Recombinant Thermotoga maritima ATP synthase subunit c (atpE)

What is Thermotoga maritima ATP synthase subunit c (AtpE) and what is its basic function?

ATP synthase subunit c (AtpE) is an essential component of the F-type ATP synthase in the hyperthermophilic bacterium Thermotoga maritima. This enzyme catalyzes the production of ATP from ADP in the presence of a sodium or proton gradient . In T. maritima, AtpE forms part of the membrane-embedded F0 portion of the F1F0-ATP synthase complex. The subunit c proteins assemble into a cylindrical c-ring structure (c-oligomer) that plays a crucial role in ion translocation across the membrane during ATP synthesis .

How does the T. maritima ATP synthase differ from other bacterial ATP synthases?

T. maritima ATP synthase is unique in being Na⁺-dependent (Na⁺-F₁F₀-type) rather than proton-dependent like many other bacterial ATP synthases . Key differences include:

This Na⁺ dependency is likely an adaptation to the hyperthermophilic lifestyle of T. maritima, which grows optimally at temperatures around 80°C .

What are the optimal conditions for expressing recombinant T. maritima AtpE in E. coli?

For successful expression of recombinant T. maritima AtpE in E. coli:

• Vector selection: pET-based expression vectors with T7 promoter systems are commonly used for thermophilic proteins .

• Host strain: E. coli BL21(DE3) or similar strains are suitable for expression .

• Expression conditions:

  • Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

  • Temperature: 30-37°C for 3-4 hours (lower temperatures may improve solubility)

  • Media: Enriched media (LB or TB) supplemented with appropriate antibiotics

• Fusion tag: N-terminal His-tag facilitates purification while maintaining protein function .

What purification strategies are most effective for T. maritima AtpE?

Given the thermostable nature of T. maritima proteins, the following purification strategy is effective:

• Heat treatment: Initial clarification at 70-75°C for 15-20 minutes to precipitate host proteins while T. maritima proteins remain soluble .

• Affinity chromatography: His-tagged AtpE can be purified using Ni-NTA chromatography with imidazole gradient elution .

• Ion exchange chromatography: Q-sepharose or similar can be used as a secondary purification step .

• Size exclusion chromatography: Final polishing step to obtain highly pure protein and determine oligomeric state .

The typical yield is >90% purity as determined by SDS-PAGE . For functional studies, reconstitution into liposomes may be necessary for activity assays .

How should purified T. maritima AtpE be stored to maintain stability and activity?

Based on product recommendations for recombinant T. maritima AtpE :

• Storage temperature: Store at -20°C/-80°C for long-term storage.

• Buffer composition: Tris/PBS-based buffer with 6% Trehalose, pH 8.0.

• Aliquoting: Prepare small aliquots to avoid repeated freeze-thaw cycles.

• Working stocks: For frequent use, store working aliquots at 4°C for up to one week.

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Cryoprotectant: Add glycerol to a final concentration of 50% for long-term storage at -20°C/-80°C.

Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity .

How can the ATPase activity of T. maritima ATP synthase containing AtpE be measured?

The ATPase activity can be measured using the following methods:

• Coupled enzyme assay:

  • ATP hydrolysis is coupled to NADH oxidation through pyruvate kinase and lactate dehydrogenase

  • Measure the decrease in NADH absorbance at 340 nm

  • Reaction conditions: 50 mM Tris-HCl (pH 7.5-8.0), 5 mM MgCl₂, 2-10 mM NaCl, 2-5 mM ATP at 37-80°C

• Colorimetric phosphate release assay:

  • Measure inorganic phosphate released from ATP hydrolysis using malachite green or similar reagents

  • Reaction conditions similar to above

• Luciferin-luciferase assay for ATP synthesis:

  • Measure ATP production in reconstituted proteoliposomes

  • Reaction triggered by creating Na⁺ gradient across the membrane

The specific activity of the purified T. maritima ATP synthase is approximately 5 U/mg with a strong Na⁺ dependency, following Michaelis-Menten kinetics with K_m of 1.2 ± 0.2 mM Na⁺ and v_max of 4.7 U/mg .

What is the Na⁺ specificity of T. maritima AtpE and how can it be experimentally verified?

The Na⁺ specificity of T. maritima AtpE can be experimentally verified through:

• Na⁺-dependent ATPase activity assays:

  • Measure ATPase activity at varying Na⁺ concentrations (0-20 mM)

  • Plot activity vs. Na⁺ concentration to determine K_m and v_max

• DCCD inhibition studies:

  • N,N'-dicyclohexylcarbodiimide (DCCD) inhibits the activity

  • Na⁺ and DCCD compete for a common binding site

  • Na⁺ relieves DCCD inhibition in Na⁺-dependent ATP synthases

• Na⁺ transport assays in proteoliposomes:

  • Reconstitute ATP synthase into liposomes

  • Incubate with ²²Na⁺

  • Measure Na⁺ accumulation in proteoliposomes upon ATP addition

  • Use ionophores (ETH2120 for Na⁺ or CCCP for H⁺) to verify specificity

These experiments have confirmed that T. maritima ATP synthase uses Na⁺ as its coupling ion rather than H⁺ .

What methods are used to determine the thermostability of T. maritima AtpE?

The thermostability of T. maritima AtpE can be assessed through:

• Differential scanning calorimetry (DSC):

  • Measures the heat capacity changes during protein unfolding

  • Determines the melting temperature (T_m)

• Circular dichroism (CD) spectroscopy:

  • Monitors changes in secondary structure with increasing temperature

  • Can show structural transitions and unfolding events

• Activity assays at different temperatures:

  • Measure enzyme activity after incubation at various temperatures

  • Determine half-life at elevated temperatures (e.g., 90°C)

  • Establish temperature optimum for activity

• Thermal shift assays:

  • Using fluorescent dyes that bind to hydrophobic regions exposed during unfolding

  • Allows high-throughput screening of stabilizing conditions

Typically, T. maritima AtpE retains structure and function at temperatures up to 80-90°C, reflecting the hyperthermophilic nature of the organism .

How does T. maritima AtpE contribute to the Na⁺ translocation mechanism of ATP synthase?

The AtpE subunit plays a central role in Na⁺ translocation through:

• Formation of the c-ring rotor: Multiple copies of AtpE assemble into a cylindrical c-ring structure in the membrane .

• Na⁺ binding sites: Each AtpE monomer contains a Na⁺ binding site with the conserved motif Px₃Qx₂₈,₃₂ET that coordinates Na⁺ ions .

• Rotary mechanism: During ATP synthesis:

  • Na⁺ ions bind to AtpE at the a-c interface

  • The c-ring rotates, carrying Na⁺ ions across the membrane

  • Na⁺ ions are released on the opposite side of the membrane

  • This rotation is coupled to conformational changes in the F₁ sector, driving ATP synthesis

• Electrogenic transport: Na⁺ transport is a primary and electrogenic event, generating membrane potential that can be measured experimentally .

The c-ring in T. maritima likely contains 10-12 AtpE subunits, each capable of transporting one Na⁺ ion per 360° rotation of the c-ring .

What is the relationship between T. maritima AtpE function and the organism's energy metabolism?

The Na⁺-dependent ATP synthase containing AtpE integrates into T. maritima's energy metabolism in several ways:

• Fermentative metabolism: T. maritima converts sugars to acetate, CO₂, and H₂ according to the equation:
  1 glucose → 2 acetate + 2 CO₂ + 4 H₂

• Respiratory chain connection: The Rnf complex (a Na⁺-pumping respiratory enzyme) and ATP synthase form the two components of T. maritima's respiratory chain, connected by an electrochemical Na⁺ gradient .

• Energy conservation during fermentation: During carbohydrate fermentation:

  • Approximately 3.7 ATP are generated by substrate-level phosphorylation

  • Additional 0.035 ATP are generated by electron transport phosphorylation via Rnf and ATP synthase

  • This represents about 1% of total ATP produced

• Role in redox balance: The ATP synthase works alongside other membrane protein complexes like the membrane-bound hydrogenase to maintain redox balance during growth .

In sulfur-containing environments, ATP yield by chemiosmosis could increase to 1.79 per mol glucose (46% of substrate-level phosphorylation), highlighting the adaptability of T. maritima's energy metabolism .

How does the Na⁺ dependency of T. maritima ATP synthase represent an adaptation to extreme environments?

The Na⁺ dependency of T. maritima ATP synthase represents several evolutionary adaptations to extreme environments:

• Membrane permeability: Na⁺ leakage across membranes is lower than H⁺ leakage at high temperatures, making Na⁺ bioenergetics more efficient in hyperthermophiles.

• pH homeostasis: Using Na⁺ instead of H⁺ allows better maintenance of intracellular pH in extreme pH environments.

• Thermostability: The Na⁺-binding motif in AtpE provides additional structural stability to the c-ring at high temperatures.

• Integration with Na⁺-based energetics: The presence of other Na⁺-dependent enzymes in T. maritima (Rnf complex, Na⁺-translocating oxaloacetate decarboxylase) creates a coordinated Na⁺-based energy metabolism suited to high-temperature environments .

• Adaptation to variable salinity: Na⁺-coupled ATP synthesis is advantageous in marine or saline hot springs where T. maritima thrives.

This adaptation appears convergent across various hyperthermophilic organisms, suggesting it provides significant selective advantages in extreme environments .

How can site-directed mutagenesis of T. maritima AtpE be used to investigate ion specificity and catalytic mechanism?

Site-directed mutagenesis offers powerful insights into AtpE function:

• Target residues for mutagenesis:

  • Na⁺-binding motif residues (Px₃Qx₂₈,₃₂ET)

  • Conserved carboxylates that coordinate the coupling ion

  • Interface residues between adjacent c-subunits

  • Residues at the a-c subunit interface

• Experimental approaches:

  • Create single or multiple mutations using PCR-based methods

  • Express wild-type and mutant proteins in E. coli

  • Purify and reconstitute proteins in liposomes

  • Compare ATPase activity, Na⁺ binding, and Na⁺ transport

• Key structure-function studies:

  • Converting Na⁺ specificity to H⁺ specificity by mutating Na⁺-binding motif

  • Altering the stoichiometry of Na⁺/ATP by modifying c-ring stability

  • Investigating the role of specific residues in Na⁺ coordination

• Analysis techniques:

  • Enzyme kinetics to determine changes in K_m and V_max

  • Isothermal titration calorimetry to measure ion binding affinity

  • Transport assays with radioactive Na⁺

  • Structural studies of mutants using X-ray crystallography or cryo-EM

These approaches can reveal the molecular basis of ion selectivity and the mechanism of energy coupling in this enzyme.

How can recombinant T. maritima AtpE be used in structural biology studies?

Recombinant T. maritima AtpE is valuable for structural biology due to its inherent thermostability:

• X-ray crystallography:

  • Express and purify His-tagged AtpE in E. coli

  • Use detergent solubilization to isolate the c-ring

  • Apply crystallization screens optimized for membrane proteins

  • Collect diffraction data and solve structure

• Cryo-electron microscopy:

  • Purify intact ATP synthase complexes

  • Prepare cryo-EM grids and collect images

  • Determine high-resolution structure of the c-ring in context of the whole complex

• NMR spectroscopy:

  • Isotopically label the protein with ¹⁵N and ¹³C

  • Perform solution NMR for structural characterization

  • Study dynamics of Na⁺ binding and protein conformational changes

• Advantages of T. maritima AtpE for structural studies:

  • High thermostability improves sample handling and storage

  • Good expression levels in E. coli

  • Na⁺-dependent mechanism provides opportunity for comparative studies

Structural information can provide insights into the organization of the c-ring, Na⁺ binding sites, and conformational changes during the catalytic cycle.

What are the challenges and strategies for functional reconstitution of T. maritima ATP synthase containing AtpE?

Functional reconstitution of T. maritima ATP synthase faces several challenges:

• Challenges:

  • Maintaining protein stability during purification

  • Achieving correct orientation in liposomes

  • Creating appropriate Na⁺ gradients

  • Measuring activity at high temperatures

  • Ensuring complete complex assembly

• Strategies for successful reconstitution:

  • Lipid composition: Use thermostable lipids or synthetic lipids stable at high temperatures

  • Reconstitution method: Detergent removal by dialysis or adsorbent beads

  • Activity measurement:

    • Use thermostable coupling enzymes for activity assays

    • Test at moderate temperatures (45-60°C) to balance activity with liposome stability

    • Use radioactive Na⁺ (²²Na⁺) for transport studies

  • Buffer optimization: Include osmolytes that enhance thermostability

• Verification of functional reconstitution:

  • Measure ATP-dependent Na⁺ uptake into proteoliposomes

  • Confirm inhibitor sensitivity (DCCD)

  • Verify Na⁺ dependency of activity

  • Use ionophores to collapse Na⁺ gradients as controls

Successful reconstitution has been achieved showing 2-2.5 fold Na⁺ accumulation in proteoliposomes upon ATP addition, demonstrating functional integration of the AtpE-containing complex .

How does T. maritima AtpE compare with AtpE subunits from other organisms in terms of structure and function?

Comparative analysis reveals important differences:

Functional differences include:

• T. maritima AtpE is strictly Na⁺-dependent

• E. coli and human c-subunits are H⁺-dependent

• Human c-subunit isoforms differ in their targeting peptides, which play additional roles in respiratory chain maintenance

What insights does T. maritima AtpE provide about the evolution of ATP synthases?

T. maritima AtpE offers valuable evolutionary insights:

These features make T. maritima AtpE a valuable model for understanding the evolution of biological energy conversion systems.

What are the key considerations when designing experiments with recombinant T. maritima AtpE?

When working with recombinant T. maritima AtpE, consider:

• Temperature optimization:

  • Expression: Balance protein expression level with correct folding

  • Purification: Exploit thermostability for heat treatment steps (70-75°C)

  • Activity assays: Consider both optimal temperature for T. maritima proteins (80°C) and stability of assay components

• Buffer considerations:

  • pH: Optimal pH range is typically 7.5-8.5

  • Salt: Include Na⁺ (1-10 mM) for structural stability

  • Stabilizing agents: Trehalose or glycerol to maintain stability

• Experimental controls:

  • Ion specificity: Compare Na⁺ vs. K⁺, Li⁺, or H⁺

  • Inhibitors: DCCD as specific inhibitor

  • Ionophores: ETH2120 (Na⁺-specific) and CCCP (H⁺-specific) for transport studies

• Handling recombinant protein:

  • Avoid repeated freeze-thaw cycles

  • Prepare single-use aliquots

  • Consider liposome stability when working at high temperatures

• Protein tagging considerations:

  • N-terminal His-tag is preferable as C-terminus may be involved in c-ring assembly

  • Verify tag does not interfere with function through activity assays

How can researchers troubleshoot common issues with T. maritima AtpE expression and activity?

Common issues and troubleshooting approaches:

• Low expression levels:

  • Optimize codon usage for E. coli

  • Try different expression strains (C41/C43 for membrane proteins)

  • Adjust induction conditions (lower IPTG, longer expression at lower temperature)

  • Use fusion partners to improve solubility

• Protein aggregation:

  • Include appropriate detergents (DDM, CHAPS) during extraction

  • Add stabilizing agents (glycerol, trehalose)

  • Consider expressing as fusion with solubility-enhancing tags

• Low activity:

  • Ensure sufficient Na⁺ in reaction buffer (1-10 mM)

  • Verify correct orientation in liposomes

  • Check pH optimization (7.5-8.5)

  • Ensure complete reconstitution of complex

• Protein degradation:

  • Add protease inhibitors during purification

  • Utilize the thermostability for heat purification step

  • Store with stabilizing agents at appropriate temperature

• Incorrect assembly:

  • Co-express with other subunits of the ATP synthase

  • Verify oligomeric state by native PAGE or size exclusion chromatography

  • Optimize detergent:protein:lipid ratios during reconstitution

What are the most promising future research directions involving T. maritima AtpE?

Exciting future research directions include:

• Structural studies:

  • High-resolution structure of the complete T. maritima ATP synthase

  • Dynamics of c-ring rotation using single-molecule techniques

  • Conformational changes during Na⁺ binding and transport

• Biotechnological applications:

  • Development of thermostable ATP-regenerating systems for high-temperature biocatalysis

  • Design of Na⁺-powered molecular motors based on c-ring principles

  • Engineering of hybrid ATP synthases with modified ion specificity

• Medical relevance:

  • Comparative studies with human ATP synthase for understanding ATP synthase disorders

  • Use as a model system for drug design targeting bacterial ATP synthases

  • Structure-based inhibitor design targeting AtpE

• Ecological and evolutionary studies:

  • Understanding the role of Na⁺-dependent bioenergetics in thermal environments

  • Investigating the co-evolution of ATP synthase with other energy-conserving systems

  • Exploration of ATP synthases from other extremophiles

• Synthetic biology applications:

  • Development of minimal ATP synthase systems

  • Creation of artificial c-rings with altered stoichiometry or ion specificity

  • Integration into artificial cell systems for energy generation