Recombinant Petrotoga mobilis ATP synthase subunit c (atpE)

What is the amino acid sequence and structure of P. mobilis ATP synthase subunit c?

The full-length Petrotoga mobilis ATP synthase subunit c (atpE) consists of 96 amino acids with the following sequence: MDLATMLQNLVTEGGSIGWGLYYLGKLLGAGVAMGIGAIGPGVGEGNIGAHAMDAMARQPEMSGNLTTRMLLAMAVTESTGLYSLVVALILLFVLP . This membrane protein functions as part of the F0 complex of ATP synthase, forming the proton-conducting channel that drives ATP synthesis. The structure is characterized by predominantly hydrophobic residues arranged in transmembrane helices that form a ring structure within the membrane.

What expression systems are most effective for producing recombinant P. mobilis atpE?

E. coli expression systems have proven effective for recombinant production of P. mobilis atpE, particularly when using His-tags for purification . When designing expression constructs, researchers should consider the hydrophobic nature of this membrane protein. Optimization strategies include:

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

• Testing various induction temperatures (typically lower than standard)

• Employing fusion partners that enhance solubility

• Optimizing codon usage for E. coli expression

The recombinant protein can be successfully expressed with an N-terminal His-tag, which facilitates purification while maintaining protein functionality .

What are the optimal conditions for storage and handling of recombinant P. mobilis atpE?

Based on established protocols, recombinant P. mobilis atpE should be stored as a lyophilized powder at -20°C to -80°C for long-term stability . For working solutions, the following guidelines are recommended:

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

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Avoid repeated freeze-thaw cycles, as these significantly reduce protein activity

• Use Tris/PBS-based buffer with 6% Trehalose at pH 8.0 for optimal stability

How can researchers verify the purity and functionality of recombinant P. mobilis atpE?

Verification of recombinant P. mobilis atpE should employ multiple complementary methods:

• Purity Assessment:

  • SDS-PAGE analysis (should demonstrate >90% purity)

  • Western blot using anti-His antibodies for tagged protein verification

  • Mass spectrometry for precise molecular weight determination

• Functional Verification:

  • Reconstitution into liposomes to measure proton conductance

  • ATP synthesis assays in reconstituted systems

  • Binding assays with known ATP synthase inhibitors

• Structural Integrity:

  • Circular dichroism spectroscopy to confirm secondary structure composition

  • Thermal stability assays to verify the thermostable properties expected of proteins from thermophilic organisms

What approaches are most effective for studying protein-protein interactions involving P. mobilis atpE?

To investigate protein-protein interactions involving P. mobilis atpE, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against atpE or its binding partners to pull down protein complexes from reconstituted systems or membrane preparations.

• Cross-linking studies: Employing chemical cross-linkers of varying lengths to capture transient interactions between atpE and other ATP synthase subunits.

• Förster Resonance Energy Transfer (FRET): Tagging atpE and potential interaction partners with appropriate fluorophores to detect proximity-based energy transfer.

• Surface Plasmon Resonance (SPR): Immobilizing atpE on a sensor chip and flowing potential interaction partners to measure binding kinetics.

• Native gel electrophoresis: Analyzing ATP synthase complexes under non-denaturing conditions to preserve protein-protein interactions.

How can homology modeling be used to predict the structure of P. mobilis atpE when crystallographic data is unavailable?

In the absence of crystallographic data, homology modeling provides a valuable approach for predicting the structure of P. mobilis atpE. Based on established protocols in structural biology research, the following methodology is recommended:

• Template Selection: Identify suitable templates from the Protein Data Bank (PDB) using BLASTP. Select templates with high sequence identity (>70%), high resolution, and appropriate completeness .

• Sequence Alignment: Align the P. mobilis atpE sequence with the template sequence using tools like ClustalW to establish the correspondence between residues .

• Model Building: Generate multiple structural models using software like MODELLER, which employs satisfaction of spatial restraints to create protein structures .

• Model Selection: Select the model with the lowest Discrete Optimized Protein Energy (DOPE) score, indicating the most energetically favorable conformation .

• Energy Minimization: Refine the selected model through energy minimization and molecular dynamics simulations using tools like AMBERTOOLS10 to resolve steric clashes and optimize the structure .

• Model Validation: Evaluate the model quality using:

  • Ramachandran plot analysis to verify backbone conformations

  • ERRAT for non-bonded interaction assessment

  • Verify_3D for compatibility of the model with its sequence

  • Root mean square deviation (RMSD) measurement by superposition with the template

This approach has been successfully applied to similar proteins, with typical RMSD values below 1.0 Å indicating reliable models .

What computational approaches can be used to identify potential inhibitor binding sites on P. mobilis atpE?

Identifying potential inhibitor binding sites on P. mobilis atpE can be accomplished through several computational approaches:

• Molecular Docking: Using software like AutoDock4.2 to determine the interaction between atpE and potential ligands, following these steps:

  • Convert both protein and ligands to PDBQT format

  • Calculate gasteiger charges

  • Determine free binding energy using Lamarckian genetic algorithms

  • Set appropriate grid map parameters (e.g., 60 × 60 × 60 with spacing of 0.375 Å)

  • Analyze docking results using visualization tools like PyMOL and Ligplot+

• Molecular Dynamics Simulations: Running simulations of atpE in a membrane environment to identify transient binding pockets and conformational changes that might reveal cryptic binding sites.

• Binding Site Prediction Algorithms: Applying algorithms that analyze surface topography, electrostatic potential, and hydrophobicity to predict potential binding cavities.

• Conservation Analysis: Examining conserved regions across multiple species' atpE sequences, as functionally important binding sites often show evolutionary conservation.

• Fragment-Based Screening in silico: Docking small molecular fragments to identify hotspots on the protein surface that could be exploited for inhibitor design.

How does the structure of P. mobilis atpE contribute to its thermostability and function under extreme conditions?

P. mobilis atpE has evolved specific structural features that contribute to its thermostability and function under extreme conditions:

• Amino Acid Composition: The protein sequence shows a higher proportion of hydrophobic residues (particularly in the transmembrane regions) and charged residues that can form stabilizing salt bridges. The sequence MDLATMLQNLVTEGGSIGWGLYYLGKLLGAGVAMGIGAIGPGVGEGNIGAHAMDAMARQPEMSGNLTTRMLLAMAVTESTGLYSLVVALILLFVLP reveals this pattern of hydrophobic clustering .

• Secondary Structure Stability: The alpha-helical transmembrane domains are packed tightly, minimizing the exposure of hydrophobic surfaces to the aqueous environment.

• Ionic Interactions: Strategic positioning of charged residues likely forms salt bridges that maintain structural integrity at high temperatures.

• Hydrogen Bonding Networks: Enhanced hydrogen bonding networks within the protein structure contribute to thermal stability.

• Reduced Flexibility: Compared to mesophilic homologs, P. mobilis atpE likely has reduced flexibility in loop regions, contributing to its resistance to thermal denaturation.

These adaptations ensure that the c-ring structure remains intact and functional even under the extreme temperature conditions of P. mobilis' natural habitat, while still allowing the necessary conformational changes for proton translocation and rotational catalysis.

What assays can be used to measure the proton translocation activity of reconstituted P. mobilis atpE?

To measure proton translocation activity of reconstituted P. mobilis atpE, researchers can employ several complementary approaches:

• pH-Sensitive Fluorescent Probes: Incorporate pH-sensitive fluorophores (e.g., ACMA, pyranine) into liposomes containing reconstituted atpE to monitor pH changes in real-time:

• Proton Leak Assays: Create a pH gradient across liposomal membranes and measure the rate of gradient dissipation with and without specific inhibitors.

• Patch-Clamp Electrophysiology: For direct measurement of proton currents through single channels or channel populations in artificial membranes.

• ATP Synthesis Coupling Assays: Reconstitute the complete ATP synthase complex and measure ATP production rates under varying proton gradient conditions using luciferase-based ATP detection.

• Ion-selective Microelectrodes: Direct measurement of proton concentration changes in compartmentalized systems containing reconstituted atpE.

The rotational catalysis mechanism proposed by Paul Boyer emphasizes the importance of conformational changes in ATP synthase function, where a complete 360° turnover requires three conformational states at each catalytic site, with a cycle concluding with a 120° rotation at a separate catalytic site .

How can researchers investigate the binding of inhibitors to P. mobilis atpE?

To investigate inhibitor binding to P. mobilis atpE, researchers should employ multiple complementary techniques:

• Isothermal Titration Calorimetry (ITC): Provides direct measurement of binding thermodynamics (ΔH, ΔG, ΔS) and stoichiometry. This technique can determine:

  • Binding affinity (Kd) values

  • Enthalpy changes upon binding

  • Number of binding sites per protein molecule

• Surface Plasmon Resonance (SPR): Allows real-time monitoring of inhibitor binding kinetics:

  • Association rate constants (kon)

  • Dissociation rate constants (koff)

  • Equilibrium dissociation constants (KD)

• Fluorescence-based Binding Assays: Using either:

  • Intrinsic tryptophan fluorescence quenching upon inhibitor binding

  • Fluorescently labeled inhibitors to monitor binding directly

• Competitive Binding Assays: Employing radioligands or fluorescent probes to conduct displacement studies with potential inhibitors.

• Structural Studies: Using X-ray crystallography or cryo-EM of atpE-inhibitor complexes to determine binding modes and interaction details.

• Molecular Dynamics Simulations: Computational analysis of inhibitor binding stability and conformational effects on protein structure.

• Functional Inhibition Assays: Measuring the effect of inhibitors on proton translocation or ATP synthesis activities in reconstituted systems.

What approaches can be used to study the assembly of P. mobilis atpE into the complete ATP synthase complex?

Studying the assembly of P. mobilis atpE into the complete ATP synthase complex requires specialized techniques addressing both the process and the final structure:

• Sequential Co-expression Systems: Express different ATP synthase subunits in controlled ratios to monitor assembly progression:

  • Use inducible promoters with varying strengths

  • Monitor assembly intermediates via tagged subunits

  • Track assembly kinetics through time-course sampling

• Native Mass Spectrometry: Analyze intact membrane protein complexes and subcomplexes to determine:

  • Subunit stoichiometry

  • Assembly intermediate composition

  • Stability of protein-protein interactions

• Blue Native PAGE: Separate intact membrane protein complexes under non-denaturing conditions to:

  • Identify discrete assembly intermediates

  • Compare wild-type vs. mutant assembly patterns

  • Track changes in complex formation under different conditions

• Förster Resonance Energy Transfer (FRET): Monitor protein-protein interactions during assembly using fluorescently labeled subunits:

  • Determine proximity relationships between subunits

  • Track dynamic changes during assembly process

  • Measure interaction kinetics in real-time

• Cryo-electron Microscopy: Visualize the structure of assembled complexes at near-atomic resolution:

  • Determine the arrangement of subunits within the complex

  • Identify conformational changes associated with assembly

  • Characterize the c-ring stoichiometry and architecture

• Chemical Cross-linking coupled with Mass Spectrometry: Identify interaction interfaces between atpE and other subunits:

  • Map specific residues involved in subunit interactions

  • Detect conformational changes during assembly

  • Identify assembly intermediates via crosslinking patterns

How does P. mobilis atpE differ from ATP synthase subunit c in other extremophiles?

Comparative analysis reveals several distinctive features of P. mobilis atpE when compared to ATP synthase subunit c from other extremophiles:

• Sequence Conservation and Divergence: While the essential functional residues involved in proton translocation are conserved, P. mobilis atpE exhibits unique sequence adaptations:

  • Higher proportion of certain amino acids associated with thermostability

  • Distinctive hydrophobic patterning in transmembrane regions

  • Specific charged residue positioning that likely contributes to ion interactions

• Structural Adaptations:

  • The number of c-subunits in the ring structure may differ between species, affecting the bioenergetic properties

  • Altered loop regions that may provide additional stability

  • Modified subunit interfaces that enhance complex stability under extreme conditions

• Functional Properties:

  • Potentially different coupling ratios (H+/ATP) compared to mesophilic organisms

  • Altered inhibitor sensitivity profiles

  • Modified pH optima for function, reflecting adaptation to specific environmental niches

• Environmental Adaptations: Unlike some extremophiles adapted solely to temperature extremes, P. mobilis exists in environments with multiple stressors:

  • Hyperthermophilic conditions requiring thermostable proteins

  • Potentially high salt concentrations necessitating halotolerant adaptations

  • Possible pressure adaptations from deep subsurface habitats

The comparative analysis of atpE across different extremophiles provides valuable insights into convergent and divergent evolutionary strategies for maintaining ATP synthase function under extreme conditions.

What role does P. mobilis atpE play in the organism's adaptation to its extreme environment?

Petrotoga mobilis atpE plays a crucial role in the organism's adaptation to its extreme environment through several mechanisms:

• Thermostability of Energy Production: The structural adaptations of atpE ensure that the ATP synthase complex remains functional at the high temperatures (up to 80°C) of P. mobilis' natural habitat, maintaining efficient energy production under conditions that would denature proteins from mesophilic organisms.

• Stress Response Integration: ATP synthase activity is closely linked to stress responses in extremophiles. When P. mobilis encounters environmental stressors, it accumulates compatible solutes like mannosylglucosylglycerate (MGG) in response to hyperosmotic conditions and supraoptimal growth temperatures . The maintenance of ATP synthesis under these conditions is essential for powering these adaptive responses.

• Membrane Integrity: The c-ring formed by atpE subunits must maintain appropriate interactions with membrane lipids, which have distinct compositions in thermophiles. These interactions help maintain membrane integrity and function at high temperatures.

• Bioenergetic Efficiency: Adaptations in atpE likely contribute to optimized bioenergetic parameters (such as H+/ATP ratio) that maximize energy conservation under the resource-limited conditions often found in extreme environments.

• Proton Gradient Maintenance: The proton-conducting channel formed by atpE must function efficiently despite the increased proton permeability of membranes at elevated temperatures, ensuring that P. mobilis can maintain the proton motive force necessary for ATP synthesis.

The binding change mechanism, as postulated by Paul Boyer, is particularly relevant here, as the rotational catalysis mechanism must be preserved while accommodating the structural adaptations needed for thermostability .

What can the study of P. mobilis atpE contribute to our understanding of ATP synthase evolution?

The study of P. mobilis atpE offers valuable insights into ATP synthase evolution across domains of life:

• Evolutionary Conservation and Divergence: Analyzing the sequence and structure of P. mobilis atpE in comparison with homologs from other organisms reveals:

  • Core functional elements that have remained invariant despite billions of years of evolution

  • Lineage-specific adaptations that reflect environmental specialization

  • Potential horizontal gene transfer events in the evolution of ATP synthase components

• Molecular Adaptation Mechanisms: P. mobilis atpE demonstrates how a fundamental biological machine can be modified for different environmental conditions while preserving its core function, providing a natural experiment in protein engineering.

• Ancestral State Reconstruction: As a member of the deeply branching bacterial lineage Thermotogae, P. mobilis atpE characteristics may reflect features closer to the ancestral state of ATP synthases, offering clues about early bioenergetic systems.

• Structure-Function Relationships: Comparative analysis of P. mobilis atpE with homologs from mesophiles helps identify which structural elements are essential for function versus those that provide environmental adaptation.

• Convergent Evolution: By comparing P. mobilis atpE with those from unrelated thermophilic organisms, researchers can identify instances of convergent evolution where similar adaptive solutions evolved independently.

• Coevolution Patterns: Studying how changes in atpE correlate with changes in other ATP synthase subunits provides insights into coevolutionary constraints in multisubunit enzyme complexes.

These evolutionary insights not only enhance our understanding of ATP synthase as a molecular machine but also inform potential biotechnological applications requiring thermostable energy-transducing systems.

How can P. mobilis atpE be used as a model system for studying membrane protein assembly and function?

P. mobilis atpE offers several advantages as a model system for studying membrane protein assembly and function:

• Thermostability Advantages: The inherent stability of P. mobilis atpE facilitates:

  • Extended experimental manipulation without degradation

  • Higher tolerance to detergents during purification

  • Greater resistance to unfolding during reconstitution procedures

  • Compatibility with a wider range of experimental conditions

• Structural Features for Assembly Studies:

  • The c-ring structure formed by multiple atpE subunits provides a model for homooligomeric membrane protein assembly

  • Interactions between atpE and other ATP synthase subunits model heteromeric membrane protein complex formation

  • The well-defined topology allows tracking of assembly intermediates

• Functional Readouts:

  • Clear activity assays through proton translocation or ATP synthesis

  • Quantifiable inhibition by various compounds

  • Defined role in energy transduction

• Experimental Approaches Facilitated:

  • Site-directed mutagenesis to probe structure-function relationships

  • Chimeric constructs with mesophilic homologs to identify thermostability determinants

  • Label incorporation for spectroscopic and microscopic studies

• Biotechnological Applications:

  • Development of thermostable membrane protein expression systems

  • Creation of robust biosensors based on proton translocation

  • Engineering of novel energy-transducing systems

The rotational catalysis mechanism, where a complete 360° turnover requires three conformational states to be achieved and changed, with a cycle concluding with a 120° rotation at a separate catalytic site, provides a fascinating framework for studying mechanical energy transduction in membrane proteins .

What are the challenges in expressing and purifying functional P. mobilis atpE, and how can they be overcome?

Expressing and purifying functional P. mobilis atpE presents several challenges, with corresponding strategies for overcoming them:

• Membrane Protein Expression Challenges:

• Purification Challenges:

• Functional Reconstitution Challenges:

• Thermostability Considerations:

  • Take advantage of the protein's natural thermostability during purification

  • Consider heat treatment steps to remove less stable contaminating proteins

  • Use higher temperatures during functional assays to reflect native conditions

Current best practices include expression in E. coli with an N-terminal His-tag, which has been successfully used to produce recombinant P. mobilis atpE with >90% purity .

How can structural information about P. mobilis atpE be applied to the design of novel ATP synthase inhibitors?

Structural information about P. mobilis atpE can be strategically applied to design novel ATP synthase inhibitors through several approaches:

• Structure-Based Drug Design:

  • Identification of binding pockets through homology modeling and molecular dynamics simulations

  • Virtual screening of compound libraries against identified binding sites

  • Fragment-based approaches to develop high-affinity ligands

  • Structure-activity relationship studies to optimize lead compounds

• Targeting Unique Features:

  • Identification of residues unique to bacterial ATP synthases but absent in mammalian homologs for selectivity

  • Exploitation of thermophile-specific structural elements for targeted inhibition

  • Design of compounds that disrupt c-ring assembly or rotation

• Rational Modification of Known Inhibitors:

  • Analysis of binding modes of established inhibitors (e.g., oligomycin, venturicidin)

  • Chemical modification to enhance affinity or selectivity

  • Development of hybrid molecules combining features of multiple inhibitor classes

• Exploitation of Dynamic Properties:

  • Identification of conformationally variable regions during the catalytic cycle

  • Design of inhibitors that lock the protein in non-productive conformational states

  • Targeting of transient binding pockets that appear during rotational catalysis

• Application of Computational Methods:

  • Molecular docking studies using software like AutoDock4.2 to predict binding modes and energetics

  • Molecular dynamics simulations to assess inhibitor stability and effects on protein dynamics

  • Quantitative structure-activity relationship (QSAR) modeling to guide inhibitor optimization

The binding change mechanism and rotational catalysis model provide crucial insights into potential inhibition strategies, as compounds that interfere with the required conformational changes during the 120° rotational steps could effectively block ATP synthesis .

What are the current knowledge gaps in our understanding of P. mobilis atpE and how might they be addressed?

Despite advances in our understanding of P. mobilis atpE, several significant knowledge gaps remain:

• High-Resolution Structure: No crystal structure or cryo-EM structure of P. mobilis atpE currently exists in the PDB. This could be addressed through:

  • X-ray crystallography of the isolated c-ring

  • Cryo-EM studies of the complete ATP synthase complex

  • Advanced NMR techniques for membrane protein structural determination

• Proton Pathway Details: The precise mechanism of proton translocation through the c-ring remains incompletely characterized. Research approaches include:

  • Molecular dynamics simulations with explicit proton transfer events

  • Site-directed mutagenesis of key residues followed by functional studies

  • Time-resolved spectroscopy to capture protonation state changes

• Thermostability Determinants: The specific structural features responsible for the thermostability of P. mobilis atpE require further elucidation:

  • Comparative mutagenesis studies with mesophilic homologs

  • Thermal unfolding studies coupled with structural analysis

  • Computational analysis of stabilizing interactions

• Integration with Other ATP Synthase Components: The specific interactions between atpE and other subunits that enable function at high temperatures:

  • Cross-linking studies to map interaction interfaces

  • Hybrid ATP synthase studies with components from different organisms

  • Assembly studies under varying temperature conditions

• Physiological Regulation: How ATP synthesis is regulated in response to environmental changes in P. mobilis:

  • Transcriptomic and proteomic studies under various stress conditions

  • Post-translational modification analysis

  • In vivo studies of ATP synthase activity under varying conditions

Addressing these knowledge gaps will require integrating structural biology, biochemistry, biophysics, and computational approaches in a multidisciplinary research program.

How might comparative studies between P. mobilis atpE and its homologs inform protein engineering efforts?

Comparative studies between P. mobilis atpE and its homologs can significantly inform protein engineering efforts in several ways:

• Thermostability Engineering:

  • Identification of amino acid substitutions that contribute to thermostability

  • Recognition of structural motifs that enhance protein rigidity at high temperatures

  • Development of design principles for engineering thermostable variants of mesophilic proteins

• Functional Optimization:

  • Understanding the sequence-function relationship by comparing homologs with different activities

  • Identifying residues that can be modified to alter substrate specificity or catalytic efficiency

  • Engineering proteins with optimal function under specific environmental conditions

• Membrane Protein Design:

  • Elucidating principles of membrane protein folding and stability

  • Developing rules for designing artificial membrane proteins

  • Creating chimeric proteins with desired properties from different natural homologs

• Protein-Protein Interaction Engineering:

  • Understanding the determinants of c-ring assembly and stability

  • Designing interfaces for controlled oligomerization

  • Creating novel protein complexes with defined stoichiometries

• Bioenergetic Applications:

  • Engineering ATP synthases with altered H+/ATP ratios for biotechnological applications

  • Developing synthetic biology systems with customized energy conversion properties

  • Creating hybrid systems combining features from different organisms

Such comparative studies, particularly between P. mobilis atpE and homologs from both thermophilic and mesophilic organisms, provide a natural laboratory for understanding how nature has solved the challenge of maintaining protein function under extreme conditions.

What emerging technologies might advance our understanding of P. mobilis atpE structure, function, and applications?

Several emerging technologies promise to significantly advance our understanding of P. mobilis atpE:

• Advanced Structural Biology Techniques:

  • Microcrystal electron diffraction (MicroED) for structural determination of membrane proteins resistant to traditional crystallization

  • Time-resolved cryo-EM to capture different conformational states during the catalytic cycle

  • Integrative structural biology approaches combining multiple data sources (SAXS, NMR, XL-MS, cryo-EM)

• Single-Molecule Techniques:

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

  • Single-molecule FRET to track conformational changes during catalysis

  • Optical tweezers to measure forces involved in ATP synthesis

• Computational Advances:

  • AI-based protein structure prediction (AlphaFold2, RoseTTAFold) to model P. mobilis atpE structure with high accuracy

  • Enhanced sampling molecular dynamics to simulate rare events in catalysis

  • Quantum mechanics/molecular mechanics (QM/MM) simulations for proton transfer energetics

• Synthetic Biology Approaches:

  • Cell-free expression systems optimized for membrane protein synthesis

  • Minimal cell systems for studying ATP synthase function in simplified contexts

  • Directed evolution platforms for engineering atpE variants with novel properties

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize ATP synthase distribution and dynamics

  • Correlative light and electron microscopy to connect structure and function

  • Cryo-electron tomography of whole cells to visualize ATP synthase in its native environment

• Novel Membrane Mimetics:

  • Nanodiscs with precisely controlled lipid compositions

  • Polymer-based membrane mimetics with enhanced stability

  • 3D-printed artificial membranes with defined geometries