Recombinant Moorella thermoacetica ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Moorella thermoacetica?

ATP synthase subunit c (atpE) in Moorella thermoacetica is a 74-amino acid protein that forms part of the F0 sector of ATP synthase. The protein has a highly hydrophobic amino acid sequence (MATIGFIGVGLAIGLAALGSGLGQGIASRGALEGMARQPEASGDIRTTLLLALAFMEALT LFSFVIAILMWTKL) consistent with its role as a membrane-embedded component . As part of the F0 sector, it forms the proton channel through which protons flow, driving the conformational changes in F1 that lead to ATP synthesis. In M. thermoacetica, ATP synthase plays a crucial role in energy conservation during both heterotrophic and autotrophic growth conditions .

Why is Moorella thermoacetica significant for bioenergetic research?

Moorella thermoacetica holds particular significance in bioenergetic research due to its thermophilic nature (growth optimum at 55°C) and its ability to fix carbon dioxide through the Wood-Ljungdahl pathway . This acetogenic bacterium can utilize various substrates including CO2, CO, and syngas, making it valuable for studying ATP synthesis under different metabolic conditions. The organism's ability to grow at temperatures higher than the boiling point of some volatile compounds (like acetone at 58°C) makes it particularly interesting for developing consolidated bioprocesses with simultaneous product separation . Its ATP synthase components, including subunit c, operate efficiently at high temperatures, offering insights into thermostable bioenergetic mechanisms.

How is recombinant M. thermoacetica ATP synthase subunit c typically produced?

The recombinant production of M. thermoacetica ATP synthase subunit c (atpE) is typically achieved through heterologous expression in E. coli . The process involves:

• Cloning the atpE gene (Q2RFX4) into an expression vector

• Adding an N-terminal His-tag for purification purposes

• Transforming the construct into E. coli expression strains

• Inducing protein expression under controlled conditions

• Cell lysis and protein purification using affinity chromatography

• Quality assessment (typically by SDS-PAGE with >90% purity standard)

• Lyophilization for storage stability

The resulting protein is supplied as a lyophilized powder and requires reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL before use .

How does the sequence and structure of ATP synthase subunit c from M. thermoacetica compare to those from mesophilic organisms?

The ATP synthase subunit c from M. thermoacetica shows several adaptations consistent with thermophilic proteins. Comparative analysis reveals:

These structural differences are crucial for maintaining functional protein conformation at the elevated temperatures (45-65°C) at which M. thermoacetica operates . The thermostability of this protein makes it valuable for understanding mechanisms of thermal adaptation in membrane proteins.

What are the challenges in expressing and purifying functional M. thermoacetica ATP synthase subunit c?

Expression and purification of functional M. thermoacetica ATP synthase subunit c present several technical challenges:

• Membrane protein solubility: As a highly hydrophobic membrane protein, subunit c tends to aggregate when expressed in heterologous systems, requiring optimization of solubilization conditions.

• Proper folding: Ensuring the protein adopts its native conformation during expression in E. coli can be challenging, as the thermophilic protein may not fold correctly at lower temperatures.

• Functionality assessment: Unlike enzymatic proteins, direct functional assays for ATP synthase subunit c are complex, often requiring reconstitution into liposomes or nanodiscs.

• Stability during purification: Maintaining the stability of the protein during extraction from membranes requires careful selection of detergents that preserve structure without denaturing the protein.

• Oligomerization: In native environments, multiple copies of subunit c form a ring structure; achieving proper oligomerization in recombinant systems is challenging but essential for functional studies.

The recommended approach involves using mild detergents for solubilization, maintaining pH and ionic strength within narrow ranges, and potentially co-expressing with chaperones to facilitate proper folding .

How does the genetic context of the atpE gene in M. thermoacetica influence its expression and regulation?

The atpE gene in M. thermoacetica exists within the ATP synthase operon, where it is co-regulated with other ATP synthase subunits. Genomic analysis reveals:

• The atpE gene (Moth_2383) is located in proximity to other ATP synthase genes, suggesting coordinated expression .

• Regulation is likely influenced by cellular energy status, with expression levels responding to changes in carbon source availability.

• In metabolic engineering contexts, manipulation of ATP synthase expression impacts energy conservation pathways. For example, engineering M. thermoacetica for acetone production by manipulating carbon flux pathways affects ATP availability and, consequently, atpE expression .

• Comparative genomic analysis of different M. thermoacetica isolates shows high conservation of the atpE genomic region (98.97-99.06% identity), indicating the functional importance of maintaining this gene's sequence and context .

What are the optimal buffer conditions for functional studies of recombinant M. thermoacetica ATP synthase subunit c?

For functional studies of recombinant M. thermoacetica ATP synthase subunit c, buffer optimization is critical:

When reconstituting from lyophilized powder, it's recommended to centrifuge the vial briefly before opening and reconstitute to 0.1-1.0 mg/mL. For long-term storage, adding glycerol to a final concentration of 5-50% and aliquoting for storage at -20°C/-80°C is advised to prevent repeated freeze-thaw cycles .

How can researchers effectively incorporate recombinant ATP synthase subunit c into proteoliposomes for functional studies?

Incorporating recombinant M. thermoacetica ATP synthase subunit c into proteoliposomes for functional studies requires careful attention to several parameters:

• Lipid composition selection:

  • Use thermostable lipids such as archaeal tetraether lipids or synthetic equivalents

  • Maintain a physiologically relevant lipid:protein ratio (typically 50:1 to 100:1 by weight)

  • Consider incorporating cardiolipin (1-5%) to enhance ATP synthase function

• Proteoliposome preparation protocol:
  a. Dissolve lipids in chloroform and create a thin film by evaporation
  b. Hydrate lipid film with buffer containing solubilized recombinant protein
  c. Subject to freeze-thaw cycles (5-10 cycles) to form multilamellar vesicles
  d. Extrude through polycarbonate membranes (100-200 nm pore size) to form unilamellar vesicles
  e. Remove detergent using biobeads or dialysis at controlled rate

• Functional validation:

  • Assess proton transport using pH-sensitive fluorescent dyes

  • Measure membrane potential generation using voltage-sensitive probes

  • When combined with other ATP synthase components, measure ATP synthesis activity

• Thermostability considerations:

  • Perform quality control at both room temperature and at M. thermoacetica physiological temperature (55°C)

  • Use differential scanning calorimetry to verify thermal stability of the proteoliposome system

This methodology allows for detailed biophysical characterization of the membrane protein in an environment that mimics its native membrane context.

What strategies can be employed to study the interaction between ATP synthase subunit c and other components of the ATP synthase complex?

To study interactions between ATP synthase subunit c and other components of the ATP synthase complex, several complementary approaches can be employed:

• Co-expression and co-purification strategies:

  • Design constructs for co-expression of subunit c with interacting partners

  • Use orthogonal affinity tags for sequential purification of intact complexes

  • Employ crosslinking agents with defined spacer lengths to capture transient interactions

• Biophysical characterization methods:

  • Förster Resonance Energy Transfer (FRET) using strategically labeled components

  • Surface Plasmon Resonance (SPR) for binding kinetics and affinity determination

  • Analytical ultracentrifugation to assess complex formation and stoichiometry

• Structural biology approaches:

  • Cryo-electron microscopy of reconstituted complexes

  • X-ray crystallography of co-crystallized components

  • Solid-state NMR of membrane-embedded complexes

• Computational modeling:

  • Molecular dynamics simulations of subunit c ring in membrane environment

  • Protein-protein docking to predict interaction interfaces

  • Evolutionary coupling analysis to identify co-evolving residues between subunits

• Functional assays:

  • Proton conductance measurements with systematically varied subunit compositions

  • ATP synthesis/hydrolysis assays with reconstituted complexes

  • Mutational analysis of predicted interaction interfaces

When studying thermophilic ATP synthase components like those from M. thermoacetica, these experiments should be performed at physiologically relevant temperatures (45-65°C) to capture authentic interaction dynamics .

How can researchers address protein aggregation issues when working with recombinant M. thermoacetica ATP synthase subunit c?

Protein aggregation is a common challenge when working with highly hydrophobic membrane proteins like ATP synthase subunit c. Researchers can implement the following strategies:

• Optimization of expression conditions:

  • Lower induction temperature (16-20°C) despite working with a thermophilic protein

  • Reduce inducer concentration to slow protein production rate

  • Consider specialized E. coli strains designed for membrane protein expression

• Solubilization approaches:

  • Screen multiple detergents at varying concentrations (starting with DDM, LMNG, or Digitonin)

  • Use lipid-like peptides or amphipols as alternatives to traditional detergents

  • Apply on-column solubilization during purification

• Buffer optimization:

  • Include stabilizing agents such as trehalose (6%) as used in commercial preparations

  • Adjust ionic strength to minimize non-specific interactions

  • Consider adding specific lipids that may stabilize the native conformation

• Quality control methods:

  • Use size exclusion chromatography to separate aggregates from properly folded protein

  • Apply dynamic light scattering to monitor aggregation state

  • Employ circular dichroism to assess secondary structure integrity

If aggregation persists despite these measures, consider refolding approaches or co-expression with molecular chaperones specific for membrane proteins. Additionally, storing the protein at recommended concentrations with glycerol (5-50%) can help prevent aggregation during storage .

What controls and validation steps should be included when studying the function of recombinant ATP synthase subunit c?

Robust controls and validation steps are essential when studying recombinant ATP synthase subunit c function:

• Protein quality controls:

  • SDS-PAGE to confirm purity (>90% as standard)

  • Mass spectrometry to verify protein identity and intact mass

  • Circular dichroism to confirm secondary structure elements

  • Thermal stability assays to verify thermophilic properties

• Negative controls:

  • Heat-denatured protein preparations

  • Preparations with specific inhibitors (e.g., oligomycin or DCCD)

  • Liposomes without incorporated protein

  • Mutated versions lacking key functional residues

• Positive controls:

  • Well-characterized ATP synthase components from model organisms

  • Native membrane preparations from M. thermoacetica

  • Synthetic proton carriers with defined conductance properties

• Functional validation approaches:

  • Proton flux measurements under defined pH gradients

  • Membrane potential generation/dissipation kinetics

  • ATP synthesis measurements when combined with F1 components

  • Structural integrity assessment via electron microscopy

• Specificity controls:

  • Competition assays with unlabeled protein

  • Dose-response relationships for functional parameters

  • Side-by-side comparison with other F-type ATP synthase c subunits

Each experiment should include these controls to ensure that observed effects are specifically attributable to the functional properties of M. thermoacetica ATP synthase subunit c rather than experimental artifacts.

How should researchers interpret differential effects of temperature on recombinant M. thermoacetica ATP synthase subunit c function?

Interpreting temperature effects on M. thermoacetica ATP synthase subunit c function requires consideration of several factors:

• Thermodynamic considerations:

  • Establish Arrhenius plots for activities measured at different temperatures (25-70°C)

  • Calculate activation energies for specific processes (e.g., proton conductance)

  • Identify temperature optima and compare to the organism's growth temperature range (45-65°C)

• Structural transitions:

  • Monitor for cooperative transitions using methods like differential scanning calorimetry

  • Assess lipid phase transitions in reconstituted systems that may affect protein function

  • Consider separate thermal effects on protein-protein interactions versus catalytic activities

• Comparative framework:

  • Compare temperature profiles with mesophilic homologs to identify thermoadaptations

  • Establish whether observed effects match in vivo performance of M. thermoacetica

  • Consider temperature effects on the entire ATP synthase complex versus isolated subunit c

• Data interpretation framework:

• Context-specific considerations:

  • Consider buffer stability at high temperatures

  • Account for temperature effects on pH (ΔpKa/°C for buffers)

  • Evaluate temperature effects on detergents or lipids in reconstituted systems

Remember that M. thermoacetica is adapted to function at temperatures that would denature mesophilic proteins, so apparent "low activity" at room temperature may simply reflect evolutionary adaptation rather than experimental issues.

How might ATP synthase subunit c be utilized in synthetic biology applications involving thermophilic systems?

ATP synthase subunit c from M. thermoacetica offers several promising applications in synthetic biology:

• Thermostable energy harvesting modules:

  • Development of heat-stable ATP-generating systems for biocatalysis

  • Creation of artificial cells capable of energy transduction at elevated temperatures

  • Design of thermostable proton gradient-driven molecular motors

• Metabolic engineering applications:

  • Integration into engineered thermophilic microorganisms for enhanced ATP yield

  • Manipulation of c-ring stoichiometry to alter H+/ATP ratios and energetic efficiency

  • Coupling to alternative cellular processes requiring proton gradients

• Biomimetic materials and devices:

  • Development of temperature-resistant proton-conductive membranes

  • Creation of nanoscale rotary devices stable at elevated temperatures

  • Inspiration for synthetic molecular machines with thermostable components

• Protein engineering opportunities:

  • Use as a scaffold for creating hybrid proteins with novel functions

  • Template for designing de novo thermostable membrane proteins

  • Platform for evolutionary optimization of membrane protein stability

The thermostability of M. thermoacetica ATP synthase components makes them particularly valuable for applications requiring operation at elevated temperatures, such as industrial biocatalysis or processes coupled to thermochemical reactions .

What are the emerging techniques for studying ATP synthase subunit c dynamics in native-like environments?

Several cutting-edge approaches are emerging for studying ATP synthase subunit c dynamics:

• Advanced microscopy methods:

  • High-speed atomic force microscopy to observe c-ring rotation in real-time

  • Single-molecule FRET to track conformational changes during function

  • Cryo-electron tomography of whole ATP synthase complexes in native membranes

• Spectroscopic approaches:

  • Site-specific infrared spectroscopy using unnatural amino acids

  • Electron paramagnetic resonance with site-directed spin labeling

  • Solid-state NMR of isotopically labeled proteins in native-like membranes

• Computational methodologies:

  • Coarse-grained molecular dynamics to access longer timescales

  • Quantum mechanics/molecular mechanics (QM/MM) to study proton transfer events

  • Machine learning approaches to identify patterns in conformational dynamics

• Hybrid systems:

  • Nanodiscs containing native lipid compositions from M. thermoacetica

  • Microfluidic platforms for single-molecule studies at elevated temperatures

  • Cell-free expression systems coupled to direct functional assays

• Time-resolved structural methods:

  • Time-resolved X-ray free-electron laser crystallography

  • Temperature-jump experiments coupled to spectroscopic readouts

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

These approaches promise to reveal the molecular details of how ATP synthase subunit c contributes to proton translocation and energy conservation in thermophilic organisms like M. thermoacetica.

How might comparative studies of ATP synthase subunit c from different Moorella species contribute to our understanding of bioenergetic adaptation?

Comparative studies of ATP synthase subunit c across Moorella species and strains can provide valuable insights:

• Evolutionary adaptation insights:

  • Identification of conserved residues essential for function versus variable positions

  • Correlation between growth temperature optima and protein sequence features

  • Reconstruction of ancestral sequences to understand evolutionary trajectories

• Structure-function relationships:

  • Mapping sequence variations to specific functional properties (proton conductance, stability)

  • Understanding how subtle sequence changes affect c-ring stoichiometry

  • Identifying co-evolutionary patterns with other ATP synthase subunits

• Ecological adaptation mechanisms:

  • Correlation between environmental niche and ATP synthase properties

  • Adaptations specific to different carbon sources or energy conservation strategies

  • Variations related to pH or ion concentration differences in natural habitats

• Experimental approach:

  • Whole genome sequencing of diverse Moorella isolates, building on existing genomic data showing 98.49-99.18% identity between strains

  • Heterologous expression and functional characterization of variant subunit c proteins

  • Creation of chimeric proteins to map functional domains

  • In situ studies of ATP synthase performance in different Moorella species

Such comparative studies would be particularly valuable given the metabolic diversity within the Moorella genus and their adaptations to various thermophilic environments, providing a natural laboratory for studying bioenergetic adaptation.

What are the key technical recommendations for researchers beginning work with recombinant M. thermoacetica ATP synthase subunit c?

For researchers new to working with recombinant M. thermoacetica ATP synthase subunit c, the following technical recommendations can help ensure successful experiments:

• Storage and handling:

  • Store lyophilized protein at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles by preparing working aliquots

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol for long-term storage

  • Store working aliquots at 4°C for up to one week

• Experimental design:

  • Include appropriate thermostability controls

  • Consider temperature effects on all components (buffers, detergents, lipids)

  • Use parallel approaches to validate observations

  • Design experiments with both mesophilic and thermophilic controls

• Technical approach:

  • Begin with established protocols for membrane protein handling

  • Optimize detergent conditions empirically for your specific experimental needs

  • Consider native M. thermoacetica lipid composition when possible

  • Validate protein functionality before complex experimental setups

• Collaborative strategy:

  • Connect with researchers experienced in ATP synthase biochemistry

  • Partner with groups studying thermophilic proteins

  • Utilize shared resources for specialized techniques (e.g., cryo-EM, advanced spectroscopy)

Following these recommendations will help new researchers overcome the technical challenges associated with this specialized protein while maximizing experimental success.

How does research on M. thermoacetica ATP synthase subunit c connect to broader questions in bioenergetics and synthetic biology?

Research on M. thermoacetica ATP synthase subunit c connects to several broader scientific questions:

• Fundamental bioenergetics:

  • Mechanisms of energy conservation in diverse organisms

  • Evolutionary adaptations of the chemiosmotic machinery

  • Structure-function relationships in proton-translocating complexes

  • Thermodynamic constraints on biological energy transduction

• Synthetic biology applications:

  • Development of artificial photosynthetic systems with thermostable components

  • Creation of minimal cells with defined bioenergetic capabilities

  • Engineering of novel metabolic pathways for bioproduction at elevated temperatures

  • Design of biomimetic energy-harvesting devices

• Biotechnology relevance:

  • Connection to M. thermoacetica's capabilities for gas fermentation and bioproduction

  • Potential applications in consolidated bioprocesses operating at elevated temperatures

  • Relationship to metabolic engineering efforts for acetone production and carbon capture

  • Insights for designing thermostable industrial enzymes and processes

• Evolutionary biology:

  • Understanding convergent evolution in thermophilic adaptations

  • Insights into the early evolution of bioenergetic systems

  • Clues about adaptation to different environmental niches

This research thus sits at the intersection of multiple scientific disciplines, contributing to both fundamental understanding and applied technology development.

What interdisciplinary collaborations might enhance research on M. thermoacetica ATP synthase subunit c?

Productive interdisciplinary collaborations for M. thermoacetica ATP synthase subunit c research include:

• Structural biology partnerships:

  • Cryo-electron microscopy experts for high-resolution structural studies

  • NMR spectroscopists specializing in membrane proteins

  • Computational structural biologists for molecular dynamics simulations

• Systems biology collaborations:

  • Metabolic engineers working on M. thermoacetica for bioproduction

  • Systems modelers to integrate ATP synthase function into whole-cell models

  • Synthetic biologists designing minimal cells or artificial organelles

• Materials science connections:

  • Biomaterials researchers developing temperature-resistant membranes

  • Nanotechnology experts for single-molecule techniques

  • Surface scientists for protein-surface interactions and immobilization

• Applied biotechnology partners:

  • Industrial biotechnology groups working on thermophilic bioprocesses

  • Bioenergy researchers developing novel energy transduction systems

  • Environmental biotechnologists interested in carbon capture technologies

• Evolutionary biology collaborators:

  • Microbial ecologists studying thermophilic environments

  • Comparative genomics experts analyzing Moorella species diversity

  • Phylogenetics specialists reconstructing evolutionary relationships