ATP Synthase Mechanism: Used to study proton translocation kinetics and ATP synthesis coupling in thermophilic systems .
Inhibitor Screening: Serves as a target for developing antimicrobial agents, particularly against pathogens like Mycobacterium tuberculosis .
Thermozyme Engineering: The heat-stable nature of this subunit makes it valuable for industrial applications in high-temperature environments .
Structural Biology: Crystallography and cryo-EM studies leverage this protein to elucidate F-type ATP synthase dynamics .
Thermal Stability: While thermostable, repeated freeze-thaw cycles degrade activity, necessitating strict storage protocols .
Functional Specificity: Unlike human ATP synthase isoforms, this subunit lacks functional redundancy, making it a distinct model for bacterial ATP synthesis .
KEGG: ate:Athe_1429
STRING: 521460.Athe_1429
ATP synthase subunit c (atpE) from Anaerocellum thermophilum is a critical component of the F₀ sector of ATP synthase, functioning as part of the membrane rotor that facilitates proton/sodium translocation during ATP synthesis. Its significance stems from its role in an ancient ATP synthase that operates efficiently at low driving forces. This efficiency is particularly notable as the ATP synthases of many anaerobic archaea have an unusual motor subunit c that is otherwise primarily found in eukaryotic V₁V₀ ATPases . Studying this protein provides insights into the evolutionary adaptations of energy-producing systems in extremophiles and advances our understanding of bioenergetic mechanisms that function near the thermodynamic limit of ATP synthesis .
The recombinant full-length Anaerocellum thermophilum ATP synthase subunit c (atpE) is a 70-amino acid protein (1-70aa) with the sequence: MTALAAGIAMLAGLGVGIGIGIATGKASESIGRQPEAFGRIFPLFLIGAALAEAVAIYSLVIAFMLISKI . When expressed as a recombinant protein, it is typically fused to an N-terminal histidine tag to facilitate purification. The protein is hydrophobic in nature, consistent with its role as a membrane-embedded component of the ATP synthase complex. This hydrophobicity necessitates special handling procedures during protein expression, purification, and reconstitution experiments .
ATP synthase subunit c serves as the building block of the membrane rotor in the F₀ complex. Multiple copies of subunit c assemble to form a ring (c-ring) that rotates during ATP synthesis. This rotation is driven by the flow of ions (either H⁺ or Na⁺, depending on the organism) across the membrane through the a-c interface .
In the case of Anaerocellum thermophilum atpE, the protein participates in a Na⁺-driven ATP synthesis mechanism. When an electrochemical sodium gradient (ΔμNa⁺) is established across the membrane, the movement of Na⁺ through the a-c interface causes rotation of the c-ring, which is mechanically coupled to conformational changes in the F₁ sector that drive ATP synthesis . Remarkably, this system can synthesize ATP at physiologically relevant driving forces of 90 to 150 mV, demonstrating efficient energy conversion even at low electrochemical potential .
Anaerocellum thermophilum has been reclassified as Caldicellulosiruptor bescii . It is an extremely thermophilic, anaerobic bacterium capable of growing at temperatures up to 90°C. This organism belongs to the phylum Firmicutes and is known for its ability to degrade complex plant biomass, including crystalline cellulose. The ATP synthase of C. bescii has attracted research interest due to its adaptation to extreme conditions and its potential applications in bioenergy production.
To analyze ATP synthesis capability using recombinant atpE incorporated into proteoliposomes, researchers can apply an electrochemical gradient and measure ATP production. A detailed methodology involves:
Preparing proteoliposomes with reconstituted ATP synthase containing the atpE subunit
Creating a potassium diffusion potential (Δψ) by:
Incubating proteoliposomes with low internal K⁺ (0.5 mM) in buffer with high K⁺ (200 mM)
Adding valinomycin to allow K⁺ entry, generating an electrical field (positive inside, approximately 160 mV)
Establishing a Na⁺ concentration gradient:
Maintaining internal Na⁺ at 200 mM and external Na⁺ at 15 mM (creating a ΔpNa of 70 mV)
This creates a total ΔμNa⁺/F of 230 mV
Initiating the reaction by adding ADP
Measuring ATP synthesis rates (typically linear for about 2 minutes)
Control experiments should include:
Addition of protonophores like 3,3′,4′,5-tetrachlorosalicylanilide (TCS) to abolish Δψ
Use of Na⁺ ionophore ETH2120 to destroy the Na⁺ gradient
Using this approach, maximum ATP synthesis rates of approximately 99.2 nmol·min⁻¹·mg protein⁻¹ have been observed with similar ATP synthase systems .
Optimizing expression and purification of recombinant Anaerocellum thermophilum atpE requires addressing its hydrophobic nature and membrane-embedded characteristics:
Expression Optimization:
Use E. coli as the expression host (the protein has been successfully expressed in E. coli systems)
Consider codon optimization for the expression host
Employ tightly controlled promoter systems to manage potential toxicity
Optimize induction conditions (temperature, inducer concentration, duration)
Purification Strategy:
Store at -20°C/-80°C in appropriate buffer containing 6% trehalose at pH 8.0
For reconstitution, use deionized sterile water to achieve 0.1-1.0 mg/mL concentration
Add 5-50% glycerol (final concentration) for long-term storage
Quality Control:
Verify protein identity using mass spectrometry
Assess functional activity through reconstitution experiments
Fusion proteins involving atpE and other ATP synthase subunits, particularly subunit a, provide valuable insights into the assembly and function of the ATP synthase complex:
Orientation-Dependent Incorporation:
Functional Impact:
Assembly Mechanism Insights:
These findings demonstrate that carefully designed fusion proteins can be used to study the structural and functional aspects of ATP synthase assembly while maintaining enzymatic activity.
The translational efficiency of atpE is a critical factor when using this gene in heterologous expression systems:
Enhanced Translational Initiation:
Sequence Pattern for Enhancement:
The atpE RBS contains a specific sequence pattern that enhances translational initiation efficiency
This pattern includes a U-rich sequence followed by an interrupted A-rich sequence (UUUUAACUGAAACAAA)
The enhancing effect on translation yield is not due to changes in mRNA stability or transcription rate
Application in Fusion Proteins:
This translational enhancement capability makes the atpE RBS region a valuable tool for improving the expression of difficult-to-express proteins in E. coli.
For functional studies of recombinant atpE, proper reconstitution is essential:
Reconstitution Protocol:
Centrifuge the vial containing lyophilized protein briefly before opening to bring contents to the bottom
Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL
Add glycerol to a final concentration of 5-50% (optimal: 50%) and aliquot for long-term storage at -20°C/-80°C
For proteoliposome preparation:
Use purified phospholipids (typically E. coli lipids or synthetic mixtures)
Form liposomes through detergent removal methods
Incorporate the purified ATP synthase complex or subunit c
Control internal and external buffer compositions to enable gradient formation
Functional Verification:
Establish ion gradients as described in section 2.1
Monitor ATP synthesis using luminescence-based assays or coupled enzyme systems
Verify specificity through inhibitor studies
Avoiding Common Pitfalls:
Prevent repeated freeze-thaw cycles that may denature the protein
Ensure complete detergent removal during reconstitution to prevent artifacts
Control for passive proton/sodium leakage in experimental designs
Understanding the ion specificity of Anaerocellum thermophilum ATP synthase requires carefully designed experiments:
Experimental Approaches:
Ion Gradient Manipulation:
Compare ATP synthesis rates driven by Na⁺ vs. H⁺ gradients
Test mixed gradients to assess relative contributions
Systematically vary individual gradient components (Δψ vs. ΔpH or ΔpNa)
Site-Directed Mutagenesis:
Target conserved residues in atpE involved in ion binding
Create mutations that alter ion selectivity
Analyze effects on ATP synthesis rates and ion dependency
Ion Competition Studies:
Perform experiments with varying ratios of Na⁺ and H⁺
Determine whether one ion can substitute for the other
Calculate apparent Km values for different ions
Data Analysis Framework:
Measure initial ATP synthesis rates at different ion concentrations
Plot rate vs. ion concentration to determine kinetic parameters
Create kinetic models to describe ion binding and translocation
Experimental Condition | Na⁺ Gradient | H⁺ Gradient | Δψ (mV) | ATP Synthesis Rate |
---|---|---|---|---|
Na⁺-driven synthesis | Present | Absent | 160 | High |
H⁺-driven synthesis | Absent | Present | 160 | Low/None |
Combined gradients | Present | Present | 160 | High |
No gradient (control) | Absent | Absent | 0 | None |
This systematic approach would determine whether the ATP synthase is exclusively Na⁺-dependent or can also utilize H⁺ under certain conditions.
When working with recombinant ATP synthase components, including atpE from Anaerocellum thermophilum, several biosafety considerations should be addressed:
Risk Assessment:
Recombinant DNA Safety:
Laboratory Containment:
Follow institutional biosafety guidelines for recombinant DNA work
Implement appropriate physical containment measures (typically Biosafety Level 1 or 2)
Experimental Design Safeguards:
Regulatory Compliance:
Obtain approval from institutional biosafety committees
Maintain proper documentation of risk assessments and safety measures
Ensure proper training of personnel handling recombinant materials
These biosafety considerations evolved from historical guidelines established following the Asilomar Conference on Recombinant DNA , though modern regulations have been updated based on decades of experience with recombinant DNA technology.
Several analytical techniques are particularly effective for studying the structure and interactions of atpE:
Structural Analysis:
X-ray Crystallography:
Provides high-resolution structural information
Challenging due to the hydrophobic nature of atpE
May require crystallization in lipidic environments
Cryo-Electron Microscopy:
Increasingly used for membrane protein complexes
Can reveal the arrangement of atpE in the c-ring and interactions with other subunits
Does not require crystallization
NMR Spectroscopy:
Suitable for studying dynamics and interactions in solution
Limited by the size of the protein complex
Solid-state NMR can be applied to membrane-embedded atpE
Interaction Studies:
Cross-linking coupled with Mass Spectrometry:
Identifies interaction interfaces between atpE and other subunits
Chemical cross-linkers can capture transient interactions
MS analysis identifies the cross-linked residues
Förster Resonance Energy Transfer (FRET):
Monitors real-time conformational changes
Requires strategic labeling of the protein with fluorophores
Can be used in reconstituted systems
Molecular Fusion Approaches:
Functional Analysis:
Patch-clamp Electrophysiology:
Measures ion conductance through reconstituted ATP synthase
Can determine ion selectivity and gating properties
Surface Plasmon Resonance:
Quantifies binding affinities between atpE and other components
Provides kinetic data on association and dissociation
These techniques, used in combination, provide comprehensive understanding of atpE structure and function within the ATP synthase complex.