Recombinant Escherichia coli O45:K1 ATP synthase subunit c (atpE)

What is the complete amino acid sequence of E. coli O45:K1 ATP synthase subunit c?

The full-length E. coli O45:K1 ATP synthase subunit c (atpE) protein consists of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA. This hydrophobic protein has several transmembrane domains that are crucial for its functional assembly in the ATP synthase complex . The high proportion of hydrophobic residues explains its insertion and stability within the membrane environment, which is essential for the rotational mechanism of ATP synthase.

What is the functional significance of atpE in the ATP synthase complex?

The atpE gene encodes the c-subunit of the F0 sector of ATP synthase, which forms an oligomeric ring in the membrane. This c-ring plays a crucial role in the rotational mechanism that couples proton translocation across the membrane to ATP synthesis. During ATP synthesis, protons move through the interface between the a-subunit and c-ring, causing the c-ring to rotate. This rotation is mechanically coupled to conformational changes in the F1 sector, driving ATP synthesis . Importantly, studies with Bacillus PS3 ATP synthase have demonstrated that mutations in the c-subunit significantly impair both ATP synthesis and proton pump activities, confirming its essential role in energy conversion.

What are the optimal conditions for recombinant expression of E. coli O45:K1 atpE?

For optimal expression of recombinant E. coli O45:K1 atpE, a heterologous E. coli expression system with an N-terminal His-tag is commonly employed . The following protocol has been established:

• Clone the atpE gene (coding for residues 1-79) into an expression vector with an N-terminal His-tag

• Transform into an E. coli expression strain (BL21(DE3) or equivalent)

• Grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5-1.0 mM IPTG

• Continue incubation at a reduced temperature (16-25°C) for 4-16 hours to enhance proper folding

• Harvest cells by centrifugation and proceed to lysis and purification

This approach typically yields sufficient protein for biochemical and structural studies, with expression levels influenced by media composition, induction timing, and post-induction temperature.

What purification strategies are most effective for recombinant atpE protein?

Purification of recombinant atpE requires specialized approaches due to its hydrophobic nature. An effective purification strategy involves:

• Cell lysis in buffer containing mild detergents (e.g., n-dodecyl-β-D-maltopyranoside or LDAO)

• Initial purification using Ni-NTA affinity chromatography, exploiting the N-terminal His-tag

• Gradient elution with imidazole (50-300 mM)

• Further purification via size-exclusion chromatography to isolate properly folded protein

• Concentration and storage in buffer containing stabilizing agents

For reconstitution studies, the protein can be solubilized in 8M urea and directly diluted into buffer containing ethanol and glycerol, similar to methods established for spinach chloroplast epsilon subunit . This approach maintains biological activity comparable to protein purified from native sources.

How can proper folding of recombinant atpE be verified?

Verification of proper folding for recombinant atpE can be assessed through multiple complementary approaches:

• SDS-PAGE analysis showing >90% purity with expected molecular weight

• Circular dichroism spectroscopy to confirm secondary structure content

• Functional assays measuring ability to inhibit ATPase activity when incorporated into ATP synthase complexes

• Proton impermeability assays using reconstituted membranes

• Size-exclusion chromatography to confirm oligomeric state

These techniques, particularly when used in combination, provide robust evidence for correctly folded and functionally active protein. The ability of recombinant atpE to restore proton impermeability to membrane preparations lacking this subunit is a particularly strong indicator of proper folding and function.

How can researchers assess the proton translocation function of atpE in experimental systems?

To assess proton translocation function of atpE, researchers can employ several complementary approaches:

• Reconstitution assays: Incorporate purified atpE into liposomes along with other Fo components and measure proton pumping using pH-sensitive dyes or electrodes

• ATP synthesis measurements: In reconstituted systems, measure ATP synthesis rates upon creation of an artificial proton gradient

• Proton leak assays: Determine the ability of atpE to maintain proton impermeability in reconstituted membranes using pH-sensitive fluorophores

• Rotation assays: Using single-molecule techniques with fluorescently labeled subunits to directly observe c-ring rotation

For quantitative assessment, researchers typically measure ATP synthesis rates under varying conditions (pH, membrane potential) and compare wild-type with mutant forms to establish structure-function relationships.

What experimental approaches reveal cooperation between c-subunits in ATP synthase?

Cooperation between c-subunits has been demonstrated through innovative experimental designs, particularly using genetically fused single-chain c-rings. Key approaches include:

• Engineered single-chain c-rings: Create fusion proteins where multiple c-subunits are connected, allowing precise introduction of mutations at specific positions

• Functional assays with heterogeneous c-rings: Measure ATP synthesis activity in systems containing both wild-type and mutant c-subunits

• Double mutation analysis: Introduce mutations in different c-subunits and analyze how their spatial relationship affects function

These results from Bacillus PS3 ATP synthase studies demonstrate that activity decreases progressively as mutations are introduced, with greater effects when mutations are spatially separated in the c-ring . This provides strong evidence for functional cooperation between c-subunits during the rotational catalysis mechanism.

How do researchers distinguish between proton uptake and release pathways in atpE function?

Distinguishing between proton uptake and release pathways requires sophisticated experimental and computational approaches:

• Site-directed mutagenesis: Target specific residues hypothesized to be involved in either proton uptake or release pathways

• pH-dependent activity assays: Measure activity across pH ranges to identify pKa shifts that affect either uptake or release

• Proton transfer-coupled molecular dynamics simulations: Model proton movement through specific pathways in the protein structure

• Structure-guided functional analysis: Use high-resolution structural data to inform mutation studies targeting specific channel regions

Analysis of simulation trajectories has revealed that in wild-type ATP synthase, two or three deprotonated carboxyl residues typically face the a-subunit, creating an environment where waiting time for proton uptake can be shared between multiple c-subunits . This sharing mechanism forms the molecular basis for cooperation among c-subunits during rotational catalysis.

What effect do specific mutations in atpE have on ATP synthase function?

Specific mutations in atpE have distinct effects on ATP synthase function, providing insights into structure-function relationships:

• E56D mutation in c-subunits: Studies in Bacillus PS3 show that this mutation reduces ATP synthesis activity. A single E56D mutation reduces activity to approximately 70% of wild-type, while double mutations further reduce activity depending on their spatial arrangement .

• N-terminal vs. C-terminal mutations: N-terminal truncations typically have more profound functional effects than C-terminal deletions, similar to what has been observed in E. coli systems .

• H37R substitution: In chloroplast ATP synthase, substitution of histidine-37 with arginine appears to uncouple ATPase inhibition from proton impermeability restoration, suggesting distinct functional domains within the protein .

These findings highlight the complex relationship between protein structure and function, with implications for understanding the evolutionary constraints on ATP synthase components.

How do researchers analyze the oligomeric assembly of c-subunits in the membrane?

Analysis of c-subunit oligomeric assembly requires specialized techniques for membrane protein complexes:

• Cryo-electron microscopy: Provides high-resolution structural information about the assembled c-ring within intact ATP synthase complexes

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry identifies specific interactions between adjacent c-subunits

• Fluorescence resonance energy transfer (FRET): Measures distances between labeled c-subunits in reconstituted systems

• Native gel electrophoresis: Preserves the oligomeric assembly for analysis of complex formation and stability

Research has revealed that the c-ring typically contains 10-15 c-subunits depending on the species, with each subunit contributing to the formation of a cylindrical structure that rotates during ATP synthesis. The precise number of c-subunits affects the bioenergetic parameters of ATP synthesis, including the H+/ATP ratio.

What computational methods are used to study proton transfer in atpE function?

Advanced computational methods have become essential for understanding the molecular mechanisms of proton transfer in atpE:

• Proton transfer-coupled molecular dynamics (MD): Simulates proton movement through the protein structure while accounting for conformational changes

• Quantum mechanics/molecular mechanics (QM/MM): Provides detailed insights into the energetics of proton transfer reactions

• pKa calculations: Predict protonation states of key residues under different conditions

• Free energy calculations: Determine energy barriers for proton translocation steps

These computational approaches have revealed that proton uptake in mutated c-subunits can be shared between adjacent subunits, with the degree of sharing decreasing as the distance between mutation sites increases . This provides a molecular explanation for the observed cooperative behavior in biochemical assays.

How does E. coli O45:K1 atpE differ from other E. coli strains and what are the functional implications?

E. coli O45:K1 represents a distinct strain with unique genetic characteristics. Comparative analysis reveals:

• The O45 antigen gene cluster in E. coli O45:K1 (strain S88) differs from that in the reference strain E. coli 96-3285 (O45:H2), suggesting that while they share some epitopes, they represent different antigens .

• The functional organization and low DNA sequence homology between these strains suggest that their O-antigen gene clusters originated from a common ancestor but have undergone multiple recombination events .

• E. coli O45:K1 (S88 strain) belongs to a highly virulent meningitis-causing clone related to the O18:K1:H7 clone but with the unusual O45 serogroup .

These differences may influence the membrane environment in which atpE functions, potentially affecting ATP synthase assembly, stability, and activity in different cellular contexts.

What evolutionary relationships exist between atpE from different bacterial species?

Evolutionary analysis of atpE reveals important relationships and conservation patterns:

• The core function of atpE in ATP synthesis is highly conserved across bacterial species, reflecting its essential role in cellular bioenergetics.

• Phylogenetic analysis based on flanking genes like gnd can help trace the evolutionary history and potential horizontal gene transfer events that shaped atpE diversity .

• Despite functional conservation, significant sequence variation exists, particularly in regions not directly involved in proton translocation or subunit interactions.

• The c-subunit structure represents an example of convergent evolution, where similar functional constraints have led to comparable structures despite sequence divergence across distant bacterial lineages.

This evolutionary perspective provides context for understanding the significance of conserved residues and the potential impact of species-specific variations on ATP synthase function and regulation.

How does bacterial atpE compare to equivalent subunits in chloroplast and mitochondrial ATP synthases?

Comparative analysis between bacterial, chloroplast, and mitochondrial ATP synthases reveals important similarities and differences:

• Structural homology: The bacterial atpE shares structural features with chloroplast and mitochondrial counterparts, reflecting their common evolutionary origin.

• Functional adaptations: In chloroplasts, the epsilon subunit (equivalent to bacterial atpE) inhibits ATPase activity and restores proton impermeability, similar to its bacterial counterpart .

• Differential regulation: Chloroplast ATP synthase shows distinct regulatory mechanisms adapted to photosynthetic energy conversion, while mitochondrial systems reflect adaptations to aerobic metabolism.

• Specific sequence features: The C-terminus of chloroplast epsilon subunit contains hydroxylated amino acids (serine/threonine) that are important for interactions with CF1 (chloroplast F1), representing a plant-specific adaptation .

These comparative insights highlight how a fundamentally conserved molecular machine has adapted to different cellular environments and energetic demands across domains of life, providing valuable perspective for researchers studying bacterial systems.