternative: Use multiple complementary assay methods

  • Validation: Include internal controls in each experiment

Data Interpretation Challenges:

• Distinguishing direct vs. indirect effects:

  • Solution: Use specific inhibitors to isolate ATP synthase function

  • Alternative: Create minimal reconstituted systems

  • Validation: Perform genetic complementation experiments

When working with ATP synthase isoforms, consider that they may have non-redundant functions despite sequence similarities. For example, research has shown that mammalian subunit c isoforms that differ only in their targeting peptides cannot cross-complement when silenced .

How can I reconcile contradictory data about ATP synthase subunit c function across different experimental systems?

Researchers often encounter seemingly contradictory results when studying ATP synthase subunit c across different experimental systems. These methodological approaches can help reconcile such discrepancies:

Systematic Comparison Approach:

• Create a comparison matrix:

  • Document experimental conditions for each study (pH, temperature, lipid composition, etc.)

  • Note the specific assays and readouts used

  • Record the exact protein constructs and expression systems

• Identify key variables:

  • Test hypotheses about which variables might explain differences

  • Systematically modify each variable while keeping others constant

  • Quantify the impact of each variable on experimental outcome

Reconciliation Strategies:

• Combined experimental approaches:

  • Perform multiple assays on the same sample

  • Use orthogonal techniques to validate findings

  • Combine in vitro and in vivo measurements when possible

• Computational modeling:

  • Develop models that can account for differences in experimental conditions

  • Use simulation to predict behavior under different conditions

  • Validate model predictions with targeted experiments

For example, when studying the functional specificity of ATP synthase subunit c isoforms, researchers discovered that the expression of targeting peptides fused to GFP variants could rescue ATP synthesis and respiratory chain defects in silenced cells, demonstrating that functional specificity resided in the targeting peptides rather than the mature protein .

What emerging technologies show promise for advancing ATP synthase subunit c research?

Several cutting-edge technologies are poised to revolutionize research on ATP synthase subunit c. Researchers should consider these methodological innovations:

Advanced Structural Biology Techniques:

• Time-resolved cryo-EM: Captures structural intermediates during the rotational cycle

• Integrative structural biology: Combines multiple techniques (cryo-EM, X-ray, NMR, mass spectrometry) for comprehensive structural understanding

• Single-particle cryo-electron tomography: Visualizes ATP synthase in its native membrane environment

Single-Molecule Approaches:

• Single-molecule FRET: Measures conformational changes and subunit interactions in real-time

• Optical tweezers: Directly measures forces and rotational motion of ATP synthase components

• Nanodiscs technology: Provides a more native-like membrane environment for single-molecule studies

Advanced Biosensors:

• Genetically encoded sensors: Next-generation versions of ATP biosensors with improved sensitivity and specificity

• Label-free detection methods: Surface plasmon resonance and other techniques for monitoring binding events

• Multiplexed sensor arrays: Simultaneous monitoring of multiple parameters (ATP levels, proton flux, conformational changes)

Recent research has demonstrated the value of ATP biosensors containing circularly permuted super-folder green fluorescent protein integrated within the ATP-binding epsilon subunit of the F0-F1 ATP synthase, which allows real-time monitoring of ATP dynamics in living cells . Such approaches can be further refined for studying specific aspects of ATP synthase function.

What are the most promising areas for future research on Pseudomonas entomophila ATP synthase subunit c?

Research on Pseudomonas entomophila ATP synthase subunit c offers several promising directions. These methodological approaches could yield significant advances:

Comparative Biology Approaches:

• Cross-species comparison: Systematic comparison of P. entomophila ATP synthase with other Pseudomonas species

• Ecological adaptation studies: Investigation of how ATP synthase variations relate to P. entomophila's ecological niche

• Host-pathogen interaction studies: Exploration of ATP synthase role in pathogenicity and host immune response

Applied Research Directions:

• Bioproduction optimization: Leverage ATP synthase engineering to enhance valuable metabolite production

• Antimicrobial development: Target P. entomophila-specific features of ATP synthase for selective inhibition

• Biosensor development: Utilize P. entomophila ATP synthase components for specialized biosensing applications

Fundamental Mechanism Studies:

• c-ring stoichiometry determination: Establish the exact number of c subunits in the P. entomophila ATP synthase

• Proton pathway mapping: Identify the complete path of proton movement through the membrane domain

• Regulatory mechanism investigation: Uncover how ATP synthase activity is regulated in response to environmental conditions

Research has shown that ATP dynamics significantly impact bioproduction in microbial strains, with transient ATP accumulations occurring during the transition from exponential to stationary growth phases . These dynamics could be leveraged to enhance production of valuable compounds in engineered P. entomophila strains.

What are the key parameters for ATP synthase activity across different experimental conditions?

The following table summarizes important parameters for ATP synthase activity measurements that researchers should consider when designing experiments:

When designing experiments to measure ATP synthase activity, researchers should carefully control these parameters to ensure reliable and reproducible results. Different experimental questions may require optimization of specific parameters within these ranges.

What are the conserved residues in ATP synthase subunit c across bacterial species?

The following table highlights the key conserved residues in ATP synthase subunit c that are critical for function across bacterial species:

*Position numbers based on E. coli ATP synthase subunit c sequence as reference

This high conservation reflects the critical nature of these residues for ATP synthase function. The sequential protonation and deprotonation of Asp61 is coupled to the stepwise movement of the c-ring, directly driving ATP synthesis . Mutations in these conserved residues typically result in significant functional consequences, from reduced ATP synthesis efficiency to complete loss of function.