Recombinant Nitrosococcus oceani ATP synthase subunit c (atpE)

What is Nitrosococcus oceani ATP synthase subunit c and what role does it play in cellular bioenergetics?

The ATP synthase subunit c (atpE) from Nitrosococcus oceani is a critical component of the F-type ATP synthase complex, specifically in the F₀ sector that spans the membrane. This protein functions as part of the proton channel that allows H⁺ ions to flow through the membrane along their electrochemical gradient, driving the rotation of the central stalk of ATP synthase and enabling ATP synthesis .

The amino acid sequence of N. oceani ATP synthase subunit c is: MDPELLVSIYASTAVSVGIILAAAGLGSALGWGLICSKYLEGIARQPEMRPQLMGQMLFTGGLMEAFPMIVLGMSMWFIFANPFTGAALAAIGS . This 94-amino acid protein is characterized by its hydrophobic nature, consistent with its role as a membrane-embedded protein that participates in forming the proton-conducting channel.

Within the ATP synthase complex, multiple c-subunits assemble into a ring structure (c-ring) that rotates as protons pass through the F₀ sector, transferring energy to the F₁ sector where ATP synthesis occurs . This mechanism is fundamental to N. oceani's energy metabolism and survival.

How is the atpE gene organized in the N. oceani genome and what is its genetic context?

The atpE gene in Nitrosococcus oceani is part of an ATP synthase operon (Noc_3073 to Noc_3080) that encodes the H⁺-translocating F₀F₁-type ATP synthase . The gene is specifically designated as Noc_3079 in the ordered locus names of the N. oceani genome . This operon organization is typical for F-type ATP synthases in bacteria, where genes encoding both the F₀ and F₁ sectors are clustered together to ensure coordinated expression.

Interestingly, the N. oceani genome contains two copies of genes necessary for functional ATP synthase assembly, which differs from many other bacterial species . In addition to the F-type ATP synthase, N. oceani also encodes a bacterial V-type ATP synthase (Noc_2081 to Noc_2089), suggesting a complex and possibly environment-specific energy metabolism strategy .

The complete genome of N. oceani consists of a single circular chromosome (3,481,691 bp with G+C content of 50.4%) and a plasmid (40,420 bp), containing a total of 3,093 candidate protein-encoding genes . This genomic context provides important insights into how ATP synthesis is integrated with other metabolic processes in this marine bacterium.

What are the optimal conditions for expressing recombinant N. oceani atpE protein in heterologous systems?

Successful expression of recombinant N. oceani ATP synthase subunit c requires careful consideration of several factors due to its hydrophobic nature and membrane association. Based on established protocols for similar proteins, the following conditions are recommended:

Expression System Selection:

• E. coli BL21(DE3) or C43(DE3) strains are preferred for membrane protein expression

• Codon optimization may be necessary due to potential codon bias between N. oceani and E. coli

Expression Vector Considerations:

• Vectors containing T7 promoter systems offer controlled induction

• Consider fusion tags that enhance solubility (e.g., MBP, SUMO) or facilitate purification (His, GST)

• Include a protease cleavage site between the tag and target protein

Induction Parameters:

• Lower temperatures (16-25°C) often yield better results than standard 37°C

• Reduced IPTG concentrations (0.1-0.5 mM) can prevent formation of inclusion bodies

• Extended expression times (overnight) at lower temperatures may improve yield of properly folded protein

For membrane proteins like ATP synthase subunit c, expression in the presence of additional phospholipids or specific detergents can improve yield and stability . A storage buffer containing Tris buffer with 50% glycerol has been successfully used for the recombinant protein .

What purification strategies are most effective for obtaining functional recombinant N. oceani ATP synthase subunit c?

Purification of recombinant N. oceani ATP synthase subunit c requires specialized approaches due to its hydrophobic nature. A successful purification strategy would include:

Initial Extraction:

• Cell lysis using either sonication or high-pressure homogenization in buffer containing mild detergents (e.g., DDM, CHAPS)

• Separation of membrane fraction by ultracentrifugation

• Solubilization of membrane proteins using appropriate detergents

Chromatography Steps:

• Affinity chromatography: If His-tagged, use Ni-NTA; if GST-tagged, use glutathione Sepharose

• Size exclusion chromatography to remove aggregates and impurities

• Ion exchange chromatography as a polishing step if needed

Buffer Optimization:

• Maintain detergent concentration above critical micelle concentration throughout purification

• Include stabilizing agents such as glycerol (15-50%)

• Consider adding lipids during purification to maintain native-like environment

Storage Conditions:

• Store at -20°C for short term or -80°C for extended periods

• Avoid repeated freeze-thaw cycles as recommended for the commercial recombinant protein

• Working aliquots can be maintained at 4°C for up to one week

Functional validation through activity assays or binding studies should be performed at each purification stage to ensure retention of biological activity.

What techniques are most appropriate for studying the structure-function relationship of N. oceani ATP synthase subunit c?

Understanding the structure-function relationship of N. oceani ATP synthase subunit c requires a multi-technique approach:

Biophysical Techniques:

• Circular Dichroism (CD) Spectroscopy - To analyze secondary structure content and thermal stability

• Nuclear Magnetic Resonance (NMR) - For solution structure determination and dynamics studies

• X-ray Crystallography - To obtain high-resolution structural information when crystals can be formed

• Cryo-Electron Microscopy - Particularly useful for visualizing the entire ATP synthase complex

Functional Analysis:

• Proton Transport Assays - Using liposomes reconstituted with purified protein

• ATP Hydrolysis/Synthesis Assays - To measure enzymatic activity when assembled with other subunits

• Membrane Potential Measurements - Using fluorescent probes to assess proton gradient formation

Molecular and Computational Approaches:

• Site-Directed Mutagenesis - To identify critical residues involved in function

• Molecular Dynamics Simulations - To understand protein dynamics and proton movement

• Homology Modeling - Using known structures of c subunits from other organisms as templates

When examining the amino acid sequence of N. oceani ATP synthase subunit c, particular attention should be paid to the GXXXG motifs that are often important for helix-helix interactions in membrane proteins, and to any conserved proton-binding sites (typically involving carboxyl groups) .

How can researchers assess the interaction between recombinant N. oceani atpE and other ATP synthase subunits?

Investigating subunit interactions within the ATP synthase complex requires specialized techniques suitable for membrane protein complexes:

Co-Purification Approaches:

• Co-expression of multiple subunits followed by tandem affinity purification

• Pull-down assays using differentially tagged subunits

• Native gel electrophoresis to analyze intact complexes

Biophysical Interaction Methods:

• Förster Resonance Energy Transfer (FRET) - For measuring distances between fluorescently labeled subunits

• Surface Plasmon Resonance (SPR) - To determine binding kinetics between purified components

• Isothermal Titration Calorimetry (ITC) - For quantitative binding thermodynamics

Cross-Linking Strategies:

• Chemical cross-linking followed by mass spectrometry (XL-MS)

• Photo-affinity labeling with photoactivatable amino acids incorporated into the sequence

• In vivo cross-linking to capture physiologically relevant interactions

Visualization Techniques:

• Single-particle cryo-EM of the assembled complex

• Negative stain EM for initial screening of complex formation

• Atomic Force Microscopy (AFM) to visualize membrane-embedded complexes

The assembled ATP synthase in N. oceani involves interactions between multiple subunits encoded by the operon (Noc_3073 to Noc_3080), with the c-subunit forming a critical ring structure that must properly interact with both the a-subunit in the membrane and the central stalk components .

How does N. oceani ATP synthase subunit c compare to homologous proteins in other bacterial species?

N. oceani ATP synthase subunit c shows both conserved features common to all F-type ATP synthases and unique adaptations potentially related to its marine, halophilic lifestyle:

Comparative Analysis Table of ATP Synthase c Subunits:

The N. oceani atpE sequence contains adaptations that may be related to its function in high-salt marine environments . Unlike many other bacteria, N. oceani contains both H⁺ and Na⁺ circuits for bioenergetics, suggesting specialized roles for its ATP synthase components . The presence of specific sodium-dependent transporters and antiporters in the genome supports this adaptation to high-salt environments .

Additionally, the evolutionary position of N. oceani in the γ-subdivision of Proteobacteria gives its ATP synthase unique phylogenetic characteristics compared to the more commonly studied β-subdivision bacterial ATP synthases .

What insights can genomic analysis provide about the evolution and adaptation of N. oceani ATP synthase?

Genomic analysis of N. oceani provides several key insights into the evolution and adaptation of its ATP synthase:

Duplication Events:
The N. oceani genome contains two copies each of the genes necessary to assemble functional complexes I and IV as well as ATP synthase . This redundancy may provide metabolic flexibility or regulatory advantages in changing marine environments.

Operon Organization:
The organization of the ATP synthase genes in an operon (Noc_3073 to Noc_3080) is consistent with other bacterial systems but shows specific adaptations. Additionally, N. oceani encodes a bacterial V-type ATP synthase (Noc_2081 to Noc_2089), suggesting acquisition through horizontal gene transfer or adaptation to specific environmental conditions .

Adaptation to Marine Environment:
The presence of several Na⁺/H⁺ antiporters (Noc_0159, Noc_0521, Noc_1282, Noc_2134, and Noc_2952) indicates adaptation to high-salt environments, possibly allowing for sodium-based bioenergetics in addition to the more common proton-based systems . This may impact the function and structure of ATP synthase components.

Evolutionary Relationships:
Comparing the ATP synthase genes of N. oceani with those of other marine bacteria can reveal patterns of convergent evolution in response to similar environmental pressures. The worldwide distribution of N. oceani suggests its energy generation mechanisms, including ATP synthase, are highly successful in marine environments .

How can recombinant N. oceani ATP synthase subunit c be used to study bioenergetics in marine chemolithoautotrophs?

Recombinant N. oceani ATP synthase subunit c offers unique opportunities for studying specialized aspects of marine bacterial bioenergetics:

Reconstitution Studies:
Purified recombinant atpE can be reconstituted into liposomes with varying lipid compositions to study how marine-specific membrane environments affect ATP synthase function. This approach can help understand adaptations to high salt conditions.

Inhibitor Development and Testing:
The unique features of N. oceani ATP synthase could be exploited to develop specific inhibitors that target marine bacteria without affecting other organisms. Such studies require pure, active recombinant protein for screening and characterization.

Proton/Sodium Selectivity:
N. oceani's adaptation to marine environments includes the potential use of both proton and sodium gradients for energy transduction . Recombinant atpE can be used to investigate ion selectivity and the molecular basis for this adaptation.

Synthetic Biology Applications:
The unique properties of N. oceani ATP synthase components could be incorporated into synthetic systems designed to function in high-salt environments or to harness energy using alternative ion gradients.

Climate Change Impact Studies:
As a marine chemolithoautotroph, N. oceani plays a role in global nitrogen and carbon cycles. Studies using its ATP synthase can help understand how ocean acidification and warming might affect the bioenergetics of these environmentally important organisms .

What challenges exist in crystallizing N. oceani ATP synthase subunit c for structural studies, and how can they be overcome?

Crystallizing membrane proteins like ATP synthase subunit c presents significant challenges, but several strategies can improve success rates:

Challenges and Solutions in Crystallization:

• Detergent Selection:

  • Challenge: Detergents must solubilize the protein while preserving native structure

  • Solution: Screen multiple detergents (DDM, LDAO, OG, etc.) and detergent mixtures

  • Approach: Perform stability assays in each detergent before crystallization trials

• Protein Stability:

  • Challenge: ATP synthase subunit c may be unstable when removed from its native environment

  • Solution: Add lipids during purification and crystallization (lipid cubic phase or bicelles)

  • Approach: Monitor protein stability using thermal shift assays or tryptophan fluorescence

• Crystal Contacts:

  • Challenge: Hydrophobic membrane proteins have limited surfaces for crystal contacts

  • Solution: Use antibody fragments, fusion partners, or crystallization chaperones

  • Approach: Engineer in additional polar residues at non-functional surfaces

• Homogeneity:

  • Challenge: Ensuring homogeneous protein preparation

  • Solution: Rigorous size exclusion chromatography and analytical ultracentrifugation

  • Approach: Use mass spectrometry to confirm protein integrity before crystallization

• Alternative Approaches:

  • If crystallization proves difficult, consider NMR for smaller constructs or cryo-EM for larger assemblies

  • Solid-state NMR may be particularly suitable for membrane proteins like ATP synthase subunit c

The successful crystallization of ATP synthase c-rings from other organisms can provide valuable protocols that may be adapted for N. oceani atpE, with appropriate modifications for its unique characteristics and the high salt conditions it naturally encounters .

How can site-directed mutagenesis be used effectively to study functional domains of N. oceani atpE?

Site-directed mutagenesis offers powerful insights into structure-function relationships of ATP synthase subunit c. A systematic approach would include:

Key Residues for Mutagenesis:

• Proton-binding site: Identify and mutate the conserved carboxyl residue essential for proton translocation

• Helix-helix interaction sites: Target conserved GXXXG motifs or other residues at subunit interfaces

• Lipid-interaction residues: Modify hydrophobic residues that may interact with membrane lipids

• Species-specific residues: Identify unique residues in N. oceani atpE compared to homologs

Mutagenesis Strategy:

• Create conservative mutations (e.g., Glu to Asp) to assess subtle functional effects

• Create non-conservative mutations (e.g., Glu to Ala) to completely disrupt function

• Generate cysteine mutants for subsequent labeling studies

• Consider creating chimeric proteins with c-subunits from other species

Functional Assessment:

• Complement ATP synthase-deficient strains to assess in vivo function

• Measure proton translocation in reconstituted systems

• Assess assembly of the c-ring using native gel electrophoresis

• Determine structural changes using spectroscopic methods

The 94-amino acid sequence of N. oceani ATP synthase subunit c provides multiple targets for mutagenesis, particularly focusing on the regions that may be involved in adaptation to marine environments .

What are the recommended protocols for assessing the functional integrity of recombinant N. oceani ATP synthase subunit c?

Assessing functional integrity requires multiple complementary approaches:

Structural Integrity Assays:

• Circular Dichroism (CD) to confirm secondary structure content

• Tryptophan fluorescence to assess tertiary structure

• Size exclusion chromatography to verify oligomeric state

• Mass spectrometry to confirm correct mass and post-translational modifications

Functional Assays:

• Proton Translocation:

  • Reconstitute protein into liposomes with pH-sensitive fluorescent dyes

  • Measure pH changes upon generation of membrane potential

• ATP Synthesis/Hydrolysis:

  • Co-reconstitute with other ATP synthase subunits

  • Measure ATP production using luciferase-based assays

  • Assess ATP hydrolysis by measuring inorganic phosphate release

• Binding Assays:

  • Assess interaction with known ATP synthase inhibitors

  • Measure binding to other ATP synthase subunits using ITC or SPR

• In vivo Complementation:

  • Express in ATP synthase-deficient strains

  • Assess restoration of growth or ATP production

When working with the recombinant protein, it's crucial to maintain the recommended storage conditions (Tris-based buffer with 50% glycerol at -20°C or -80°C) to preserve functional integrity, and avoid repeated freeze-thaw cycles .

How can studies of N. oceani ATP synthase contribute to our understanding of marine nitrogen cycling?

N. oceani is a significant marine ammonia-oxidizing bacterium with worldwide distribution, and its energy metabolism directly impacts marine nitrogen cycling :

ATP Synthase Role in Nitrogen Cycling:

• N. oceani obtains energy from oxidizing ammonia to nitrite, a critical step in the nitrogen cycle

• ATP synthase is essential for capturing the energy generated during this process

• Understanding ATP synthase efficiency helps explain N. oceani's ecological niche and distribution

Research Applications:

The worldwide distribution of N. oceani suggests its energy capture mechanisms, including ATP synthase, are well-adapted to diverse marine environments . This makes it an excellent model organism for studying bioenergetic adaptations in marine bacteria.

What techniques can be used to study the expression and regulation of atpE in natural N. oceani populations?

Studying atpE expression in natural environments requires specialized approaches:

Field Sampling and Preservation:

• Collect seawater samples using Niskin bottles at various depths

• Immediately filter onto 0.2 μm filters and preserve in RNA later or by flash freezing

• Process within optimal timeframes to prevent RNA degradation

Expression Analysis:

• Quantitative PCR (qPCR):

  • Design primers specific to N. oceani atpE

  • Use RT-qPCR to quantify transcript abundance

  • Include appropriate reference genes for normalization

• Metatranscriptomics:

  • Extract total RNA from environmental samples

  • Perform rRNA depletion and cDNA synthesis

  • Sequence using high-throughput platforms and analyze atpE expression

• Environmental Proteomics:

  • Extract proteins from environmental samples

  • Identify and quantify ATP synthase subunits using mass spectrometry

  • Compare protein abundance across different conditions

Visualization Techniques:

• Fluorescent in situ hybridization (FISH) with probes specific to N. oceani

• Immunofluorescence using antibodies against ATP synthase components

• Combining these approaches with microautoradiography to assess activity

These techniques can be applied to study how N. oceani atpE expression varies across different marine environments, seasons, and depths, providing insights into the regulation of energy metabolism in this environmentally important bacterium .

What are the major unresolved questions about N. oceani ATP synthase function and how might they be addressed?

Despite progress in understanding N. oceani ATP synthase, several key questions remain:

Unresolved Questions and Research Approaches:

• Ion Specificity Question:

  • Does N. oceani ATP synthase use both H⁺ and Na⁺ gradients for ATP synthesis?

  • Approach: Create reconstituted systems with controlled ion gradients and measure ATP synthesis rates under each condition

• Regulatory Mechanisms Question:

  • How is ATP synthase expression regulated in response to environmental changes?

  • Approach: Study transcriptional and post-translational modifications under different growth conditions

• Structural Adaptations Question:

  • What structural features allow N. oceani ATP synthase to function optimally in marine environments?

  • Approach: Comparative structural analysis with ATP synthases from non-marine organisms

• Evolutionary Origins Question:

  • Did the multiple ATP synthase systems in N. oceani arise through duplication or horizontal gene transfer?

  • Approach: Detailed phylogenetic analysis of ATP synthase components across bacterial lineages

• Environmental Resilience Question:

  • How will ocean acidification affect N. oceani ATP synthase function?

  • Approach: Measure ATP synthase activity and expression under simulated future ocean conditions

Each of these questions requires a combination of biochemical, molecular, structural, and environmental approaches, highlighting the interdisciplinary nature of research on this important protein complex .

How might emerging technologies advance our understanding of N. oceani ATP synthase structure and function?

Emerging technologies offer exciting new possibilities for ATP synthase research:

Cutting-Edge Approaches:

• Cryo-Electron Tomography:

  • Visualize ATP synthase in its native membrane environment without extraction

  • Study spatial organization and interactions with other cellular components

  • Potential to observe conformational changes during function

• Single-Molecule Techniques:

  • Use optical tweezers or magnetic tweezers to measure forces generated during ATP synthesis

  • Apply single-molecule FRET to observe conformational changes in real-time

  • Employ patch-clamp of reconstituted systems to measure proton translocation at the single-complex level

• Advanced Computational Methods:

  • Apply machine learning to predict functional impacts of sequence variations

  • Use quantum mechanics/molecular mechanics simulations to model proton transfer

  • Implement systems biology approaches to integrate ATP synthase function with cellular metabolism

• Synthetic Biology and Bioengineering:

  • Create minimal synthetic systems incorporating N. oceani ATP synthase components

  • Engineer hybrid ATP synthases with components from different organisms

  • Develop biosensors based on ATP synthase components for environmental monitoring

• In situ Structural Methods:

  • Apply cellular cryo-electron microscopy to visualize ATP synthase in intact cells

  • Use correlative light and electron microscopy to connect structure with function

  • Implement microfluidic approaches for real-time monitoring of ATP synthesis