Recombinant Escherichia fergusonii ATP synthase subunit delta (atpH)
Description
Overview of Recombinant Escherichia fergusonii ATP Synthase Subunit Delta (atpH)
The delta subunit (atpH) is part of the peripheral stalk connecting the F<sub>1</sub> (catalytic) and F<sub>0</sub> (proton-translocating) domains of ATP synthase. In E. fergusonii, the recombinant form is produced via heterologous expression in E. coli, leveraging genetic engineering to study its role in energy transduction .
Functional Role
The delta subunit acts as a stator, countering rotational torque generated during ATP synthesis. Key functions include:
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Mechanical Stabilization: Prevents co-rotation of the α<sub>3</sub>β<sub>3</sub> hexamer with the γε rotor .
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Proton Channel Regulation: Modulates proton flow through F<sub>0</sub> by conformational coupling .
Recombinant Production and Purification
Recombinant atpH is typically expressed in E. coli with tags for affinity purification. Example parameters from homologous systems:
Table 2: Recombinant Production Parameters3
| Parameter | Details |
|---|---|
| Expression System | E. coli BL21(DE3) |
| Vector | pET-based plasmid |
| Tag | GST or His-tag (determined post-production) |
| Purity | >85% (SDS-PAGE) |
| Storage | -20°C (short-term); -80°C (long-term) |
| Reconstitution | 0.1–1.0 mg/mL in deionized water with 50% glycerol |
Research Findings
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Assembly Mechanism: The delta subunit is indispensable for integrating F<sub>1</sub> and F<sub>0</sub>. Knockout studies in E. coli show incomplete ATP synthase assembly in Δδ mutants, resolvable by delayed δ-subunit expression .
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Dynamic Behavior: NMR studies reveal conformational flexibility in the C-terminal domain, suggesting adaptive binding to F<sub>0</sub> under rotational stress .
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Evolutionary Conservation: The α-helix bundle is conserved across γ-proteobacteria, underscoring its structural necessity .
Applications and Implications
Product Specs
Target Background
KEGG: efe:EFER_4034
Q&A
What is the structural arrangement of the ATP synthase delta subunit in E. fergusonii and related species?
The ATP synthase delta subunit forms part of the stator complex in F1F0 type ATPases. Based on structural studies of related bacterial species, the delta subunit features a six alpha-helix bundle that constitutes its N-terminal domain (residues 1-134). This domain primarily interacts with the F1 core via the N-terminal region of the alpha subunit. The C-terminal domain, while less structurally defined, plays a crucial role in binding to the F0 component through direct interaction with the b subunits .
The delta subunit, together with two copies of the b subunit, forms a second stalk linking the F1 and F0 components. This arrangement functions as a critical stator that enables the energy-linked rotational movements of gamma and epsilon subunits during ATP synthesis and hydrolysis .
What are the optimal expression systems for recombinant ATP synthase delta subunit production?
Expression of recombinant ATP synthase subunits requires careful consideration of host strains. Comparative studies between E. coli strains (such as M15 and DH5α) have revealed significant differences in their capacity to express functional recombinant proteins. The E. coli M15 strain has demonstrated superior expression characteristics for certain recombinant proteins due to differences in fatty acid and lipid biosynthesis pathways .
For optimal expression of the delta subunit, the following recommendations apply:
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Select an expression system with appropriate transcriptional and translational machinery
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Consider how the host cell's metabolic state will impact protein production
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Evaluate strain-specific differences in post-translational processing capabilities
Proteomics analysis has revealed that the timing of protein synthesis induction is critical in determining the fate of recombinant proteins within host cells. Early induction may increase the metabolic burden, while later induction might better align with the host cell's capacity to produce the target protein .
What purification strategies yield the highest purity and biological activity of the delta subunit?
Purification of recombinant ATP synthase delta subunit typically employs a multi-step approach:
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Initial capture using affinity chromatography (commonly His-tag systems)
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Intermediate purification through ion exchange chromatography
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Polishing steps using size exclusion chromatography
The pH-dependent binding properties of histidine residues in proteins can be leveraged during purification, with optimal binding to metal ions like Ni²⁺ or Co²⁺ occurring at pH 8.0 and above . This principle applies to both native histidine residues and polyhistidine tags used for recombinant protein purification.
For ATP synthase components, maintaining the appropriate buffer conditions is critical to preserve structural integrity. The following table outlines recommended buffer compositions for different purification stages:
| Purification Stage | Buffer Composition | pH Range | Additives |
|---|---|---|---|
| Affinity Chromatography | 50 mM Tris-HCl | 7.5-8.5 | 300 mM NaCl, 10% glycerol |
| Ion Exchange | 20 mM phosphate | 7.0-8.0 | 50-500 mM NaCl gradient |
| Size Exclusion | 20 mM Tris-HCl | 7.5-8.0 | 150 mM NaCl, 5% glycerol |
| Storage | 20 mM Tris-HCl | 7.5-8.0 | 150 mM NaCl, 50% glycerol |
How can one assess the binding interactions of the recombinant delta subunit with other ATP synthase components?
The functional assessment of delta subunit interactions with other ATP synthase components can be approached through multiple complementary techniques:
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Fluorescence-based binding assays: Labeling the purified delta subunit with fluorescent markers such as Cy3 allows for direct measurement of binding interactions. This approach has been successfully applied to ε subunit studies, where ATP binding affinity was measured across different pH conditions .
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Surface Plasmon Resonance (SPR): This label-free technique enables real-time monitoring of binding kinetics between the delta subunit and potential interaction partners.
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Isothermal Titration Calorimetry (ITC): Provides thermodynamic parameters of binding, including association constants, enthalpy changes, and binding stoichiometry.
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Co-immunoprecipitation: Can verify protein-protein interactions under near-physiological conditions.
When designing binding experiments, researchers should consider that interactions may be pH-dependent, as demonstrated for the ε subunit, where ATP binding affinity changed 5.9-fold between pH 7.0 and pH 8.5 .
What techniques are most effective for analyzing the structural dynamics of the delta subunit?
Structural dynamics analysis of the ATP synthase delta subunit requires sophisticated biophysical methods:
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NMR spectroscopy: This technique has successfully revealed the structure of the E. coli delta subunit, identifying its six alpha-helix bundle arrangement. NMR is particularly valuable for detecting conformational changes under different conditions .
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Molecular Dynamics (MD) simulations: Can provide insights into the protonation states of key residues and their impact on protein conformation. MD simulations have successfully predicted how histidine protonation affects nucleotide binding in ATP synthase components .
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Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Useful for mapping flexibility and solvent accessibility changes in different functional states.
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Single-molecule FRET: Can track dynamic conformational changes during functional cycles.
When applying these techniques to E. fergusonii delta subunit, researchers should consider how its unique sequence characteristics might influence structural dynamics compared to model organisms like E. coli.
How does the delta subunit contribute to the regulation of ATP synthase activity in different physiological conditions?
The regulation of ATP synthase activity involves sophisticated mechanisms that include the delta subunit's structural interactions. While the delta subunit itself is not the primary regulatory component, its position in the stator complex makes it integral to maintaining proper enzyme function across varying physiological conditions.
Research on related ATP synthase components reveals pH-dependent regulatory mechanisms. For instance, the ε subunit's ATP binding affinity changes dramatically with small pH shifts, showing distinct behaviors above and below pH 7.75. At lower pH values (7.0-7.5), the binding affinity is poor (Kd values of 20.2 and 16.5 mM), while at higher pH values (8.0-8.5), the affinity increases significantly (Kd values of 5.8 and 3.4 mM) .
What are the key experimental considerations when investigating the effects of mutations in the delta subunit?
When designing mutation studies for the ATP synthase delta subunit, researchers should consider:
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Functional domain targeting: Mutations in the N-terminal domain may impact interactions with the F1 core, while C-terminal mutations would more likely affect interactions with the b subunits and F0 component .
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Conserved residue identification: Comparative sequence analysis across bacterial species can identify highly conserved residues likely critical for function.
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Structural consequences: MD simulations can predict how mutations might alter the protein's conformational stability and binding properties.
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pH sensitivity: If the delta subunit contains histidine residues in functional regions, mutations affecting their pK₍a₎ values could alter pH-dependent behaviors, similar to observations in the ε subunit where histidine protonation states significantly impact ATP binding .
The effects of mutations should be assessed through multiple approaches, including:
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Structure determination via NMR or X-ray crystallography
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Binding affinity measurements
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ATP synthase assembly efficiency
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ATP synthesis/hydrolysis activity assays
How does recombinant expression of the delta subunit impact host cell metabolism?
Recombinant protein production (RPP) places significant metabolic burden on host cells, affecting various cellular processes. Proteomics studies have revealed that RPP induces substantial changes in both transcriptional and translational machinery, which can impact growth rate and protein yield .
Key metabolic impacts include:
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Altered energy metabolism: Resources are diverted from growth to recombinant protein synthesis
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Stress responses: Induction of heat shock proteins and other stress-response elements
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Membrane composition changes: Alterations in fatty acid and lipid biosynthesis pathways
What evolutionary insights can be gained by comparing ATP synthase delta subunits across different bacterial species?
Evolutionary analysis of ATP synthase components across bacterial species provides valuable insights into both conserved functional elements and adaptations to specific environmental niches. For E. fergusonii, comparative genomic approaches similar to those used in other studies can identify unique features of its ATP synthase delta subunit.
Key evolutionary considerations include:
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Sequence conservation: Highly conserved regions likely represent functionally critical domains
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Species-specific adaptations: Unique sequence features may reflect adaptation to specific ecological niches
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Horizontal gene transfer: Assessment of whether ATP synthase components show evidence of gene transfer events
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Coevolution patterns: Correlation between changes in the delta subunit and other ATP synthase components
Such evolutionary analyses can guide the design of experiments to investigate how specific sequence features contribute to E. fergusonii's ATP synthase function under its natural physiological conditions.
What are common obstacles in recombinant ATP synthase delta subunit expression and how can they be addressed?
Researchers frequently encounter specific challenges when expressing ATP synthase components:
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Poor expression levels: This may result from codon usage bias, mRNA secondary structures, or promoter inefficiency. Solutions include codon optimization, using different promoter systems, or testing alternative host strains with different transcriptional machinery characteristics .
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Protein insolubility: ATP synthase components evolved to function within a complex and may show poor solubility when expressed individually. Approaches to improve solubility include:
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Expression at lower temperatures (16-25°C)
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Co-expression with chaperones
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Fusion with solubility-enhancing tags
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Addition of mild detergents to extraction buffers
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Improper folding: The structural integrity of the delta subunit is crucial for function. Proteomics approaches can help identify whether misfolding is occurring and which cellular pathways are responding to the recombinant protein burden .
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Host metabolic limitations: The timing of induction significantly impacts recombinant protein yield. Monitoring cellular responses through proteomics can inform optimization strategies for induction timing and conditions .
How can isotope labeling be effectively implemented for structural studies of the delta subunit?
Isotope labeling is essential for advanced structural studies such as NMR spectroscopy, which has been successfully applied to determine the structure of ATP synthase components . For effective isotope labeling of the delta subunit:
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Expression media optimization: M9 minimal media supplemented with ¹⁵N-ammonium chloride and/or ¹³C-glucose as the sole nitrogen and carbon sources, respectively.
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Expression strategy modifications:
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Lower expression temperature (typically 18-25°C)
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Extended expression time to compensate for slower growth in minimal media
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Higher inoculum densities to overcome growth limitations
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Selective labeling approaches: For larger proteins or specific structural questions, selective amino acid labeling can be more economical and provide targeted information.
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Cell-free protein synthesis: This alternative approach allows for more controlled incorporation of labeled amino acids and can be particularly valuable for difficult-to-express proteins.
The quality of isotope incorporation should be verified by mass spectrometry prior to structural studies to ensure sufficient labeling efficiency for high-quality NMR data.
