Recombinant Escherichia coli O127:H6 ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c (atpE) in E. coli O127:H6?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase in E. coli O127:H6. This hydrophobic protein consists of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The protein functions as part of the membrane-embedded proton channel that enables proton translocation across the membrane, which is coupled to ATP synthesis. In the ATP synthase complex, multiple c-subunits form a ring structure within the membrane that rotates during proton transport, driving conformational changes in the F1 sector that catalyze ATP synthesis. Additionally, subunit c plays a crucial role in maintaining proton impermeability of membranes when properly assembled with other components of the ATP synthase complex .

How does the amino acid composition of atpE affect its functional properties?

The amino acid composition of atpE is characterized by a high proportion of hydrophobic residues, reflecting its membrane-embedded nature. The protein contains critical hydroxylated amino acids (serine and threonine) in its C-terminal region that appear to be important in protein-protein interactions within the ATP synthase complex . Research has shown that the C-terminus of atpE contains six amino acids that represent the primary region of sequence divergence between spinach and pea epsilon proteins, with four of these being serine or threonine residues. When these C-terminal residues are deleted, the protein exhibits reduced ATPase inhibitory potency, suggesting their importance in functional interactions with other subunits of the complex . Additionally, specific residues like histidine-37 appear to have specialized roles in coupling ATPase inhibition with proton impermeability functions .

How is the atpE gene organized within the ATP synthase operon?

The atpE gene is part of the atpIBEFHAGDC operon in E. coli, which encodes all subunits of the ATP synthase complex. In experimental systems, this entire operon has been engineered to be IPTG-titratable, allowing researchers to control the expression level of ATP synthase . The organization of genes within this operon reflects the assembly sequence and stoichiometry of the ATP synthase complex components. The atpE gene specifically encodes the c subunit that forms the c-ring in the F0 sector of ATP synthase. The coordinated expression of all genes in this operon is critical for proper assembly and function of the complete ATP synthase complex. Expression of this operon is highly regulated and responds to growth conditions, enabling bacteria to optimize ATP production according to metabolic demands .

What are the optimal conditions for recombinant expression of E. coli O127:H6 atpE?

For optimal recombinant expression of E. coli O127:H6 atpE, researchers should consider several critical parameters. The gene can be effectively overexpressed in Escherichia coli expression systems, as demonstrated in studies with both the epsilon subunit from spinach chloroplast ATP synthase and E. coli ATP synthase . When designing expression constructs, it's advisable to include appropriate affinity tags to facilitate purification, though the specific tag type may be determined during the production process based on the protein's characteristics . Expression should be conducted under controlled temperature conditions, typically at lower temperatures (16-25°C) to minimize inclusion body formation of this membrane protein. Induction parameters such as IPTG concentration and induction time require optimization for maximal protein yield while maintaining proper folding. For membrane proteins like atpE, expression in specialized E. coli strains designed for membrane protein production (such as C41(DE3) or C43(DE3)) may improve yield and proper membrane insertion .

How can I solubilize and refold atpE to maintain its biological activity?

To effectively solubilize and refold atpE while preserving its biological activity, follow this methodological approach: First, extract the protein using 8 M urea as a denaturant, which has been shown to effectively solubilize recombinant ATP synthase subunits . For refolding, dilute the urea-solubilized protein directly into a buffer containing ethanol and glycerol, which has been demonstrated to produce biologically active protein comparable to that purified from native sources . Specifically, the research shows that recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 (the catalytic portion of chloroplast ATP synthase) that is deficient in epsilon, and restores proton impermeability to reconstituted membranes . The precise buffer composition should be optimized, but typically includes a Tris-based buffer with 50% glycerol to stabilize the protein structure . During the refolding process, maintain protein concentration between 0.1-1.0 mg/mL to prevent aggregation, and consider a step-wise dialysis to gradually remove the denaturant if direct dilution results in precipitation .

What storage conditions maximize stability of purified recombinant atpE?

For optimal stability of purified recombinant atpE, implement the following storage protocol based on empirical research findings: Store the protein at -20°C or -80°C, with the latter recommended for extended storage periods . The protein should be maintained in a Tris-based buffer containing 50% glycerol, which has been optimized specifically for this protein . The glycerol concentration is critical, as it prevents freeze-thaw damage and maintains protein solubility; a range of 5-50% glycerol is acceptable, though 50% is the default recommendation for maximum stability . Aliquot the protein into single-use volumes to avoid repeated freeze-thaw cycles, which significantly degrade protein quality and function . For short-term use (up to one week), working aliquots may be stored at 4°C . Under these conditions, the liquid formulation typically maintains stability for approximately 6 months, while lyophilized preparations can remain stable for up to 12 months . The shelf life depends on multiple factors including buffer composition, storage temperature, and the intrinsic stability of the specific protein preparation .

How can I measure the ATPase inhibitory activity of recombinant atpE?

To measure the ATPase inhibitory activity of recombinant atpE, implement a systematic approach focusing on quantifiable enzymatic parameters. First, isolate or purify ATP synthase (F1-ATPase) that is deficient in the epsilon subunit—this can be achieved from native sources or through recombinant expression of an incomplete complex. Establish a baseline ATPase activity measurement using standard colorimetric or coupled-enzyme assays that detect inorganic phosphate release or NAD(P)H oxidation . Next, add increasing concentrations of your purified recombinant atpE to these assays and measure the resulting ATPase activity. Calculate the percentage inhibition at each concentration and determine the IC50 value (concentration causing 50% inhibition) . For comprehensive characterization, perform these measurements with both soluble and membrane-bound ATP synthase, as inhibitory potency may differ between these forms. Research has shown that properly folded recombinant atpE inhibits ATPase activity of both soluble and membrane-bound CF1 that lacks the epsilon subunit, providing a functional readout of biological activity . Compare your inhibition profile with published values or with atpE from other species (such as the comparison between spinach and pea epsilon proteins) to contextualize your findings .

What methods can be used to study the role of atpE in proton translocation?

To investigate atpE's role in proton translocation, employ these methodological approaches: First, prepare thylakoid or bacterial membrane vesicles reconstituted with ATP synthase complexes lacking the atpE subunit. Measure baseline proton leakage using pH-sensitive fluorescent dyes such as ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine, which provide real-time monitoring of proton gradient dissipation . Next, add purified recombinant atpE to these membrane preparations and quantify changes in proton impermeability. Research has demonstrated that properly folded recombinant epsilon restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon, providing a functional assay for this critical activity . For more detailed analysis, conduct these experiments with mutant forms of atpE (containing specific amino acid substitutions or truncations) to identify regions essential for maintaining proton impermeability. Notably, studies have shown that substitution of histidine-37 with arginine appears to uncouple ATPase inhibition from proton impermeability functions, suggesting this residue has specialized importance in coordinating these activities . Additionally, implement patch-clamp techniques on reconstituted membranes or proteoliposomes to directly measure proton conductance at the single-channel level with and without atpE incorporation.

How does atpE expression level affect bacterial growth across different nutrient conditions?

AtpE expression level significantly impacts bacterial growth in a nutrient-dependent manner, with E. coli demonstrating remarkable optimization of ATP synthase expression across diverse conditions. Experimental evidence shows that wild-type E. coli expresses ATP synthase at levels remarkably close to optimal concentrations that maximize immediate growth rate, with deviations averaging only 6.1 ± 6.1% from optimality across 27 different growth conditions . To investigate this relationship, implement an IPTG-titratable atpIBEFHAGDC operon system that allows precise control of ATP synthase expression levels, including the atpE component . Measure growth rates across a range of expression levels (controlled by IPTG concentration) in different nutrient environments including various carbon sources (sugars, organic acids) and nitrogen sources . This methodological approach reveals that an optimal expression level exists at which growth rate is maximized for each condition, and that wild-type E. coli naturally achieves this same maximal growth rate across diverse conditions . This indicates robust evolutionary optimization of ATP synthase expression for immediate growth-rate maximization rather than preparation for future conditions, suggesting strong selection pressure on energy metabolism efficiency .

How do N-terminal versus C-terminal truncations differently affect atpE function?

N-terminal and C-terminal truncations of atpE produce distinctly different functional consequences, providing critical insights into structure-function relationships. N-terminal truncations generally exhibit more profound effects on atpE function compared to C-terminal deletions, as demonstrated in both E. coli and chloroplast ATP synthase studies . When designing truncation experiments, create a series of constructs with progressively increasing truncations from either terminus, express them recombinantly, and assess their functional properties using ATPase inhibition and proton impermeability assays. Research has shown that C-terminal truncations, particularly deletion of six amino acids from the C-terminus of spinach chloroplast ATP synthase epsilon subunit, results in reduced ATPase inhibitory potency that resembles the naturally less potent pea epsilon protein . This region represents the primary sequence divergence between these species, containing four serine or threonine residues that appear important for epsilon-CF1 interactions . The differential effects of N- versus C-terminal truncations suggest distinct functional domains within the protein, with the N-terminus potentially involved in core catalytic regulation and structural integrity, while the C-terminus may primarily mediate specific protein-protein interactions that fine-tune activity .

What is the significance of the histidine-37 residue in atpE function?

Histidine-37 represents a pivotal residue in atpE function, serving as a molecular switch that couples ATPase inhibition with proton impermeability functions. Site-directed mutagenesis studies have revealed that substitution of histidine-37 with arginine produces a unique phenotype that uncouples these two fundamental functions of the protein . To investigate this residue's importance, researchers should employ site-directed mutagenesis to create the H37R variant and other substitutions at this position, followed by comprehensive functional characterization. Unlike other mutations that affect both ATPase inhibition and proton impermeability to similar extents, the H37R mutation selectively impacts one function while preserving the other . This suggests histidine-37 serves as a critical communication node between the protein domains responsible for these distinct but coordinated functions. The unique properties of histidine—including its ability to change protonation state at physiological pH, form hydrogen bonds, and participate in both hydrophobic and hydrophilic interactions—likely contribute to its specialized role. Researchers investigating the molecular mechanisms of ATP synthase regulation should focus particular attention on this residue and its immediate structural environment, as it represents a key regulatory element within the protein .

How can I design effective site-directed mutagenesis experiments to study atpE function?

To design effective site-directed mutagenesis experiments for studying atpE function, implement a methodical approach targeting specific functional hypotheses. First, conduct comprehensive sequence alignment across diverse species to identify highly conserved residues, which often indicate functional importance . Focus on charged residues (D, E, K, R, H) that may participate in proton translocation or subunit interactions, and hydroxylated residues (S, T) that could be involved in hydrogen bonding networks or conformational changes . Based on published crystal structures or predictive modeling, identify residues at interfaces between subunits or in regions undergoing conformational changes during catalysis. Design mutations that test specific hypotheses—conservative substitutions (e.g., D→E) to maintain charge while altering side chain geometry, non-conservative substitutions (e.g., D→N) to eliminate charge while preserving size, or radical changes (e.g., D→A) to assess the importance of both properties . Include control mutations in regions predicted to have minimal functional impact. Research has successfully employed this approach to generate truncations and single amino acid substitutions in the primary structure of epsilon, revealing differential effects on ATPase inhibition and proton impermeability . Express these mutants using the same system described for wild-type protein (8 M urea solubilization followed by refolding in buffer with ethanol and glycerol) to ensure comparable protein quality .

How can recombinant atpE be used to study cellular energy homeostasis?

Recombinant atpE serves as a powerful tool for investigating cellular energy homeostasis through multiple experimental approaches. Researchers can use purified recombinant atpE to manipulate ATP synthase activity in reconstituted systems or permeabilized cells, enabling precise control over energy production pathways . This approach allows for direct measurement of how ATP synthase regulation affects ATP/ADP ratios, proton motive force, and downstream metabolic processes. By introducing wild-type or mutant forms of atpE (particularly regulatory mutants like H37R) into systems lacking endogenous protein, researchers can dissect the molecular mechanisms governing energy production feedback loops . Additionally, recombinant atpE can be employed in competitive binding assays to identify small molecules that modulate ATP synthase activity, potentially revealing new regulatory mechanisms or therapeutic targets. Research has demonstrated that E. coli optimizes ATP synthase expression to maximize growth rate across diverse nutrient conditions, indicating sophisticated regulatory control of energy metabolism . By manipulating atpE expression or activity and measuring resulting metabolic parameters, researchers can illuminate how organisms balance energy production with growth requirements and adapt to changing environmental conditions .

What insights does atpE expression optimization provide about bacterial adaptation?

The remarkable optimization of ATP synthase expression in E. coli provides profound insights into bacterial evolutionary adaptation and metabolic efficiency. Research demonstrates that wild-type E. coli expresses ATP synthase at levels remarkably close to optimal concentrations (within a few percent) that maximize immediate growth rate across 27 different nutrient conditions, indicating robust evolutionary tuning of energy production machinery . This optimization occurs despite the dramatically different energetic demands of various carbon sources—during growth on sugars, glycolysis supplies most ATP while ATP synthase becomes the primary ATP source during growth on acetate . To leverage these insights in your research, implement experimental systems with IPTG-titratable ATP synthase operons that allow manipulation of expression levels, coupled with growth rate measurements across diverse nutrient environments . This approach reveals whether your organism of interest prioritizes immediate growth rate maximization (short-term fitness) or preparation for future conditions (long-term fitness). The robust optimization observed in E. coli suggests strong selection pressure on energy metabolism efficiency and demonstrates that bacteria can achieve protein expression levels that maximize immediate growth rather than displaying suboptimal expression as might be expected in fluctuating environments .

Why might recombinant atpE show reduced activity compared to native protein?

Recombinant atpE may exhibit reduced activity compared to native protein due to several methodological challenges that can be systematically addressed. First, incorrect protein folding represents a primary concern—the hydrophobic nature of this membrane protein makes proper folding particularly challenging in recombinant systems . Research indicates that specific refolding conditions are critical; solubilization in 8 M urea followed by direct dilution into buffer containing ethanol and glycerol has proven effective in producing biologically active protein comparable to that purified from native sources . Absence of post-translational modifications present in the native system may also impact activity. Additionally, recombinant expression may produce protein lacking essential cofactors or interaction partners required for full functionality. To troubleshoot activity issues, compare different protein preparation methods, systematically testing variables like denaturant type, refolding buffer composition, and refolding kinetics . Assess protein quality using circular dichroism to confirm secondary structure, and size exclusion chromatography to detect aggregation. Functional assays should include both ATPase inhibition and proton impermeability restoration to CF1-deficient membranes, as these functions can be differentially affected . If activity remains suboptimal, consider co-expression with other ATP synthase subunits to facilitate proper folding and assembly, or expression in specialized membrane protein production systems that better mimic the native environment .

How can I resolve inconsistent results in atpE functional assays?

To resolve inconsistencies in atpE functional assays, implement a systematic troubleshooting approach addressing key variables that influence experimental outcomes. First, standardize protein quality control measures—confirm protein purity (>85% by SDS-PAGE), verify proper folding using spectroscopic methods, and assess oligomeric state through size exclusion chromatography . Protein storage conditions significantly impact stability; maintain consistent storage in Tris-based buffer with 50% glycerol at -20°C/-80°C and avoid repeated freeze-thaw cycles, which can cause progressive activity loss . For functional assays measuring ATPase inhibition or proton impermeability restoration, standardize the preparation of ATP synthase complexes lacking epsilon/atpE, as variations in these preparations can introduce significant result variability . Control environmental variables during assays, including temperature, pH, and ionic strength. Implement internal controls in each experiment, including positive controls (native protein) and negative controls (buffer only). When testing mutant proteins, include wild-type protein prepared under identical conditions as a reference standard . Assay sensitivity to atpE concentration is critical; establish complete concentration-response curves rather than single-point measurements to identify potential shifts in potency or efficacy . Finally, recognize that atpE may exhibit different activities in different experimental systems; research has shown that substitution of histidine-37 with arginine uncouples ATPase inhibition from proton impermeability functions, potentially explaining seemingly contradictory results when measuring only one function .

How should I interpret contradictory data regarding atpE's role in different experimental systems?

When confronted with contradictory data regarding atpE's role across different experimental systems, employ a structured analytical framework to reconcile these discrepancies. First, critically evaluate methodological differences between experimental systems—variations in protein preparation (solubilization conditions, refolding protocols), assay conditions (pH, temperature, ionic strength), or experimental models (in vitro reconstituted systems versus cellular models) can significantly impact functional outcomes . Research has demonstrated that proper refolding conditions are critical for obtaining biologically active protein, suggesting improper preparation could yield misleading results . Consider the multifunctional nature of atpE, which participates in both ATPase inhibition and proton impermeability maintenance—these functions can be differentially affected by mutations or experimental conditions, as evidenced by the histidine-37 to arginine substitution that uncouples these activities . Assess whether contradictions reflect genuine biological differences between systems (e.g., species-specific variations in ATP synthase regulation) or technical artifacts. The C-terminal region of atpE shows significant sequence variation between species and affects functional properties, potentially explaining system-specific findings . Additionally, recognize that ATP synthase expression is highly regulated and dependent on growth conditions, suggesting experimental growth parameters could influence outcomes . Reconcile contradictions by designing experiments that directly compare systems under identical conditions or that specifically test hypotheses explaining the observed discrepancies.

Data Table: Functional Effects of Key atpE Mutations