Recombinant Trichodesmium erythraeum ATP synthase subunit c (atpE)

What is Trichodesmium erythraeum ATP synthase subunit c (atpE) and what is its significance in marine ecosystems?

ATP synthase subunit c (atpE) in Trichodesmium erythraeum is a critical component of the F0 sector of ATP synthase, forming the membrane-embedded c-ring that facilitates proton translocation during ATP synthesis. This 81-amino acid protein (UniProt ID: Q112Z2) is essential for energy metabolism in this globally important marine cyanobacterium . Trichodesmium plays a pivotal role in the marine nitrogen cycle as a significant nitrogen fixer in oligotrophic oceans, converting atmospheric N2 into bioavailable nitrogen forms . The efficient functioning of its ATP synthase is crucial for generating the energy needed to support nitrogen fixation, which requires substantial ATP input. Understanding the structure and function of atpE provides insights into the energy metabolism that supports Trichodesmium's ecological role in marine biogeochemical cycling.

What expression systems are most effective for producing functional recombinant Trichodesmium erythraeum atpE?

E. coli expression systems have proven most effective for producing recombinant Trichodesmium erythraeum atpE. The protein can be successfully expressed as a full-length construct (1-81 amino acids) with an N-terminal His-tag to facilitate purification . While E. coli is the predominant expression system due to its ease of use and high yield, researchers should consider several optimization steps:

• Codon optimization for E. coli is essential to overcome codon bias issues between Trichodesmium and E. coli.

• Expression temperature should be maintained at 18-25°C to prevent inclusion body formation due to the hydrophobic nature of atpE.

• Induction conditions should be carefully optimized using low IPTG concentrations (0.1-0.5 mM).

Similar approaches have been successfully employed for recombinant expression of membrane proteins from other cyanobacteria, such as Synechococcus elongatus . For advanced applications requiring post-translational modifications, eukaryotic expression systems may be considered, though they typically yield lower protein amounts.

What are the optimal methods for purifying recombinant Trichodesmium erythraeum atpE while maintaining its native conformation?

Purification of recombinant Trichodesmium erythraeum atpE requires specialized techniques due to its hydrophobic nature as a membrane protein. The optimal purification protocol involves:

• Cell lysis using a combination of enzymatic (lysozyme) and mechanical (sonication) methods in a buffer containing mild detergents (typically 1% DDM or LDAO).

• Initial purification using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin, exploiting the N-terminal His-tag .

• Size exclusion chromatography for further purification and assessment of oligomeric state.

• Throughout purification, maintaining the protein in an appropriate detergent micelle environment is crucial for stability.

The purified protein is typically obtained as a lyophilized powder that requires careful reconstitution . For functional studies, researchers should verify protein integrity using SDS-PAGE (>90% purity) , circular dichroism to assess secondary structure, and mass spectrometry for precise molecular weight determination. For structural studies, detergent screening is essential to identify conditions that maintain native conformational states while allowing for crystallization or other structural analyses.

How can researchers effectively reconstitute purified Trichodesmium erythraeum atpE into liposomes for functional studies?

Reconstitution of purified Trichodesmium erythraeum atpE into liposomes is critical for functional characterization. The following methodology has proven effective:

• Liposome preparation: Create liposomes using a mixture of phospholipids (typically POPC:POPE:POPG at 3:1:1 ratio) by the film hydration method followed by extrusion through 100-200 nm filters.

• Protein incorporation: Mix purified atpE protein (0.1-1.0 mg/mL in Tris/PBS buffer with 6% trehalose) with preformed liposomes at a lipid-to-protein ratio of 50:1 to 100:1.

• Detergent removal: Remove detergents using Bio-Beads or dialysis against detergent-free buffer.

• Verification of incorporation: Confirm successful reconstitution using freeze-fracture electron microscopy or sucrose density gradient centrifugation.

For functional studies, researchers should verify that the reconstituted protein forms proper oligomeric structures. Notably, c-subunit rings from ATP synthases can form voltage-dependent channels when reconstituted into liposomes . Electrophysiological studies using patch-clamp techniques can verify channel formation and characterize conductance properties. Additionally, assays for proton translocation using pH-sensitive fluorescent dyes can confirm functional reconstitution.

What spectroscopic and biophysical methods are most informative for characterizing the structure and conformational dynamics of Trichodesmium erythraeum atpE?

Several complementary spectroscopic and biophysical methods provide valuable insights into the structure and dynamics of Trichodesmium erythraeum atpE:

For structural studies, cryo-electron microscopy has emerged as a powerful technique for visualizing the entire ATP synthase complex, including the c-ring. This approach has been successfully applied to study c-subunit ring structures from various organisms and could provide valuable insights into the unique features of Trichodesmium erythraeum atpE . X-ray crystallography remains challenging due to the difficulty in crystallizing membrane proteins but has been successful for c-rings from other organisms.

How does phosphate limitation affect ATP synthase function and expression in Trichodesmium erythraeum?

Phosphate limitation significantly impacts ATP synthase function and expression in Trichodesmium erythraeum, reflecting adaptations to the oligotrophic ocean environments where this organism thrives. Under phosphate-limited conditions:

• Trichodesmium maintains growth at reduced phosphate concentrations (as low as 12.4-16.3 nM soluble reactive phosphorus) , demonstrating adaptation to phosphate scarcity common in its natural habitat.

• The cellular N:P ratio increases significantly (to approximately 48:1 compared to 20:1 under phosphate-replete conditions) , indicating reduced phosphate incorporation into cellular components including ATP.

• ATP synthase expression and composition may be modified to conserve phosphorus, potentially affecting the stoichiometry or turnover rate of ATP production.

• Alkaline phosphatase activity increases substantially , enabling utilization of organic phosphorus sources to maintain ATP synthesis when inorganic phosphate is limited.

These adaptations enable Trichodesmium to maintain energy production even in phosphate-limited environments, although with potential changes to ATP synthase efficiency or activity that may affect nitrogen fixation capabilities. The ability to adjust ATP synthase function under nutrient limitation represents a critical adaptation for maintaining ecological function in oligotrophic oceans.

What roles might Trichodesmium erythraeum atpE play in response to acidification and other environmental stressors?

Trichodesmium erythraeum atpE likely plays crucial roles in the organism's response to ocean acidification and other environmental stressors:

• Ocean acidification response: Under acidified conditions (750 μatm CO2), Trichodesmium shows increased phosphorus requirements and decreased phosphorus-specific nitrogen fixation rates . ATP synthase, including atpE, must adapt to maintain proton gradients against altered external pH, potentially requiring structural or conformational adjustments of the c-ring.

• UV radiation tolerance: While not directly addressed in the search results for atpE, cyanobacteria like Synechococcus elongatus require specific adaptations for UV tolerance . ATP synthase must maintain functionality during UV stress to provide energy for repair mechanisms.

• Thermal stress adaptation: As ocean temperatures rise, membrane fluidity changes may affect the c-ring structure and function, requiring compensatory modifications to maintain proton translocation efficiency.

The c-subunit's role as part of the proton channel makes it particularly sensitive to environmental pH changes. As the c-ring rotates during ATP synthesis, proper protonation/deprotonation cycles are essential for function. Ocean acidification could alter these cycles, potentially requiring adaptive responses in the c-subunit structure or composition to maintain ATP production efficiency under changing conditions.

How does the interaction between Trichodesmium and its associated bacteria affect ATP synthase function and nitrogen metabolism?

The Trichodesmium microbiome significantly influences the host's energy metabolism and nitrogen cycling:

• Trichodesmium colonies host diverse heterotrophic bacteria, particularly from the marine Roseobacter clade (MRC) of Alphaproteobacteria, creating microscale nutrient-rich oases in oligotrophic waters .

• These bacterial associates metabolize trimethylamine (TMA), converting it to more accessible nitrogen forms including ammonium, which Trichodesmium can then utilize .

• This nitrogen transfer from associated bacteria suppresses nitrogen fixation in Trichodesmium , potentially reducing the ATP demand since nitrogen fixation is energetically costly.

• When Trichodesmium obtains nitrogen from bacterial associates rather than fixing N2, ATP demand decreases, potentially affecting ATP synthase expression or regulation.

This mutualistic relationship represents a sophisticated adaptation where Trichodesmium benefits from bacterial nitrogen metabolism, reducing its energetic investment in nitrogen fixation. The resulting energy conservation has implications for ATP synthase function and regulation, as the enzyme may operate under different demands depending on nitrogen source. This interaction exemplifies the complex ecological adaptations that influence core metabolic processes in Trichodesmium .

How can site-directed mutagenesis of Trichodesmium erythraeum atpE be used to investigate proton translocation mechanisms and c-ring assembly?

Site-directed mutagenesis of Trichodesmium erythraeum atpE enables detailed investigation of proton translocation mechanisms and c-ring assembly through targeted modification of key residues:

• Proton-binding site mutations: The conserved carboxylic acid residue (typically glutamate or aspartate) in the middle of the second transmembrane helix is critical for proton binding. Mutations at this position (E/D → Q/N/A) would eliminate proton binding, allowing assessment of this residue's importance in Trichodesmium's ATP synthase function.

• Inter-subunit interaction mutations: Residues at the interface between adjacent c-subunits can be mutated to investigate factors affecting c-ring assembly and stability. Based on knowledge from similar c-subunits, mutations in glycine-rich motifs often disrupt proper ring formation.

• Functional channel investigation: As shown in the provided research, c-subunit rings can form voltage-dependent channels . Mutations in the central pore region of the c-ring can help identify residues critical for channel formation and conductance properties.

For expression and functional characterization of these mutants, researchers should follow the same techniques used for wild-type protein, including E. coli expression, purification with appropriate detergents, and reconstitution into liposomes for electrophysiological studies . Comparing wild-type and mutant proteins through techniques such as blue native PAGE and analytical ultracentrifugation can reveal differences in oligomerization properties.

What experimental approaches can be used to study potential channel formation by Trichodesmium erythraeum atpE oligomers?

Several experimental approaches can effectively characterize potential channel formation by Trichodesmium erythraeum atpE oligomers:

• Electrophysiological characterization: After reconstitution into liposomes or planar lipid bilayers, patch-clamp techniques can detect and characterize ion channels formed by atpE oligomers. This approach has successfully demonstrated channel activity in mammalian ATP synthase c-subunits .

• Ion flux assays: Liposomes containing reconstituted atpE can be loaded with fluorescent dyes sensitive to specific ions (H+, Ca2+, K+) to measure ion transport across membranes. Changes in fluorescence intensity indicate channel activity.

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry can reveal the oligomeric state and structural arrangement of atpE complexes in membranes.

• Voltage-dependent behavior analysis: As demonstrated with mammalian ATP synthase c-subunits, channel properties may be voltage-dependent . Applying varying membrane potentials during electrophysiological recordings can characterize voltage sensitivity.

• Pharmacological profiling: Testing channel activity in the presence of various inhibitors can provide insights into the channel properties and potential regulatory mechanisms.

These methods can determine if Trichodesmium erythraeum atpE forms channels similar to those observed in mammalian systems, where the c-subunit ring can function as a permeability transition pore component . Such findings would have significant implications for understanding energy coupling and potential uncoupling mechanisms in Trichodesmium's ATP synthase.

How can computational modeling enhance our understanding of Trichodesmium erythraeum atpE structure and function?

Computational modeling provides powerful tools for understanding Trichodesmium erythraeum atpE structure and function:

• Homology modeling: Using the known structures of c-subunits from other organisms as templates, researchers can build three-dimensional models of Trichodesmium erythraeum atpE. The amino acid sequence (MDPLIAAASVVAAALAVGLGAIGPGIGQGNAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVSLVLLFANPFA) provides the primary input for such modeling.

• Molecular dynamics simulations: These simulations can reveal:

  • Conformational dynamics of the c-ring in lipid bilayers

  • Proton binding and release mechanisms

  • Structural changes under different environmental conditions (pH, temperature)

  • Interaction with other ATP synthase subunits

• Quantum mechanical/molecular mechanical (QM/MM) approaches: These methods can provide detailed insights into the energetics of proton transfer within the c-ring structure.

• Protein-protein docking: Modeling interactions between atpE and other ATP synthase subunits can illuminate the structural basis of rotary catalysis.

• Evolutionary analysis: Comparative genomics and evolutionary trace methods can identify conserved regions critical for function versus regions that may represent adaptations to Trichodesmium's specific ecological niche.

These computational approaches complement experimental methods and are particularly valuable for membrane proteins like atpE, which present challenges for experimental structural determination. Models can generate testable hypotheses about structure-function relationships and guide the design of site-directed mutagenesis experiments to investigate specific aspects of atpE function.

What techniques can be used to study the integration of ATP synthase function with nitrogen fixation in Trichodesmium erythraeum?

Investigating the integration of ATP synthase function with nitrogen fixation in Trichodesmium erythraeum requires multi-faceted approaches:

• Metabolic flux analysis: Isotope labeling (13C, 15N) combined with mass spectrometry can trace energy flow between ATP synthesis and nitrogen fixation. This approach has revealed how nitrogen sources like trimethylamine affect nitrogen metabolism in Trichodesmium .

• Real-time ATP measurements: Luciferase-based ATP sensors or fluorescent ATP biosensors can monitor ATP levels during nitrogen fixation under various conditions.

• Transcriptomics and proteomics integration:

  • RNA-Seq analysis during nitrogen fixation and under different environmental stressors

  • Quantitative proteomics to measure changes in ATP synthase subunit abundance

  • Phosphoproteomics to identify regulatory modifications

• Bioenergetic profiling: Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements can assess respiratory capacity and ATP production during nitrogen fixation.

• In situ protein-protein interaction studies:

  • Förster resonance energy transfer (FRET) between labeled ATP synthase and nitrogenase components

  • Proximity labeling techniques to identify proteins interacting with ATP synthase during nitrogen fixation

Research has shown that when Trichodesmium obtains nitrogen from bacterial associates rather than fixing N2, nitrogen fixation is suppressed . This metabolic shift likely affects ATP demand and potentially ATP synthase regulation. Understanding these dynamics requires integrative approaches that connect energy metabolism with nitrogen cycling at the molecular, cellular, and community levels.

How might emerging technologies advance our understanding of the structure and function of Trichodesmium erythraeum atpE?

Emerging technologies offer exciting opportunities to advance our understanding of Trichodesmium erythraeum atpE:

• Cryo-electron microscopy (cryo-EM): Recent advances in cryo-EM technology now enable determination of membrane protein structures at near-atomic resolution without crystallization. This approach could reveal the complete structure of Trichodesmium ATP synthase, including the c-ring composed of atpE subunits.

• Single-molecule techniques:

  • High-speed atomic force microscopy (HS-AFM) to visualize c-ring rotation in real-time

  • Single-molecule FRET to track conformational changes during function

  • Magnetic tweezers to measure forces generated by ATP synthase rotation

• Microfluidics and organ-on-chip technologies: These could enable real-time monitoring of ATP synthase function under precisely controlled environmental conditions mimicking oceanic changes (pH, temperature, nutrient levels).

• CRISPR-Cas9 genome editing: Development of genetic manipulation tools for Trichodesmium would enable in vivo studies of atpE function through precise genomic modifications.

• Artificial intelligence and machine learning: These approaches can identify subtle patterns in large datasets, potentially revealing new insights about structure-function relationships in atpE and its response to environmental variables.

These technologies, especially when combined in integrative approaches, promise to overcome current limitations in studying membrane proteins in cyanobacteria and could reveal how Trichodesmium's ATP synthase has adapted to its unique ecological niche.

What are the implications of understanding Trichodesmium erythraeum atpE for climate change research and marine ecosystem modeling?

Understanding Trichodesmium erythraeum atpE has significant implications for climate change research and marine ecosystem modeling:

• Ocean acidification effects: As atmospheric CO2 increases, resulting ocean acidification directly affects ATP synthase function. Research shows that acidification increases phosphorus requirements and decreases phosphorus-specific nitrogen fixation rates in Trichodesmium . Understanding how atpE responds to pH changes is crucial for predicting future nitrogen fixation capacity in warming oceans.

• Energy-nutrient coupling in biogeochemical models: Detailed knowledge of how ATP synthase function relates to nutrient limitation (especially phosphorus) can improve parameterization of biogeochemical models that predict marine primary production and nitrogen fixation.

• Microbial interactions in ecosystem function: The discovery that bacterial associates affect Trichodesmium's nitrogen metabolism highlights the importance of incorporating microbial interactions into ecosystem models, rather than treating species as isolated entities.

• Evolutionary adaptation capacity: Understanding the molecular basis of ATP synthase adaptation in Trichodesmium provides insights into potential evolutionary responses to climate change, informing predictions about ecosystem resilience.

This research bridges molecular biology and global biogeochemical processes, contributing to more accurate predictions of how marine nitrogen fixation—a process significantly influenced by Trichodesmium—will respond to ongoing climate change, with implications for marine productivity and carbon sequestration.