Recombinant Natranaerobius thermophilus ATP synthase subunit c (atpE)

What is Natranaerobius thermophilus ATP synthase subunit c (atpE) and what are its key characteristics?

Natranaerobius thermophilus ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex found in this extremophilic bacterium. The full-length protein consists of 86 amino acids with the sequence: MIDGQSLVLAASAIGAGLAMIAGIGAGIGQGFAAGKGAESVGRQPDAQGDIIRTMLLGAAVAETTGIYALVIALLLLFANPLIGML . This protein functions as part of the F0 sector of ATP synthase, forming the membrane-embedded proton channel. Unlike typical ATP synthases that synthesize ATP, the Natranaerobius thermophilus enzyme primarily catalyzes ATP hydrolysis to expel cytoplasmic Na+ ions, helping the organism avoid Na+ toxicity in its extreme habitat . The protein is notable for its stability under extreme conditions, making it valuable for structural and functional studies of ATP synthases from extremophiles.

How does the function of Natranaerobius thermophilus ATP synthase differ from conventional ATP synthases?

Interestingly, the ATP synthase from Natranaerobius thermophilus represents a biological adaptation to extreme environmental conditions. While conventional ATP synthases primarily function in ATP synthesis by utilizing proton gradients across membranes, the Natranaerobius thermophilus enzyme operates predominantly in reverse, catalyzing ATP hydrolysis to expel cytoplasmic Na+ ions . This reversed functionality plays a crucial role in maintaining ion homeostasis within the cell, preventing Na+ toxicity that would otherwise be detrimental to cellular processes. This functional adaptation contrasts with other bacterial ATP synthases (such as those in H. modesticaldum) that maintain traditional ATP synthesis roles. The structural modifications that enable this specialized function provide valuable insights into the evolutionary adaptability of essential enzyme complexes across diverse bacterial species in extreme environments.

What expression systems are most effective for producing recombinant Natranaerobius thermophilus atpE protein?

E. coli expression systems have demonstrated high efficiency for the recombinant production of Natranaerobius thermophilus ATP synthase subunit c (atpE) . When designing expression protocols, researchers should optimize codon usage for E. coli while preserving the native protein sequence. The recombinant protein is typically expressed with affinity tags (such as His-tags) to facilitate purification through affinity chromatography. After induction (commonly with IPTG for T7-based expression systems), cells should be cultured under controlled conditions (typically 37°C initially, followed by expression at 16-25°C to enhance protein folding). Lysis should be performed with appropriate buffers containing mild detergents to extract the membrane-associated protein without denaturation. Purification using nickel affinity chromatography followed by size exclusion chromatography produces the highest purity samples. For functional studies, careful detergent selection is critical as harsh detergents can denature the protein and disrupt its native conformation and activity.

What experimental approaches can resolve the structural determinants of atpE's ion specificity in Natranaerobius thermophilus?

Investigating the structural determinants of ion specificity in Natranaerobius thermophilus atpE requires a multi-faceted experimental approach. Site-directed mutagenesis targeting conserved residues in the transmembrane regions can identify amino acids critical for Na+ binding and translocation. Researchers should focus particularly on residues that differ between Na+-specific and H+-specific ATP synthases. A systematic approach would involve:

• Creating a library of point mutations in key residues predicted to form the ion-binding pocket

• Expressing and purifying these mutants in E. coli expression systems with His-tags for efficient purification

• Reconstituting the purified proteins into liposomes for functional assays

• Measuring ion transport rates using radiolabeled ions or fluorescent probes

• Conducting electrophysiological studies (patch-clamp) on reconstituted proteins

For structural determination, cryo-electron microscopy is particularly effective for membrane proteins like atpE, providing high-resolution structures in near-native environments. X-ray crystallography of the c-ring, though challenging, can provide atomic-level resolution of the ion-binding sites. Molecular dynamics simulations using these structural data can further elucidate the ion translocation pathway and energetics, providing insights into the ion specificity mechanisms that cannot be directly observed experimentally.

How does the ATP hydrolysis activity of Natranaerobius thermophilus ATP synthase compare to ATP synthesis capabilities under varying environmental conditions?

The ATP synthase from Natranaerobius thermophilus exhibits a strong preference for ATP hydrolysis over synthesis, contrasting with typical F-type ATP synthases . To investigate this functional bias, researchers should employ a comparative biochemical approach:

• Prepare purified ATP synthase complexes from both Natranaerobius thermophilus and a conventional ATP synthase (e.g., from E. coli) for direct comparison

• Measure ATP synthesis rates using luciferin/luciferase assays under various conditions:

  • pH gradients (pH 6.0-10.0, reflecting the alkaliphilic nature of N. thermophilus)

  • Temperature ranges (30-65°C, covering mesophilic to thermophilic conditions)

  • Salt concentrations (0-2M NaCl, mimicking halophilic environments)

  • Na+/H+ ratios to determine coupling ion preferences

• Assess ATP hydrolysis rates through phosphate release assays under the same conditions

The bidirectionality of the enzyme can be quantified by calculating the ratio of ATP synthesis to hydrolysis rates under identical conditions. This experimental design should include thermodynamic analyses to determine if the preference for hydrolysis is due to structural adaptations in the catalytic sites or altered coupling between the F1 and F0 domains. Importantly, reconstitution of the ATP synthase into liposomes with controlled internal and external environments allows for precise manipulation of electrochemical gradients to determine the minimum gradient required for ATP synthesis versus the threshold for ATP hydrolysis.

What methodologies can effectively distinguish between the functional roles of atpE in membrane organization versus its catalytic contributions to ATP synthase activity?

Distinguishing between the structural and catalytic roles of atpE requires sophisticated biochemical and biophysical approaches. Researchers should employ differential functional reconstitution experiments:

• Prepare three experimental systems:

  • Complete ATP synthase complexes containing native atpE

  • Complexes with catalytically inactive atpE mutants (identified through site-directed mutagenesis)

  • Artificial membranes containing only atpE (no other ATP synthase components)

• Analyze membrane properties using:

  • Fluorescence anisotropy to measure membrane fluidity

  • Differential scanning calorimetry to assess phase transition temperatures

  • Atomic force microscopy to visualize membrane organization

  • Electron paramagnetic resonance spectroscopy with spin-labeled lipids to measure ordering effects

• Evaluate catalytic function through:

  • ATP hydrolysis assays with isolated F1 portions in the presence of different c-subunit variants

  • Proton/sodium pumping assays using pH-sensitive or Na+-sensitive fluorescent dyes

Cross-linking studies between atpE and other subunits can map interaction surfaces involved in either structural organization or catalytic coupling. Additionally, reconstitution of hybrid ATP synthases, containing atpE from Natranaerobius thermophilus and remaining components from conventional ATP synthases, can reveal which functional properties are directly attributable to atpE. This comprehensive approach will elucidate whether atpE primarily contributes to membrane organization, ion translocation, catalytic regulation, or a combination of these functions.

What purification protocols maximize both yield and activity of recombinant Natranaerobius thermophilus atpE?

Optimizing purification of recombinant Natranaerobius thermophilus atpE requires balancing yield with preserved activity. Based on established protocols, a comprehensive purification strategy should include:

• Initial Expression Optimization:

  • Express in E. coli BL21(DE3) or C41/C43 strains specially designed for membrane proteins

  • Induce with 0.1-0.5 mM IPTG at OD600 0.6-0.8

  • Shift to lower temperature (18-25°C) post-induction

  • Harvest cells after 16-20 hours expression

• Membrane Fraction Isolation:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, protease inhibitors

  • Perform differential centrifugation (10,000g then 100,000g) to isolate membrane fractions

  • Solubilize membranes with gentle detergents (0.5-1% n-dodecyl-β-D-maltoside or digitonin)

• Two-Stage Chromatography:

  • Immobilized metal affinity chromatography using Ni-NTA resin with imidazole gradient elution

  • Size exclusion chromatography for high purity using Superdex 200 column

• Activity Preservation:

  • Maintain 6% trehalose as a stabilizing agent in all buffers

  • Avoid freeze-thaw cycles and store at 4°C for short-term use

  • For long-term storage, add 30-50% glycerol and store at -80°C in small aliquots

This protocol typically yields >90% pure protein as confirmed by SDS-PAGE , with preserved structural integrity and functional activity. For functional assays, the protein should be reconstituted into liposomes composed of E. coli polar lipids or synthetic lipid mixtures that mimic the native membrane environment of Natranaerobius thermophilus.

What experimental design best elucidates the evolutionary adaptations of Natranaerobius thermophilus atpE compared to mesophilic homologs?

To systematically investigate the evolutionary adaptations of Natranaerobius thermophilus atpE, a comprehensive experimental design should incorporate both comparative sequence analysis and functional characterization:

• Phylogenetic Analysis Framework:

  • Construct a multi-sequence alignment of atpE homologs from diverse bacterial species

  • Select representatives spanning extremophiles (thermophiles, alkaliphiles, halophiles) and mesophiles

  • Generate phylogenetic trees using maximum likelihood methods

  • Identify conserved residues across all species versus those unique to extremophiles

• Structural Stability Assessment:

  • Express recombinant atpE from Natranaerobius thermophilus and selected mesophilic species

  • Subject proteins to thermal shift assays across temperature ranges (25-80°C)

  • Perform circular dichroism spectroscopy to assess secondary structure stability

  • Evaluate resistance to denaturants (urea, guanidine HCl) through activity retention assays

• Functional Characterization:

  • Reconstitute purified proteins into liposomes

  • Measure ion translocation rates at varying pH (6.0-10.0) and temperatures (30-70°C)

  • Assess ATP hydrolysis coupling efficiency under different conditions

  • Perform electrophysiological recordings to determine ion selectivity and gating properties

• Domain Swapping Experiments:

  • Create chimeric proteins with domains exchanged between Natranaerobius thermophilus atpE and mesophilic homologs

  • Evaluate which domains confer extremophilic properties (temperature stability, alkaline pH optimum)

  • Use site-directed mutagenesis to confirm the role of specific amino acid substitutions

This experimental design follows the principles of effective research by systematically varying conditions while controlling for extraneous variables . The combination of sequence analysis, structural characterization, and functional assays provides a comprehensive framework for understanding the molecular basis of evolutionary adaptations in atpE for extreme environments.

What structural features of Natranaerobius thermophilus atpE contribute to its function in extreme environments?

Natranaerobius thermophilus atpE exhibits specific structural adaptations that enable functionality in extreme conditions. The protein's 86-amino acid sequence (MIDGQSLVLAASAIGAGLAMIAGIGAGIGQGFAAGKGAESVGRQPDAQGDIIRTMLLGAAVAETTGIYALVIALLLLFANPLIGML) reveals several key features:

• Membrane Integration Domains:

  • High proportion of hydrophobic residues (A, I, L, V) forming transmembrane helices

  • Glycine-rich regions (particularly GIGAGIGQ) providing conformational flexibility within the membrane environment

  • Strategic positioning of aromatic residues (F) at membrane interfaces for anchoring

• Ion Coordination Sites:

  • Conserved polar residues within transmembrane regions for ion binding

  • Specialized ion-binding pocket architecture favoring Na+ over H+ coordination

  • Unique spacing of ion-binding residues optimized for the alkaliphilic environment

• Stability-Enhancing Elements:

  • Increased alanine content providing thermostability through efficient packing

  • Reduced frequency of thermolabile residues (C, M, Q, N) compared to mesophilic homologs

  • Enhanced hydrophobic core interactions stabilizing tertiary structure

These structural features collectively contribute to the protein's capacity to maintain functional integrity in the extreme halophilic, thermophilic, and alkaliphilic conditions of Natranaerobius thermophilus's native environment. The unique arrangement of transmembrane helices creates an atpE c-ring that optimally functions in ATP hydrolysis rather than synthesis, facilitating Na+ expulsion to maintain cellular homeostasis under high salt conditions . X-ray crystallography and cryo-electron microscopy studies of similar ATP synthase c-subunits suggest that these structural adaptations directly influence the thermodynamic equilibrium of the enzyme, favoring ATP hydrolysis over synthesis in Natranaerobius thermophilus.

How do post-translational modifications affect the functionality of recombinant Natranaerobius thermophilus atpE compared to native protein?

Post-translational modifications (PTMs) can significantly impact the functionality of recombinant Natranaerobius thermophilus atpE compared to its native counterpart. A systematic analysis reveals several considerations:

• Potential PTMs in Native vs. Recombinant Systems:

  • N-terminal processing: Native proteins often undergo N-terminal methionine excision, while recombinant systems may retain this residue

  • Lipid modifications: Native membrane proteins may undergo lipidation, which is often absent in recombinant expression

  • Glycosylation patterns: E. coli lacks the machinery for complex glycosylation present in some extremophiles

• Functional Consequences:

  • Membrane integration: Absence of native lipid modifications in recombinant atpE may affect proper membrane insertion and orientation

  • Oligomerization: PTMs can influence c-ring assembly, potentially altering the stoichiometry and ion conductance properties

  • Protein-protein interactions: Modified interaction surfaces may affect coupling with other ATP synthase subunits

• Experimental Assessment Methods:

  • Mass spectrometry to identify and compare PTMs between native and recombinant proteins

  • Site-directed mutagenesis to mimic or prevent specific modifications

  • Activity assays comparing native (isolated from Natranaerobius thermophilus) versus recombinant protein

To address these differences, researchers can employ advanced expression systems with engineered post-translational modification capabilities or perform in vitro modifications of purified recombinant proteins. When expressed in E. coli, the recombinant protein maintains high purity (>90% by SDS-PAGE) but may lack specific modifications that influence native function. This underscores the importance of functional validation when using recombinant systems for mechanistic studies of atpE and highlights potential limitations in directly extrapolating findings to the native protein behavior.

What statistical approaches best quantify structural differences between Natranaerobius thermophilus atpE and related proteins from non-extremophiles?

Quantitative analysis of structural differences between Natranaerobius thermophilus atpE and non-extremophile homologs requires sophisticated statistical methodologies:

• Sequence-Based Statistical Approaches:

  • Position-specific scoring matrices to identify conservation patterns

  • Information entropy analysis at each amino acid position

  • Z-score analysis of amino acid composition biases

  • Multiple sequence alignment followed by principal component analysis to identify clustering of extremophile vs. mesophile sequences

• Structural Comparison Methodologies:

  • Root mean square deviation (RMSD) calculations from superimposed structures

  • Distance matrix analysis to identify regions of structural divergence

  • Statistical analysis of secondary structure content (α-helix, β-sheet percentages)

  • Solvent accessibility surface area (SASA) comparison for exposed vs. buried residues

• Molecular Dynamics-Based Analysis:

  • Comparative flexibility analysis using B-factor analysis or root mean square fluctuation (RMSF)

  • Principal component analysis of conformational sampling

  • Free energy landscape comparison to identify stability differences

  • Statistical analysis of hydrogen bond and salt bridge networks

• Bayesian Statistical Framework:

  • Bayesian model comparison to identify which structural features best predict extremophile adaptation

  • Machine learning approaches (Random Forest, Support Vector Machines) to classify structural features associated with extremophilic properties

When applying these methods, researchers must account for phylogenetic relationships to distinguish adaptation from ancestry and implement appropriate multiple testing corrections for statistical significance. This multi-parameter statistical approach provides a robust framework for quantifying the structural adaptations that enable Natranaerobius thermophilus atpE to function in extreme environments, revealing insights beyond what qualitative structural comparisons alone can provide.

How do experimental results from recombinant Natranaerobius thermophilus atpE models translate to in vivo function predictions?

Translating experimental findings from recombinant Natranaerobius thermophilus atpE studies to in vivo predictions requires careful consideration of biological context:

• Reconstitution System Limitations:

  • In vitro systems lack the complex lipid composition of native membranes

  • Recombinant proteins expressed in E. coli may have different post-translational modifications

  • The stoichiometry of ATP synthase components may differ between reconstituted and native systems

• Validation Approaches:

  • Comparative activity measurements between purified native complexes and reconstituted recombinant systems

  • Live-cell imaging using fluorescently tagged ATP synthase components to track localization and dynamics

  • Mutagenesis studies in both systems to verify structure-function relationships

  • Correlation analysis between in vitro measured parameters and in vivo phenotypes

• Predictive Modeling Framework:

  • Develop mathematical models incorporating experimentally determined parameters (ion binding affinities, catalytic rates)

  • Simulate cellular energetics under varying environmental conditions

  • Conduct sensitivity analysis to identify which parameters most strongly influence in vivo function

  • Validate model predictions through targeted in vivo experiments

Key Properties of Natranaerobius thermophilus ATP Synthase Subunit c (atpE)