Recombinant Anaerocellum thermophilum ATP synthase subunit c (atpE)

What is Anaerocellum thermophilum ATP synthase subunit c (atpE) and why is it significant for research?

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 .

What are the structural characteristics of recombinant Anaerocellum thermophilum atpE?

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 .

How does atpE function within the ATP synthase complex?

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 .

What is known about the taxonomic classification of Anaerocellum thermophilum?

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.

How can functional studies of ATP synthesis be conducted using recombinant atpE in proteoliposomes?

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

• Omission of ADP

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 .

What strategies can be employed to optimize expression and purification of recombinant atpE protein?

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:

• Utilize the N-terminal His-tag for affinity chromatography

• Lyophilize the purified protein to increase stability

• 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:

• Confirm purity using SDS-PAGE (should be >90%)

• Verify protein identity using mass spectrometry

• Assess functional activity through reconstitution experiments

How do fusion proteins of atpE with other ATP synthase subunits affect assembly and function?

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:

  • Fusion proteins with correct orientation of transmembrane helices can be successfully inserted into the membrane and incorporated into the F₀ complex

  • The orientation of the helical hairpin of subunit c is critical for proper assembly

• Functional Impact:

  • When fused c subunits have correct orientation, they can be incorporated into the c-ring, but may tether the ATP synthase rotor to the stator

  • Fusions with incorrect orientation of c-helices remain on the periphery of the c-ring and do not interfere with rotor movement

• Assembly Mechanism Insights:

  • Interaction with monomeric subunit c triggers insertion of subunit a into the membrane

  • This interaction initiates formation of the a-c complex, which functions as the ion-translocating module of ATP synthase

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.

What role does the translational efficiency of atpE play in heterologous expression systems?

The translational efficiency of atpE is a critical factor when using this gene in heterologous expression systems:

• Enhanced Translational Initiation:

  • The ribosome binding site (RBS) upstream of the E. coli atpE gene promotes highly efficient translational initiation

  • A segment of 30 bp or less upstream from the atpE Shine-Dalgarno (SD) sequence plays a primary role in determining high translation levels

• 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:

  • When fused with heterologous genes (such as human IL2 and IFNβ), the atpE translational initiation region significantly increases protein expression

  • The maximal levels of synthesis achieved using the atpE sequence are at least as high as those previously reported for these genes in E. coli

This translational enhancement capability makes the atpE RBS region a valuable tool for improving the expression of difficult-to-express proteins in E. coli.

What reconstitution methods are recommended for functional studies of atpE?

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

• Store working aliquots at 4°C for no more than one week

• Ensure complete detergent removal during reconstitution to prevent artifacts

• Control for passive proton/sodium leakage in experimental designs

How can researchers design experiments to study the ion specificity of Anaerocellum thermophilum ATP synthase?

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

This systematic approach would determine whether the ATP synthase is exclusively Na⁺-dependent or can also utilize H⁺ under certain conditions.

What biosafety considerations should be addressed when working with recombinant ATP synthase components?

When working with recombinant ATP synthase components, including atpE from Anaerocellum thermophilum, several biosafety considerations should be addressed:

Risk Assessment:

• Recombinant DNA Safety:

  • The experiments involving recombinant ATP synthase components generally fall into the moderate risk category according to historical guidelines

  • Consider potential for gene transfer to other organisms

• Laboratory Containment:

  • Follow institutional biosafety guidelines for recombinant DNA work

  • Implement appropriate physical containment measures (typically Biosafety Level 1 or 2)

• Experimental Design Safeguards:

  • Use well-characterized laboratory strains of E. coli as hosts

  • Employ biological containment through the use of attenuated strains

  • Avoid creating constructs that might confer antibiotic resistance transfer to pathogenic strains

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.

What analytical techniques are most effective for studying atpE structure and interactions?

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:

  • Generation of fusion proteins between atpE and other subunits

  • Analyze functional consequences of tethering subunits

  • Provides insights into assembly mechanisms

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.