Recombinant Aromatoleum aromaticum ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Aromatoleum aromaticum?

ATP synthase subunit c (atpE) in Aromatoleum aromaticum is a critical component of the F0 sector of the F-type ATP synthase complex. It is a small hydrophobic protein that forms a cylindrical oligomer in the membrane domain of ATP synthase. The recombinant form of this protein (UniProt ID: Q5P4E7) consists of 82 amino acids (1-82aa) and is typically expressed with an N-terminal His tag for purification purposes . ATP synthase subunit c is also known as lipid-binding protein and plays a central role in the proton pumping mechanism that couples the proton gradient generated by the respiratory chain to ATP synthesis .

What expression systems are used for producing recombinant A. aromaticum atpE?

Recombinant A. aromaticum ATP synthase subunit c is predominantly expressed in Escherichia coli expression systems. The recombinant protein is designed with a His tag (typically N-terminal) to facilitate purification using affinity chromatography techniques . The expression in E. coli allows for high-yield protein production while maintaining the functional properties of the native protein. For optimal expression, codon optimization may be necessary to account for differences in codon usage between A. aromaticum and E. coli.

What are the recommended storage and handling conditions for recombinant atpE?

The recommended storage and handling conditions for recombinant Aromatoleum aromaticum ATP synthase subunit c are:

These conditions help maintain protein stability and functional integrity. The addition of glycerol prevents ice crystal formation during freezing, which can damage protein structure .

How does atpE contribute to the proton-pumping mechanism in ATP synthase?

ATP synthase subunit c plays a fundamental role in the proton-pumping mechanism by forming a cylindrical oligomer (typically c10) in the membrane that functions as a rotor. In this process:

• Protons enter the c-ring through a half-channel in subunit a and bind to a conserved carboxylate residue (typically glutamate or aspartate) in subunit c.

• This binding neutralizes the negative charge, allowing the c-ring to rotate within the hydrophobic membrane environment.

• After rotation, the proton is released through another half-channel in subunit a.

• The rotation of the c-ring is mechanically coupled to conformational changes in the F1 sector, driving ATP synthesis .

In A. aromaticum, subunit c directly cooperates with subunit a in this proton translocation process, which is essential for energy conservation during both aerobic and anaerobic metabolism. This mechanism allows A. aromaticum to efficiently utilize energy from the degradation of aromatic compounds .

What role does ATP synthase play in A. aromaticum's ability to metabolize aromatic compounds?

ATP synthase plays a central role in A. aromaticum's energy metabolism during the degradation of aromatic compounds. This bacterium thrives in diverse habitats by utilizing a broad range of recalcitrant organic molecules coupled to either denitrification (under anaerobic conditions) or oxygen respiration (under aerobic conditions) .

The ATP synthase complex (AtpA-H) in A. aromaticum is expressed at high levels under all tested growth conditions, regardless of the carbon source (aromatic compounds or acetate) or respiratory electron acceptor (nitrate or oxygen) . This constitutive high expression suggests that maintaining efficient ATP production is critical for:

• Supporting the energy-intensive initial activation of aromatic compounds

• Powering transport systems for substrate uptake and metabolite export

• Fueling biosynthetic pathways for cellular components

• Maintaining cellular homeostasis during metabolism of potentially toxic aromatic compounds

The versatility of A. aromaticum in degrading aromatic compounds is reflected in its sophisticated energy conservation machinery, with ATP synthase serving as the final component in converting the proton gradient into usable chemical energy in the form of ATP .

How do the isoforms of ATP synthase subunit c differ in their functions?

While A. aromaticum appears to have a single atpE gene encoding ATP synthase subunit c, studies in mammalian systems have revealed important insights into functional differentiation of subunit c isoforms that may have parallels in bacterial systems:

Mammals have three isoforms of F1F0-ATP synthase subunit c (P1, P2, and P3) that differ only in their mitochondrial targeting peptides while having identical mature peptides. Despite this apparent redundancy, these isoforms are not functionally interchangeable. Experimental evidence shows that:

• Silencing any individual subunit c isoform results in ATP synthesis defects

• The P2 isoform specifically affects cytochrome oxidase assembly and function

• Cross-complementation experiments demonstrate that P1 cannot substitute for P2, indicating functional specificity

• The targeting peptides play additional roles beyond protein import, including maintenance of respiratory chain integrity

These findings suggest that ATP synthase components may have specialized functions beyond their direct role in ATP synthesis. For bacterial systems like A. aromaticum, this raises questions about potential specialized roles of ATP synthase in different metabolic contexts or growth conditions .

What experimental approaches are used to study the function of ATP synthase in A. aromaticum?

The function of ATP synthase in A. aromaticum has been studied using several integrated experimental approaches:

Multi-omics studies have revealed that while the transcription and protein formation of ATP synthase in A. aromaticum appear to be constitutive across different carbon sources and respiratory conditions, the actual metabolite profiles show significant differences between anaerobic (nitrate-reducing) and aerobic conditions .

For in-depth functional studies of ATP synthase components like subunit c, researchers often employ techniques such as:

• Site-directed mutagenesis to alter key residues involved in proton translocation

• ATP synthesis assays in membrane vesicles or reconstituted systems

• Membrane potential measurements using fluorescent probes

• Structural studies using cryo-electron microscopy or X-ray crystallography

What insights have multi-omics studies revealed about ATP synthase regulation in A. aromaticum?

Integrated multi-omics studies of A. aromaticum have yielded several significant insights about ATP synthase regulation:

• Constitutive Expression Pattern: Transcripts and proteins of the ATP synthase complex (AtpA-H, including atpE) are observed at high levels under all tested growth conditions, suggesting that maintaining ATP synthesis capacity is a core requirement regardless of substrate or electron acceptor .

• Contrast with Substrate-Specific Modules: Unlike the degradation modules for aromatic compounds that show substrate-dependent regulation (ranging from highly specific for 3-(4-hydroxyphenyl)propanoate to relatively relaxed for benzoate), the respiration network, including ATP synthase, appears to be constitutively expressed .

• Differential Regulation at Metabolite Level: Despite constitutive expression at transcript and protein levels, metabolomic analyses revealed distinct anoxia-specific versus oxia-specific metabolite profiles, indicating that regulation may occur primarily at the metabolic level rather than through transcriptional or translational control .

• Coordination with Respiratory Chain Components: The respiration network components are generally constitutively expressed, with the exception of nitrate reductase (with narGHI expression occurring only under nitrate-reducing conditions), suggesting coordination between terminal electron acceptor usage and ATP synthesis capacity .

These findings highlight the sophisticated regulatory network that coordinates energy conservation with substrate utilization in A. aromaticum, positioning ATP synthase as a central, constitutively maintained component of the cell's energy metabolism machinery .

What purification methods are most effective for recombinant A. aromaticum atpE?

The most effective purification strategy for recombinant Aromatoleum aromaticum ATP synthase subunit c leverage the N-terminal His tag that is typically incorporated into the recombinant construct. A recommended purification protocol includes:

• Cell Lysis: Using appropriate detergents to solubilize the membrane-associated protein

• Immobilized Metal Affinity Chromatography (IMAC): Using Ni-NTA or similar matrices to capture the His-tagged protein

• Size Exclusion Chromatography: To remove aggregates and achieve higher purity

• Quality Control: SDS-PAGE analysis to confirm purity (>90% is typically achieved)

The purified protein is usually obtained as a lyophilized powder after buffer exchange and freeze-drying. For functional studies, careful reconstitution in appropriate lipid environments may be necessary to maintain the native conformation of this highly hydrophobic membrane protein .

How can researchers assess the functional integrity of purified recombinant atpE?

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Size exclusion chromatography to verify oligomeric state

  • Limited proteolysis to assess proper folding

• Lipid Binding Assays:

  • Since subunit c is also known as lipid-binding protein, lipid binding assays using fluorescently labeled lipids can assess functionality

• Reconstitution Experiments:

  • Incorporation into liposomes or nanodiscs

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Complementation of depleted membrane preparations

• Assembly into c-ring Structures:

  • Electron microscopy to visualize c-ring formation

  • Native gel electrophoresis to assess oligomerization

• Functional Assays in Reconstituted Systems:

  • When combined with other ATP synthase subunits, ATP synthesis or hydrolysis activity measurements

These methodological approaches provide complementary information about the functional state of the recombinant protein, which is essential for interpreting experimental results in studies of ATP synthase function .

What are the challenges in studying the proton translocation function of ATP synthase subunit c?

Studying the proton translocation function of ATP synthase subunit c presents several significant challenges:

• Membrane Protein Isolation: As a highly hydrophobic membrane protein, subunit c requires careful handling with appropriate detergents to maintain its native conformation while removing it from the membrane environment.

• Functional Reconstitution: The function of subunit c depends on its assembly into the c-ring structure and interaction with other ATP synthase subunits, particularly subunit a. Reconstituting this complex system in vitro while maintaining functionality is technically demanding .

• Measuring Proton Movements: Detecting and quantifying proton translocation requires specialized techniques such as:

  • pH-sensitive fluorescent probes

  • Membrane potential measurements

  • Patch-clamp electrophysiology

  • Isotope exchange studies

• Distinguishing Direct Effects: Because subunit c functions as part of a larger complex, distinguishing direct effects of this subunit from indirect effects mediated through other components can be difficult .

• Structural Studies: Obtaining high-resolution structural information of subunit c in its native lipid environment is challenging, though advances in cryo-electron microscopy have improved capabilities in this area.

Researchers typically address these challenges through an integrated approach, combining biochemical, biophysical, and structural methods to build a comprehensive understanding of ATP synthase subunit c function .

How can researchers incorporate A. aromaticum atpE into liposomes for functional studies?

Incorporating Aromatoleum aromaticum ATP synthase subunit c into liposomes for functional studies requires a carefully optimized protocol:

• Protein Preparation:

  • Reconstitute the lyophilized recombinant protein in a buffer containing appropriate detergents (e.g., n-dodecyl β-D-maltoside or CHAPS)

  • Ensure complete solubilization while maintaining protein stability

• Liposome Preparation:

  • Prepare liposomes using lipids that mimic the bacterial membrane composition (typically a mixture of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin)

  • Form uniform unilamellar vesicles through extrusion

• Protein Incorporation:

  • Mix solubilized protein with detergent-destabilized liposomes

  • Remove detergent using bio-beads, dialysis, or gel filtration

  • Monitor incorporation efficiency using density gradient centrifugation

• Functional Assessment:

  • Verify proper orientation using protease protection assays

  • Measure proton permeability using pH-sensitive fluorescent dyes

  • Assess ATP synthesis/hydrolysis if co-reconstituted with other ATP synthase components

• Quality Control:

  • Freeze-fracture electron microscopy to visualize protein distribution

  • Dynamic light scattering to assess vesicle size and homogeneity

For more comprehensive studies, co-reconstitution with other ATP synthase subunits, particularly subunit a, may be necessary to recapitulate the native proton translocation function .

How does A. aromaticum atpE contribute to our understanding of ATP synthase evolution?

Studying Aromatoleum aromaticum ATP synthase subunit c provides valuable insights into ATP synthase evolution:

• Comparative Genomics: The atpE gene in A. aromaticum can be compared with homologs across bacterial species, archaea, and eukaryotes to trace evolutionary relationships. The conserved regions likely represent functionally critical domains that have been maintained through selective pressure.

• Adaptations to Different Energy Metabolisms: A. aromaticum's ability to thrive in both aerobic and anaerobic conditions may be reflected in adaptations of its ATP synthase components. The ATP synthase complex, including subunit c, is expressed at high levels regardless of growth conditions, suggesting fundamental importance across metabolic modes .

• Functional Conservation: The cooperative functioning of subunit c with subunit a in proton pumping represents a conserved mechanism that has been maintained throughout the evolution of F-type ATP synthases, from bacteria to mitochondria in higher organisms .

• Structural Variations: Comparing the structure of A. aromaticum atpE with other bacterial species may reveal adaptations to specific membrane environments or energy coupling requirements. The number of c-subunits in the c-ring can vary between species, affecting the bioenergetic efficiency of ATP synthesis.

• Horizontal Gene Transfer Assessment: Analysis of atpE sequence and its genomic context can provide evidence for potential horizontal gene transfer events that may have contributed to A. aromaticum's metabolic versatility.

These evolutionary insights contribute to our fundamental understanding of bioenergetics across the tree of life and may inform synthetic biology approaches to engineer energy-efficient biological systems .

What role might ATP synthase play in A. aromaticum's adaptation to different environmental conditions?

ATP synthase likely plays a critical role in A. aromaticum's remarkable adaptability to diverse environmental conditions:

• Metabolic Flexibility: The constitutive high-level expression of ATP synthase (AtpA-H) across different growth conditions suggests that maintaining robust ATP production capability is essential for A. aromaticum's ability to utilize diverse carbon sources, including recalcitrant aromatic compounds .

• Adaptation to Oxygen Availability: As a facultative anaerobe, A. aromaticum must adjust its energy metabolism between aerobic respiration and denitrification. The ATP synthase complex appears to be a constant component across these respiratory modes, though its operational efficiency may vary depending on the proton motive force generated by different electron transport chains .

• Response to Energy Demands: During degradation of aromatic compounds, which often requires energy-intensive activation steps, efficient ATP synthesis is crucial. The high expression of ATP synthase components ensures that energy captured in the proton gradient can be efficiently converted to ATP.

• Stress Response: Under environmental stress conditions, maintaining energy homeostasis becomes critical. ATP synthase may play a role in stress adaptation by ensuring sufficient ATP production even when primary metabolic pathways are challenged.

• Growth Rate Modulation: The efficiency of ATP synthesis directly impacts growth rates and biomass yields. Multi-omics and metabolic modeling approaches have shown good agreement between predicted and experimental growth parameters for A. aromaticum on various substrates .

Understanding the role of ATP synthase in environmental adaptation may provide insights for biotechnological applications, such as bioremediation of aromatic pollutants or engineering of stress-resistant bacterial strains .

How can the study of A. aromaticum atpE inform the development of novel antibiotics or bioenergetic inhibitors?

The study of Aromatoleum aromaticum ATP synthase subunit c can inform the development of novel antibiotics or bioenergetic inhibitors through several approaches:

• Structural Uniqueness Assessment: Comparative analysis of A. aromaticum atpE with human ATP synthase homologs can identify structural differences that might be exploited for selective targeting. These differences may include:

  • Amino acid variations in the proton-binding site

  • Differences in c-ring stoichiometry

  • Unique protein-protein interaction interfaces

• Functional Mechanism Insights: Understanding the detailed mechanism of proton translocation through the c-ring can reveal potential points for intervention that might disrupt ATP synthesis without affecting other cellular processes.

• Design of c-subunit Inhibitors: Knowledge of the specific binding sites and critical residues in A. aromaticum atpE can guide rational design of inhibitors that selectively target bacterial ATP synthase. Several existing antibiotics, such as bedaquiline, target mycobacterial ATP synthase subunit c, demonstrating the viability of this approach.

• Cross-Species Applicability: Insights from A. aromaticum atpE studies may be applicable to pathogenic bacteria with similar ATP synthase structures, potentially leading to broad-spectrum antibiotics targeting energy metabolism.

• Resistance Mechanism Prediction: Understanding the molecular details of atpE function can help predict potential resistance mechanisms and guide the development of inhibitor combinations or alternative targeting strategies.

This research direction is particularly valuable given the increasing problem of antibiotic resistance and the need for antibiotics with novel mechanisms of action targeting essential bacterial processes .

What implications does the multi-omics research on A. aromaticum have for systems biology approaches to studying bacterial metabolism?

The multi-omics research on Aromatoleum aromaticum has significant implications for systems biology approaches to bacterial metabolism:

• Integrated Data Analysis Framework: The successful integration of transcriptomics, proteomics, and metabolomics data from A. aromaticum demonstrates a powerful framework for holistic understanding of bacterial metabolism. This approach revealed that while ATP synthase expression is constitutive at transcript and protein levels, metabolic responses show condition-specific patterns .

• Genome-Scale Metabolic Modeling: The development of a genome-scale metabolic model for A. aromaticum, comprising 655 enzyme-catalyzed reactions and 731 distinct metabolites, provides a quantitative platform for predicting metabolic behaviors. This model successfully predicted growth rates and biomass yields for most tested substrates .

• Regulatory Network Mapping: The matrix factorization of transcriptomic data revealed that anaerobic modules accounted for most of the variance across the degradation network, highlighting the importance of studying metabolism under diverse conditions rather than focusing solely on standard laboratory conditions .

• Predictive Modeling Refinement: The discrepancy between model predictions and experimental results for growth on 3-(4-hydroxyphenyl)propanoate (where 4-hydroxybenzoate was exported) demonstrates how systems biology approaches can identify gaps in our understanding and guide further research .

• Transferable Methodologies: The multi-tiered analytical approach used for A. aromaticum provides a template for studying other bacteria with complex metabolic capabilities, particularly those with environmental or biotechnological significance.

These systems biology approaches offer a more complete understanding of bacterial metabolism beyond individual pathways, revealing emergent properties that cannot be discerned from reductionist approaches alone .

What future research directions might yield the most significant insights about ATP synthase function in A. aromaticum?

Several promising future research directions could yield significant insights about ATP synthase function in Aromatoleum aromaticum:

• Structure-Function Studies: High-resolution structural determination of A. aromaticum ATP synthase, particularly focusing on the c-ring architecture and its interaction with subunit a, would provide crucial insights into the specific adaptations of this enzyme to A. aromaticum's diverse energy metabolism.

• Single-Cell Bioenergetics: Developing methods to measure ATP synthesis rates and proton motive force at the single-cell level would reveal how individual A. aromaticum cells modulate energy conservation in heterogeneous environments or during metabolic transitions.

• Synthetic Biology Approaches: Engineering modified versions of ATP synthase components, including atpE, could test hypotheses about the relationship between ATP synthase structure and the efficiency of aromatic compound degradation under various respiratory conditions.

• Protein-Protein Interaction Networks: Comprehensive mapping of ATP synthase interactions with other cellular components might reveal unexpected connections between energy conservation and other aspects of A. aromaticum metabolism.

• Comparative Bioenergetics: Systematic comparison of ATP synthase function across different Aromatoleum species that specialize in different aromatic substrates could reveal adaptations of the energy conservation machinery to specific metabolic niches.

• Environmental Adaptation Studies: Investigating how ATP synthase expression and function change in response to environmental stressors (pH, temperature, toxicants) would provide insights into A. aromaticum's resilience mechanisms.

• Spatiotemporal Organization: Examining the localization and dynamics of ATP synthase complexes within A. aromaticum cells during different growth phases and metabolic states could reveal additional regulatory mechanisms.

These research directions would contribute to a more comprehensive understanding of how ATP synthase function is integrated with A. aromaticum's remarkable metabolic versatility and environmental adaptability .