Recombinant Escherichia coli ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in bacterial energy metabolism?

ATP synthase subunit c is an essential component of the membrane-embedded F₀ domain of ATP synthase that participates in transmembrane proton conduction. It forms a ring structure (c-ring) that rotates during proton translocation across the membrane, which drives the synthesis of ATP in the F₁ domain. The c-subunit is critical for coupling proton movement to the mechanical rotation that powers ATP synthesis in E. coli and other organisms .

How is the c-subunit organized in the ATP synthase complex?

The c-subunit assembles into a ring structure within the membrane domain of ATP synthase. In E. coli, these c-rings typically consist of 8-10 identical c-subunits arranged in a circular formation. This annular architecture has been documented across different species, though the exact number of c-subunits varies (between 8-17) depending on the organism . The assembled c-ring directly interacts with other membrane components of the ATP synthase complex, particularly the a-subunit, which forms the proton channel .

How does the primary structure of subunit c influence its assembly?

The primary amino acid sequence of subunit c is the key determinant for its self-assembly into ring structures. Research has demonstrated that subunit c can self-assemble into annular structures even in the absence of other ATP synthase components when purified in non-ionic detergent solutions . This inherent self-assembly property suggests that the specific amino acid sequence contains all the information necessary for proper oligomerization, including the stoichiometry of the resulting c-rings .

What are the recommended expression systems for E. coli ATP synthase subunit c?

For recombinant production of E. coli ATP synthase subunit c, the pET expression system is most commonly used due to its high-level protein expression capability. This system employs T7 RNA polymerase under the control of the lac operator, allowing for IPTG-inducible expression. For optimal results, lower IPTG concentrations (<0.1 mM) may reduce toxicity effects while maintaining reasonable protein yields .

When designing your expression construct, consider the following parameters:

• Strong, inducible promoter (T7 is standard)

• Appropriate fusion tags (His-tag at C-terminus works well)

• Codon optimization if yield is insufficient

• Temperature reduction post-induction (to 25-30°C)

What purification strategies yield the most homogeneous c-subunit preparations?

Table 1: Comparison of Purification Methods for ATP Synthase Subunit c

For high-purity c-subunit preparations, a multi-step approach is recommended. The protein can be first extracted from membranes using detergents like DDM or Triton X-100, followed by affinity purification using a C-terminal His-tag . Further purification by size exclusion chromatography helps separate different oligomeric states. Silver staining of SDS-PAGE gels can confirm purity, while clear native PAGE can validate the oligomeric assembly state of the c-ring .

How can oligomeric integrity of c-rings be preserved during purification?

Maintaining the integrity of c-rings during purification requires careful attention to detergent selection and concentration. Non-ionic detergents like DDM (n-dodecyl-β-D-maltoside) at concentrations just above CMC are preferred for extraction and purification to preserve native c-ring structures . Avoid harsh detergents like SDS during purification steps, as these cause partial to complete disassembly of higher-order oligomeric states .

Critical factors for preserving c-ring integrity include:

• Use non-ionic detergents throughout purification

• Maintain detergent above CMC but not excessively high

• Avoid freeze-thaw cycles of the purified protein

• Include lipids during purification to stabilize the complex

• Keep pH within physiological range (6.5-8.0)

What methods can determine the oligomeric state of recombinant c-rings?

Multiple complementary approaches can determine the oligomeric state of purified c-rings:

• Clear native PAGE: Identifies higher-order structures without denaturation. Human c-subunit has been detected at ~250 kDa, suggesting tetramers of octameric rings .

• Size exclusion chromatography: Separates c-rings based on their hydrodynamic radius, allowing estimation of the oligomeric state.

• Cross-linking followed by SDS-PAGE: Chemical cross-linkers stabilize interactions between adjacent subunits before analysis.

• Analytical ultracentrifugation: Provides information about the molecular weight and shape of the complex in solution.

• Cryo-EM or X-ray crystallography: Offers direct visualization of the ring structure and precise subunit counting.

How does one evaluate the functional integrity of purified c-rings?

Functional assessment of purified c-rings involves several complementary approaches:

• Channel activity measurement: Reconstitution into planar lipid membranes or liposomes allows measurement of ion conductance. Functional c-rings form large multi-conductance, voltage-gated channels with conductance of approximately 1.5 nS .

• ATP sensitivity testing: Channel activity of properly assembled c-rings shows sensitivity to adenine nucleotides, with different binding affinities depending on the preparation method .

• Inhibitor binding assays: Specific inhibitors like oligomycin or DCCD can be used to confirm proper folding and functional state.

• Proton translocation assays: Using pH-sensitive fluorescent dyes in liposome reconstitution systems.

How can recombinant c-subunits be used to investigate the mechanism of proton translocation?

Recombinant c-subunits provide a valuable platform for investigating proton translocation mechanisms:

• Site-directed mutagenesis of key residues (particularly the conserved carboxylate residue in the middle of the second transmembrane helix) can identify amino acids essential for proton binding and transfer.

• Introduction of non-canonical amino acids at specific positions can provide unique biophysical probes for monitoring conformational changes during proton translocation.

• Reconstitution of purified c-rings with different lipid compositions can reveal how membrane environment influences proton conduction.

• Single-molecule FRET experiments using labeled c-subunits can track rotational movements during proton flow.

• Combination with a-subunit in reconstituted systems enables study of the complete proton pathway.

What is known about the dual role of ATP synthase c-ring in energy production and cell death?

The ATP synthase c-ring serves dual functions in cellular physiology:

• In normal conditions, it participates in energy production through ATP synthesis by coupling proton movement to F₁ rotation.

• Under stress conditions (particularly calcium overload), the c-ring can form a leak channel known as the ATP synthase c-subunit leak channel (ACLC) that contributes to mitochondrial permeability transition (mPT) .

This leak channel has the following characteristics:

• Large conductance (~1.5 nS)

• Voltage-gated properties

• Sensitivity to adenine nucleotides

• Inhibition by the ATP synthase F₁ subcomplex

The c-ring channel likely plays a significant role in excitotoxic neuronal death, as knocking down c-subunit prevents osmotic changes in response to high calcium and eliminates the large conductance, Ca²⁺ and CsA-sensitive channel activity of mPT .

How do post-translational modifications affect c-ring assembly and function?

Although research on post-translational modifications (PTMs) of bacterial ATP synthase c-subunits is limited, several modifications may influence assembly and function:

• Disulfide bond formation between adjacent c-subunits can stabilize the ring structure under oxidative conditions.

• Phosphorylation of specific residues may modulate protein-protein interactions within the ATP synthase complex.

• Lipid modifications could influence membrane integration and stability of the c-ring.

For eukaryotic systems, PTMs are better characterized and include:

• O-methylation of glutamate residues involved in proton binding

• Phosphorylation at multiple sites

• Oxidative modifications affecting channel properties

Why does expression of recombinant c-subunit sometimes result in low yields or toxicity?

Several factors may contribute to low yields or toxicity when expressing recombinant ATP synthase subunit c:

• Membrane protein overexpression burden: As a highly hydrophobic membrane protein, excessive production can overwhelm the membrane insertion machinery and trigger stress responses .

• T7 RNA polymerase toxicity: High levels of T7 RNA polymerase activity can lead to excessive mRNA production that outcompetes endogenous mRNA, impairing synthesis of essential cellular proteins .

• Protein aggregation: Improper folding or insufficient membrane integration can lead to toxic aggregates.

Solutions include:

• Reducing inducer (IPTG) concentration to <0.1 mM

• Lowering culture temperature after induction

• Using strains with regulated expression of T7 RNA polymerase

• Including membrane-stabilizing components in growth media

• Co-expressing chaperones that assist membrane protein folding

What strategies can prevent c-ring disassembly during purification and analysis?

To maintain c-ring integrity throughout purification and analysis:

• Detergent selection is critical - use mild non-ionic detergents (DDM, LMNG) at minimal effective concentrations.

• Include stabilizing lipids (particularly cardiolipin) in purification buffers.

• Avoid exposure to SDS or other harsh detergents that cause disassembly.

• For SDS-PAGE analysis, samples should not be boiled unless complete denaturation is desired.

• Consider amphipol or nanodisc reconstitution for long-term stability studies.

• For structural analysis, cross-linking agents can be employed to stabilize subunit interactions before exposure to potentially disruptive conditions.

How can researchers distinguish between monomeric and oligomeric forms of the c-subunit?

Table 2: Analytical Methods for Distinguishing c-Subunit Oligomeric States

When using SDS-PAGE, partial to complete disassembly of higher-order oligomeric states can be observed in the presence of SDS, with bands appearing at ~66 kDa and ~8 kDa positions . In contrast, clear native PAGE without detergent denaturation shows intact c-rings at approximately 250 kDa, representing tetramers of octameric rings .

How can synthetic biology approaches modify c-ring stoichiometry for biotechnological applications?

The stoichiometry of c-rings (number of c-subunits per ring) varies naturally between species and is determined primarily by the amino acid sequence of the protein . Researchers can exploit this property through:

• Rational design of the c-subunit interface: Modifying key residues at the subunit-subunit interface can alter the preferred ring size.

• Chimeric protein engineering: Creating fusion proteins with segments from species with different native stoichiometries.

• Directed evolution approaches: Selecting for variants with altered ring sizes through appropriate screening methods.

• Integration of non-canonical amino acids: Introducing novel chemistries at the interface to enforce different geometries.

Potential applications include:

• Creating ATP synthases with altered H⁺/ATP ratios for synthetic biology circuits

• Developing nanopores with defined conductance properties for sensing applications

• Engineering proton-selective channels with tunable gating properties

What is the relationship between c-ring assembly and bacterial adaptation to different energy environments?

The assembly characteristics of ATP synthase c-rings reflect evolutionary adaptations to different energetic constraints:

• The c-ring stoichiometry directly affects the H⁺/ATP ratio, with larger rings requiring more protons per ATP synthesized, influencing energy efficiency.

• Bacteria from different environments show variations in c-ring size that correlate with their typical proton motive force magnitudes.

• Expression levels and assembly efficiency may be regulated in response to energy availability.

Future research directions include:

• Comparative studies of c-ring assembly across extremophiles

• Investigation of regulatory mechanisms that might modulate c-ring composition

• Analysis of how c-ring variants confer selective advantages in specific ecological niches

• Examining whether c-ring composition changes in response to environmental stressors

How might modern structural biology techniques advance our understanding of c-ring dynamics?

Recent advances in structural biology offer unprecedented opportunities to study c-ring dynamics:

• Time-resolved cryo-EM can potentially capture different conformational states during rotation.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map dynamic regions and conformational changes.

• Molecular dynamics simulations based on atomic structures can model proton movement and ring rotation.

• Single-molecule FRET approaches can track rotational dynamics in real-time.

• Microfluidic approaches combined with structural methods can assess c-ring behavior under different energetic states.

• Native mass spectrometry can reveal the assembly pathways and intermediate states during c-ring formation.

These techniques could resolve longstanding questions about proton binding, release, and the coupling mechanism between proton flow and mechanical rotation of the c-ring during ATP synthesis.