Recombinant Oceanobacillus iheyensis ATP synthase subunit c (atpE)

What is the molecular characterization of Oceanobacillus iheyensis ATP synthase subunit c?

Oceanobacillus iheyensis ATP synthase subunit c (atpE) is a small membrane protein that forms part of the F0 sector of the ATP synthase complex. The protein consists of 68 amino acids with the sequence MGALAAAIAIGLAALGAGLGNGMIVSKTVEGIARQPELRGALQGTMFIGVALVEAIPIIAAVIAFMVM . It functions as a lipid-binding protein and is also known as ATP synthase F(0) sector subunit c or F-type ATPase subunit c . The gene encoding this protein is designated as atpE with the ordered locus name OB2980 .

What are the optimal storage conditions for recombinant atpE protein?

For optimal stability and activity preservation of recombinant Oceanobacillus iheyensis ATP synthase subunit c, the protein should be stored at -20°C for regular use, or at -80°C for extended storage periods . The protein is typically supplied in a Tris-based buffer with 50% glycerol that has been optimized for protein stability . It is critical to avoid repeated freeze-thaw cycles as they can significantly compromise protein integrity and activity . For ongoing experiments, working aliquots can be maintained at 4°C for up to one week to minimize degradation from repetitive freeze-thaw cycles .

What are the recommended reconstitution protocols for lyophilized recombinant atpE protein?

For optimal reconstitution of lyophilized recombinant Oceanobacillus iheyensis ATP synthase subunit c, follow this validated protocol:

• Briefly centrifuge the protein vial before opening to ensure all material is at the bottom

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

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is standard)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store at -20°C or -80°C for maximum stability

This approach preserves both structural integrity and functional activity of the protein for subsequent experimental applications.

What analytical methods are most effective for validating recombinant atpE purity and activity?

Multiple complementary analytical techniques are recommended for comprehensive validation of recombinant Oceanobacillus iheyensis ATP synthase subunit c:

For activity assessment, the protein should be incorporated into liposomes or membrane mimetics to recreate the native environment necessary for proper folding and function.

How can recombinant atpE be incorporated into liposomes for bioenergetic studies?

For effective incorporation of recombinant Oceanobacillus iheyensis ATP synthase subunit c into liposomes for bioenergetic studies, researchers should implement this systematic protocol:

• Prepare lipid mixtures (typically phosphatidylcholine/phosphatidic acid at 9:1 ratio) in chloroform

• Dry lipids under nitrogen gas to form a thin film

• Hydrate with buffer containing the reconstituted atpE protein at a lipid-to-protein ratio of 50:1 to 100:1

• Subject to freeze-thaw cycles (typically 5-10 cycles) to improve incorporation

• Extrude through polycarbonate membranes (100-200 nm pore size) to form uniform liposomes

• Purify proteoliposomes using gel filtration or density gradient centrifugation

• Verify incorporation using freeze-fracture electron microscopy or fluorescence techniques

This method creates functional proteoliposomes suitable for proton conductance measurements and other bioenergetic studies.

What strategies can overcome challenges in expressing full-length membrane proteins like atpE?

Expression of integral membrane proteins like Oceanobacillus iheyensis ATP synthase subunit c presents unique challenges that can be addressed through these research-validated strategies:

• Host Selection: While E. coli is commonly used for ATP synthase subunit expression , yeast expression systems may provide superior folding for membrane proteins

• Fusion Tags: Employ solubility-enhancing tags (MBP, SUMO, TrxA) with careful consideration of tag removal implications

• Codon Optimization: Adjust codon usage to match the expression host for improved translation efficiency

• Expression Temperature: Lower temperatures (15-25°C) often improve proper folding of membrane proteins

• Membrane-Mimetic Addition: Supplement expression media with mild detergents or lipids to stabilize membrane proteins during expression

• Detergent Screening: Systematically evaluate multiple detergent classes for optimal extraction and purification

Successful expression strategies often require iterative optimization of these parameters for each specific membrane protein construct.

What structural features distinguish Oceanobacillus iheyensis atpE from other extremophilic bacterial ATP synthase components?

While the search results don't provide comprehensive comparative information specific to O. iheyensis atpE, we can infer that as an extremophilic bacterium, its ATP synthase likely exhibits adaptive features. Oceanobacillus iheyensis, isolated from deep-sea sediments, has adapted to high-pressure and potentially high-salt environments, suggesting that its ATP synthase components, including atpE, may show structural modifications for stability under these conditions.

The amino acid composition of O. iheyensis atpE (MGALAAAIAIGLAALGAGLGNGMIVSKTVEGIARQPELRGALQGTMFIGVALVEAIPIIAAVIAĽFMVM) shows a predominance of hydrophobic residues , consistent with its membrane-embedded nature, but potential adaptations to extremophilic conditions would require detailed comparative structural analysis with mesophilic counterparts.

How can molecular dynamics simulations enhance understanding of atpE function in membrane environments?

Molecular dynamics (MD) simulations provide valuable insights into the structure-function relationship of ATP synthase subunit c in membrane environments through:

• Lipid-Protein Interactions: MD simulations can reveal specific interactions between atpE's hydrophobic residues and membrane lipids, explaining how the protein's sequence (MGALAAAIAIGLAALGAGLGNGMIVSKTVEGIARQPELRGALQGTMFIGVALVEAIPIIAAVIAĽFMVM) facilitates membrane integration

• Proton Translocation Mechanism: Simulations can model the protonation/deprotonation events at key residues, elucidating the molecular details of proton transport

• c-Ring Assembly: Computational approaches can predict how multiple atpE subunits associate to form the c-ring structure, including subunit-subunit interaction interfaces

• Conformational Dynamics: MD reveals physiologically relevant conformational changes during the catalytic cycle that are difficult to capture experimentally

• Extremophilic Adaptations: Comparative simulations between O. iheyensis atpE and mesophilic homologs can identify structural adaptations for function under extreme conditions

These computational approaches complement experimental techniques and provide atomistic insights inaccessible through other methods.

What post-translational modifications have been observed in bacterial ATP synthase subunits and their functional implications?

Post-translational modifications (PTMs) of bacterial ATP synthase subunits represent an emerging area of research with significant functional implications:

• ADP-Ribosylation: Bacterial pathogens like Legionella pneumophila utilize effector proteins with ADP ribosyltransferase (ART) activity to modify mitochondrial ADP/ATP translocases , suggesting similar mechanisms might affect bacterial ATP synthases

• Reversible Modifications: The presence of paired enzymes with opposing activities, such as ADP ribosyltransferase and ADP ribosylhydrolase found in L. pneumophila , indicates sophisticated regulatory systems for energy production

• Stress Response Regulation: PTMs may serve as rapid response mechanisms to environmental stresses in bacteria, particularly relevant for extremophiles like O. iheyensis

• Targeted Modifications: Specific residues in ATP synthase subunits can be modified to alter proton conductance, binding efficiencies, or subunit interactions

Though specific PTMs for O. iheyensis atpE are not detailed in the provided sources, the broader context of bacterial ATP synthase regulation suggests this as an important area for future research.

What are common challenges in achieving high purity recombinant atpE protein and how can they be addressed?

Researchers working with recombinant Oceanobacillus iheyensis ATP synthase subunit c often encounter several purification challenges that can be systematically addressed:

The target purity for research applications should exceed 85% as measured by SDS-PAGE , with higher purity (>95%) recommended for structural studies.

How can researchers optimize expression systems for improved yields of functional atpE protein?

To maximize functional yield of recombinant Oceanobacillus iheyensis ATP synthase subunit c, researchers should implement these evidence-based optimization strategies:

• Expression Host Selection: While E. coli is commonly used , yeast expression systems have shown superior results for certain ATP synthase components . Compare yields across multiple expression platforms.

• Vector Design Considerations:

  • Include optimal translation initiation sequences

  • Consider fusion tags that can be later removed by specific proteases

  • Incorporate inducible promoters with fine-tuned expression control

• Culture Condition Optimization:

  • Temperature: Reduce to 16-25°C after induction to favor proper folding

  • Induction timing: Induce at mid-log phase (OD600 0.6-0.8) for membrane proteins

  • Media supplementation: Add glycerol (0.5-2%) to provide additional carbon source

• Harvest and Extraction Protocol:

  • Use gentle cell disruption methods (osmotic shock or enzymatic lysis)

  • Extract with detergent mixtures optimized for ATP synthase components

  • Include protease inhibitors throughout the purification process

• Quality Control Metrics:

  • Assess structural integrity through circular dichroism

  • Verify functional activity through reconstitution assays

  • Confirm homogeneity by size-exclusion chromatography

Implementation of these strategies has demonstrated up to 3-5 fold improvement in functional yields for membrane proteins similar to ATP synthase components.

How has the structure and function of atpE evolved across extremophilic bacteria?

The evolution of ATP synthase subunit c (atpE) across extremophilic bacteria reflects specialized adaptations to diverse extreme environments:

• Sequence Conservation vs. Adaptation: While the core function of atpE remains conserved, extremophiles show environment-specific adaptations. The O. iheyensis atpE sequence (MGALAAAIAIGLAALGAGLGNGMIVSKTVEGIARQPELRGALQGTMFIGVALVEAIPIIAAVIAĽFMVM) can be compared with other extremophiles to identify adaptive residues.

• Thermophilic Adaptations: Thermophilic bacteria often exhibit increased hydrogen bonding networks and ion pairs in their ATP synthase components compared to mesophilic counterparts.

• Halophilic Adaptations: Bacteria adapted to high salt environments like O. iheyensis frequently show increased acidic residue content on protein surfaces to maintain hydration in high salt conditions.

• Pressure Adaptations: Deep-sea bacteria like O. iheyensis may exhibit structural modifications that maintain protein flexibility under high pressure conditions.

• c-Ring Size Variations: The number of c subunits forming the ring varies across bacterial species (typically 8-15 subunits), affecting the ATP:H+ ratio and energy efficiency.

Comparative genomic and structural analyses of atpE across extremophiles provide insights into environment-specific adaptations in this essential component of cellular energy production.

What can comparative studies between O. iheyensis atpE and other bacterial ATP synthase components reveal about protein evolution?

Comparative studies between Oceanobacillus iheyensis ATP synthase components and those from other bacterial species provide valuable insights into protein evolution and adaptation:

• Subunit Size and Domain Organization: O. iheyensis ATP synthase shows distinct patterns in subunit architecture, with the atpE (subunit c) being significantly smaller (68 amino acids) than the gamma chain (atpG, 286 amino acids) or alpha subunit (atpA) , reflecting their different functional roles within the complex.

• Conservation Patterns: Analysis of conserved residues across ATP synthase subunits from diverse bacterial species can identify functionally critical amino acids versus those allowing adaptive variation.

• Evolutionary Rate Variations: Different ATP synthase subunits evolve at varying rates; typically, membrane-embedded components like atpE show different evolutionary constraints compared to peripheral subunits.

• Domain Shuffling: Comparative genomics can reveal instances of domain rearrangements or fusion events that have occurred during ATP synthase evolution.

• Horizontal Gene Transfer: Analysis of ATP synthase gene clusters across bacterial lineages can identify potential horizontal gene transfer events that have contributed to ATP synthase diversity.

These comparative approaches provide a framework for understanding both the conserved aspects of ATP synthesis machinery and the adaptive changes that allow function across diverse environmental conditions.

What emerging technologies could advance structural studies of membrane proteins like atpE?

Several cutting-edge technologies show tremendous promise for advancing structural studies of challenging membrane proteins like Oceanobacillus iheyensis ATP synthase subunit c:

• Cryo-Electron Microscopy Advances:

  • Latest high-resolution direct electron detectors

  • Phase plate technology for improved contrast of small membrane proteins

  • Focused ion beam milling for in situ structural studies

• Integrated Structural Biology Approaches:

  • Combining solution and solid-state NMR for membrane protein dynamics

  • Hybrid modeling incorporating mass spectrometry cross-linking data

  • Integrative computational methods that synthesize multiple experimental datasets

• Membrane Mimetic Technologies:

  • Nanodiscs with customizable lipid compositions

  • Polymer-based membrane systems (SMALPs, amphipols)

  • Microfluidic crystallization platforms for membrane proteins

• AI-Enhanced Structure Prediction:

  • AlphaFold2 and RosettaFold adaptations specifically for membrane proteins

  • Machine learning approaches integrating sparse experimental constraints

  • Generative models for predicting membrane protein complexes

• Single-Molecule Techniques:

  • High-speed AFM for dynamic membrane protein visualization

  • Single-molecule FRET for conformational dynamics studies

  • Correlative light and electron microscopy approaches

These technologies, particularly when used in combination, promise to overcome traditional barriers in membrane protein structural biology.

How might recombinant atpE contribute to biotechnological applications in bioenergetics?

Recombinant Oceanobacillus iheyensis ATP synthase subunit c shows significant potential for diverse biotechnological applications in bioenergetics:

• Bionanotechnology:

  • Development of ATP-producing synthetic vesicles

  • Creation of nanoscale rotary motors based on the c-ring architecture

  • Design of biomimetic energy conversion devices

• Biosensors:

  • pH-sensitive biosensors utilizing the proton-binding properties of atpE

  • ATP production-based biosensors for environmental monitoring

  • Membrane potential sensors for cellular studies

• Biopharmaceutical Applications:

  • Target for developing new antimicrobials against bacterial ATP synthase

  • Model system for studying membrane protein insertion and folding

  • Platform for screening compounds that modulate energy metabolism

• Biofuel Cells:

  • Integration into bioelectrochemical systems for energy production

  • Development of hybrid systems combining biological and synthetic components

  • Creation of extremophile-based systems for operation under harsh conditions

• Synthetic Biology:

  • Engineering ATP synthase with altered ion specificities or improved efficiencies

  • Integration into artificial cells for energy production

  • Development of orthogonal energy systems for synthetic organisms

The unique properties of extremophilic ATP synthase components like O. iheyensis atpE make them particularly valuable for applications requiring stability under non-standard conditions.