Recombinant Thermoanaerobacter tengcongensis ATP synthase subunit c (atpE)

What is Thermoanaerobacter tengcongensis and why is its ATP synthase subunit c of interest?

Thermoanaerobacter tengcongensis is a rod-shaped, anaerobic eubacterium isolated from a freshwater hot spring in Tengchong, China. While empirically classified as gram-negative by staining, genomic analysis reveals it shares many characteristics with gram-positive bacteria . The organism grows optimally at 75°C (range 50-80°C) and pH 7-7.5 (range 5.5-9) .

Its ATP synthase subunit c (atpE) is of particular interest because of its adaptation to function under extreme thermal conditions. In thermophiles, ATP synthase components must maintain structural integrity and functional efficiency at temperatures that would denature mesophilic proteins. The atpE subunit, as part of the membrane-embedded F₀ domain that forms the proton channel, has evolved specific amino acid compositions and structural modifications that contribute to thermostability while preserving its critical role in ATP synthesis.

What are the genomic characteristics of T. tengcongensis relevant to ATP synthase genes?

T. tengcongensis possesses a single circular chromosome of 2,689,445 base pairs with a relatively low genomic G+C content of 37.6% . A distinctive feature of this genome is that 86.7% of its genes are encoded on the leading strand of DNA replication . The genome encodes 2588 predicted coding sequences, with 68.2% classified according to homology to other documented proteins .

The ATP synthase genes in T. tengcongensis would be expected to follow the general pattern of high expression genes in thermophiles - exhibiting higher G+C content than the genomic average, which contributes to thermostability through stronger G-C bonding. This pattern is evident in the organism's rRNA genes (58.2%-60.3% G+C) and tRNA genes (52.6%-69.3% G+C) . This characteristic G+C enrichment in functional RNA molecules is a common adaptation among thermophiles.

How does the expression of recombinant T. tengcongensis atpE differ from mesophilic counterparts?

Expressing recombinant T. tengcongensis atpE presents unique challenges compared to mesophilic variants. The expression system must account for:

• Codon usage bias: T. tengcongensis may utilize different codon preferences than common expression hosts like E. coli

• Protein folding: The atpE protein has evolved to fold correctly at high temperatures

• Post-translational modifications: Any thermophile-specific modifications must be considered

• Membrane integration: As a hydrophobic membrane protein, atpE requires proper membrane insertion machinery

Methodological approach: Consider using thermophilic expression systems or modified E. coli strains with chaperones adapted for thermophilic proteins. Cold-shock expression protocols may help mitigate misfolding issues, where expression at lower temperatures (15-18°C) can slow folding and improve yield of properly folded protein. For membrane integration, cell-free expression systems supplemented with lipid nanodiscs or liposomes may better accommodate the hydrophobic nature of atpE.

What purification methods are most effective for T. tengcongensis atpE?

Purifying the hydrophobic atpE subunit requires specialized techniques:

• Detergent solubilization: Screen multiple detergents (DDM, LDAO, Triton X-100) for optimal extraction while maintaining native structure

• Heat treatment: Exploit the thermostability by incorporating a heat purification step (65-70°C) to denature contaminating proteins

• Chromatography sequence: Typically utilize IMAC (if His-tagged), followed by size exclusion chromatography

• Stability buffers: Include specific lipids (often archaeal-type) and glycerol to maintain stability during purification

Methodological approach: Begin with cell disruption in a buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 10% glycerol, and protease inhibitors. Solubilize membranes with 1% DDM (w/v) for 1 hour at room temperature. After centrifugation, apply the supernatant to a pre-equilibrated Ni-NTA column, and elute with an imidazole gradient. Pool and concentrate fractions containing purified atpE, then apply to a size exclusion column for final purification in buffer containing 0.05% DDM.

How do the structural adaptations of T. tengcongensis atpE contribute to thermostability?

The atpE subunit from T. tengcongensis exhibits several structural adaptations that contribute to its remarkable thermostability:

• Amino acid composition: Increased prevalence of charged residues forming salt bridges, reduction in thermolabile residues (Asn, Gln, Cys, Met), and increased hydrophobic core packing

• Secondary structure elements: More extensive hydrogen bonding networks and α-helical stabilization

• Oligomeric interactions: The c-ring structure benefits from stronger subunit-subunit interactions

• Lipid interactions: Specialized interactions with ether-linked lipids common in thermophiles

Methodological approach: Perform comparative structural analysis between T. tengcongensis atpE and mesophilic homologs using X-ray crystallography or cryo-EM. Site-directed mutagenesis targeting potential thermostability-conferring residues followed by thermal stability assays (differential scanning calorimetry) can identify specific adaptations. Molecular dynamics simulations at different temperatures can reveal dynamic aspects of thermostability mechanisms.

What is the functional role of the ion-binding site in T. tengcongensis atpE under extreme conditions?

The ion-binding site in atpE is crucial for ATP synthase function as it coordinates the ion (typically H⁺ or Na⁺) translocation that drives ATP synthesis. In T. tengcongensis, this site likely exhibits adaptations for function under extreme conditions:

• Ion specificity: May show altered preference between H⁺ and Na⁺ compared to mesophilic counterparts

• pKa modulation: Altered pKa values of key residues to maintain function at high temperatures

• Coordination geometry: Potentially modified to maintain optimal ion binding despite thermal motion

• Binding kinetics: Likely exhibits different on/off rates optimized for thermophilic metabolism

Methodological approach: Characterize ion binding through isothermal titration calorimetry at various temperatures. Use reconstituted proteoliposomes containing purified atpE to measure ion transport rates. Compare wild-type and site-directed mutants of key binding site residues to establish structure-function relationships. Spectroscopic methods (FTIR, NMR) can provide insights into ionization states of key residues at different temperatures.

How does the c-ring stoichiometry of T. tengcongensis ATP synthase compare to other organisms, and what are the functional implications?

ATP synthase c-rings show variable stoichiometry (8-15 subunits) across species, directly affecting the ATP synthesis efficiency:

*The exact stoichiometry for T. tengcongensis would need experimental determination

Functional implications include:

• H⁺/ATP ratio: Determines thermodynamic efficiency (more c-subunits = more protons per ATP)

• Energy conversion efficiency: Affects how efficiently proton motive force is converted to ATP

• Thermal adaptation: Stoichiometry may reflect adaptation to energy availability in thermal environments

Methodological approach: Determine c-ring stoichiometry through atomic force microscopy, mass spectrometry of intact c-rings, or high-resolution cryo-EM. Reconstitute ATP synthase with different ratios of subunits to establish functional consequences. Compare ATP synthesis rates at different temperatures to correlate stoichiometry with thermal adaptation.

What experimental approaches can resolve contradictory findings regarding Na⁺ versus H⁺ specificity in T. tengcongensis ATP synthase?

Conflicting reports on ion specificity in thermophilic ATP synthases require systematic resolution:

• Ion competition assays: Measure ATP synthesis/hydrolysis rates in the presence of H⁺ or Na⁺ gradients with varying concentrations of competing ions

• Binding site mutagenesis: Create systematic mutations of key residues in the ion binding pocket

• Spectroscopic analysis: Monitor site-specific protonation states using FTIR or NMR

• Electrophysiology: Direct measurement of ion currents through reconstituted c-rings

Methodological approach: Purify recombinant T. tengcongensis ATP synthase components and reconstitute into liposomes. Establish H⁺ or Na⁺ gradients across the membrane using ionophores or buffer exchanges. Measure ATP synthesis rates under strictly controlled ion conditions (varying pH, [Na⁺], temperature). Perform parallel experiments with site-directed mutants of key ion-coordinating residues to determine their contribution to specificity.

How do the gene expression patterns of ATP synthase components in T. tengcongensis respond to environmental stressors?

T. tengcongensis must regulate ATP synthase expression in response to environmental challenges:

• Temperature fluctuations: Expression changes during heat shock or cooling

• pH variations: Acidic/alkaline stress response mechanisms

• Nutrient limitation: Metabolic reprogramming affects energy production

• Oxygen exposure: Response to oxidative stress despite anaerobic lifestyle

Methodological approach: Utilize RNA-Seq to capture transcriptome-wide expression changes under various conditions. Employ quantitative RT-PCR to specifically track ATP synthase gene expression. Complement with proteomic analysis using stable isotope labeling and LC-MS/MS to measure protein-level changes. Design reporter gene constructs fused to ATP synthase promoters to monitor expression dynamics in real-time.

What are the optimal conditions for functional reconstitution of recombinant T. tengcongensis atpE?

Functional reconstitution requires careful optimization of multiple parameters:

• Lipid composition: Test various lipid mixtures including archaeal-like tetraether lipids

• Protein:lipid ratio: Typically 1:50 to 1:100 (w/w) depending on the reconstitution method

• Buffer conditions: Consider ionic strength, pH, and temperature effects

• Reconstitution method: Compare detergent removal via dialysis, Bio-Beads, or dilution

Methodological approach: Prepare liposomes from E. coli polar lipids supplemented with synthetic tetraether lipids (10-20%). Solubilize with mild detergent (0.5% Triton X-100) and mix with purified atpE at various protein:lipid ratios. Remove detergent using SM-2 Bio-Beads with gentle agitation at room temperature. Verify reconstitution by freeze-fracture electron microscopy and functional assays measuring proton translocation using pH-sensitive fluorescent dyes (ACMA or pyranine).

How can cryo-electron microscopy be optimized for structural analysis of T. tengcongensis ATP synthase c-ring?

Optimizing cryo-EM for the c-ring requires addressing several technical challenges:

• Sample preparation: The small size (~5 nm diameter) and membrane-embedded nature complicate grid preparation

• Image acquisition: Low contrast necessitates specialized collection parameters

• Data processing: Distinguishing between closely-packed c-subunits requires high-resolution analysis

• Validation: Confirming the correct assembly and stoichiometry

Methodological approach: Purify the c-ring in amphipol A8-35 or reconstitute into nanodiscs using MSP1D1 scaffold protein. Apply sample to glow-discharged Quantifoil R1.2/1.3 grids and vitrify using Vitrobot (4 seconds blot time, 100% humidity). Collect images on a Titan Krios with K3 direct detector (0.6-0.8 Å/pixel, 40 frames/micrograph, 40-50 e-/Ų total dose). Process data using RELION with CTF correction and impose symmetry based on preliminary 2D class averages to improve resolution.

What computational models best predict the impact of mutations on T. tengcongensis atpE stability and function?

Several computational approaches can predict mutation effects:

• Molecular dynamics simulations: Provide dynamic information about protein stability at different temperatures

• Rosetta-based modeling: Enables ΔΔG calculations for stability predictions

• Evolutionary coupling analysis: Identifies co-evolving residues critical for function

• Machine learning approaches: Trained on thermostable protein datasets

Methodological approach: Build a homology model of T. tengcongensis atpE based on available c-subunit structures. Perform in silico alanine scanning to identify stabilizing residues. For key residues, conduct extended molecular dynamics simulations at elevated temperatures (300K, 350K, and 375K) to assess thermal stability. Validate computational predictions with experimental thermal stability assays of selected mutants using circular dichroism spectroscopy and differential scanning calorimetry.

How can isotope labeling strategies enhance structural studies of T. tengcongensis atpE?

Strategic isotope labeling enables advanced structural analysis:

• Uniform ¹⁵N/¹³C labeling: Enables solution and solid-state NMR studies

• Selective amino acid labeling: Targets specific structural elements

• Segmental labeling: Focuses on particular regions of interest

• Deuteration: Improves NMR spectral quality for membrane proteins

Methodological approach: Express recombinant atpE in M9 minimal media supplemented with ¹⁵NH₄Cl and ¹³C-glucose as sole nitrogen and carbon sources. For membrane proteins like atpE, deuteration is critical - grow E. coli in D₂O-based media with increasing D₂O concentration (50%, 70%, 100%) to adapt cells. For selective labeling, use amino acid auxotrophic strains and supplement with specific ¹⁵N/¹³C-labeled amino acids. Purify labeled protein using established protocols, then conduct solid-state NMR experiments optimized for membrane proteins (MAS-NMR at 55-60 kHz spinning rates).

How does T. tengcongensis atpE interact with other ATP synthase subunits in the complete enzyme complex?

The interaction network of atpE with other ATP synthase components involves:

• c-c subunit interactions: Forming the complete c-ring

• a-c subunit interface: Creating the critical proton translocation pathway

• c-ε coupling: Connecting the F₀ and F₁ domains

• Lipid-mediated interactions: Stabilizing the complex in the membrane

Methodological approach: Employ cross-linking mass spectrometry (XL-MS) using BS3 or EDC cross-linkers followed by LC-MS/MS analysis to map interaction interfaces. Complement with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify protected regions indicating subunit interactions. Validate interactions through co-immunoprecipitation studies using antibodies against different subunits. For a comprehensive view, perform single-particle cryo-EM of the intact ATP synthase complex.

How does the evolution of T. tengcongensis atpE compare with other extremophiles and what does this reveal about convergent adaptation?

Evolutionary analysis of atpE can reveal adaptation patterns:

• Sequence conservation: Identify highly conserved vs. variable regions

• Selective pressure: Calculate dN/dS ratios across thermophiles

• Ancestral sequence reconstruction: Infer evolutionary trajectory

• Horizontal gene transfer: Detect potential shared adaptations

Methodological approach: Collect atpE sequences from diverse extremophiles (thermophiles, acidophiles, alkaliphiles). Perform multiple sequence alignment using MUSCLE or MAFFT algorithms. Construct maximum likelihood phylogenetic trees using RAxML or IQ-TREE with appropriate substitution models. Conduct tests for selective pressure using PAML to identify positively selected sites. Compare structural models of atpE from different extremophiles to identify convergent structural adaptations despite divergent sequences.

What metabolic engineering approaches could improve heterologous expression of functional T. tengcongensis atpE?

Optimizing heterologous expression requires systematic metabolic engineering:

• Codon optimization: Adapt for expression host preference

• Chaperone co-expression: Include thermophilic chaperones

• Membrane composition modification: Engineer host lipid biosynthesis

• Export pathway enhancement: Optimize membrane protein insertion

Methodological approach: Design synthetic genes with codon optimization based on the expression host's preference while preserving critical RNA secondary structures. Co-express with thermophilic chaperones (GroEL/ES homologs) from T. tengcongensis. Supplement growth media with lipid precursors or modify host lipid biosynthesis genes to produce more thermophile-like membranes. Consider testing various fusion tags (MBP, SUMO) to improve solubility and membrane targeting. Implement a combinatorial approach testing different promoters, ribosome binding sites, and growth conditions to optimize expression.

How might the unique properties of T. tengcongensis atpE be applied in synthetic biology applications?

The thermostable properties of T. tengcongensis atpE offer several applications:

• Thermostable bioenergy systems: Creating heat-resistant bioenergetic modules

• Minimal cell designs: Incorporating robust ATP production systems

• Nanoscale rotary motors: Engineering molecular machines based on ATP synthase

• Biosensors: Developing temperature-resistant sensing technologies

Methodological approach: Engineer minimal synthetic cells by reconstituting purified T. tengcongensis ATP synthase components with artificial lipid bilayers. Design chimeric ATP synthases combining thermostable components with modules for specific functions. Test these constructs in extreme environments to validate functionality. Explore immobilization of engineered ATP synthases on synthetic surfaces for nanoscale energy conversion applications.

What structural features of T. tengcongensis atpE could inform the design of novel antimicrobials targeting ATP synthase?

ATP synthase is an emerging antimicrobial target, and T. tengcongensis atpE features could inform drug design:

• Structural differences: Identify unique features compared to human ATP synthase

• Binding pocket analysis: Characterize sites for selective inhibitor design

• Thermostability mechanisms: Exploit for developing drugs with broader stability profiles

• Cross-species conservation: Assess potential for broad-spectrum activity

Methodological approach: Perform detailed structural comparison between T. tengcongensis atpE and human ATP synthase subunit c. Identify unique binding pockets using computational cavity detection algorithms. Conduct virtual screening of compound libraries targeting these pockets. Validate hits through biochemical assays measuring ATP synthase activity in the presence of candidate compounds. Test promising compounds against clinically relevant pathogens to assess broad-spectrum potential.