Recombinant Rickettsia peacockii ATP synthase subunit c (atpE)
Description
Production Overview
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Storage: Lyophilized powder at -20°C/-80°C; reconstituted in deionized water with 50% glycerol recommended
Notes:
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Stability: Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for ≤1 week .
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Reconstitution: Centrifuge vials before opening; reconstitute to 0.1–1.0 mg/mL with glycerol for long-term storage .
Role in ATP Synthase
Subunit c forms a proton-conducting ring in the F₀ sector, working synergistically with subunit a to pump protons across the membrane. This process drives ATP synthesis via the F₁ sector . In Rickettsia, ATP synthase is critical for energy production, as these obligate intracellular bacteria rely on host-derived metabolites (e.g., glucose, glutamine) to fuel their TCA cycle .
Genomic and Evolutionary Context
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Non-Pathogenicity: R. peacockii lacks virulence factors (e.g., ompA, scaI) present in R. rickettsii, with mutations in atpE not directly implicated in pathogenicity loss .
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Genomic Rearrangements: ISRpe1 transposons in R. peacockii caused deletions and synteny disruptions, but atpE remains intact, suggesting its conservation across rickettsial genomes .
Research Applications and Potential Use Cases
Limitations:
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Host-Specificity: Rickettsia ATP synthase subunits may not functionally replace homologs in other bacteria due to sequence divergence .
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Secretion Pathways: While Rickettsia employs Sec and TolC pathways for protein secretion, atpE localization is cytoplasmic, limiting its role in host-pathogen interactions .
Table 2: Comparative Features of R. peacockii and R. rickettsii ATP Synthase Subunit c
| Feature | R. peacockii atpE (C4K0P2) | R. rickettsii atpE |
|---|---|---|
| Sequence Identity | ~70% (predicted) | N/A |
| Protein Length | 74 aa | Similar (72–76 aa) |
| Genomic Stability | No transposon-associated deletions | Synteny preserved |
| Pathogenicity Link | None | No direct association |
Product Specs
Please note that we will preferentially ship the format we have in stock. However, if you have a specific requirement for the format, please indicate it in your order notes. We will prepare the product according to your request.
All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance as additional charges may apply.
Generally, the shelf life of liquid formulations is 6 months at -20°C/-80°C. The shelf life of lyophilized formulations is 12 months at -20°C/-80°C.
Target Background
KEGG: rpk:RPR_01055
Q&A
How Should Researchers Design Experiments to Compare the Role of atpE in Rickettsia peacockii vs. Virulent Rickettsia Species like R. rickettsii?
To compare atpE’s functional and evolutionary roles, a multi-omics approach is critical:
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Genomic Context: Analyze transposon-driven genomic reorganization in R. peacockii (e.g., ISRpe1 transposons causing deletions) and correlate these with atpE sequence divergence from R. rickettsii .
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Functional Knockouts: Use CRISPR-Cas9 to disrupt atpE in R. peacockii and assess impacts on ATP synthesis, proton transport, and intracellular survival in tick/mammalian cells. Compare results to R. rickettsii mutants .
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Proton Motive Force (PMF) Assays: Measure ATP synthesis rates and proton translocation efficiency in purified recombinant atpE (His-tagged, expressed in E. coli) using fluorescent pH-sensitive dyes (e.g., ACMA) .
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Phylogenetic Analysis: Map atpE mutations (e.g., truncations or amino acid substitutions) across Rickettsia species to infer evolutionary pressures linked to virulence loss .
Table 1: Key Experimental Parameters for Comparative Studies
Why Do Conflicting Reports Exist Regarding atpE’s Role in Rickettsia Pathogenicity?
Discrepancies arise from differences in experimental systems and evolutionary contexts:
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Host-Specific Effects: R. peacockii atpE mutations may reduce virulence in ticks but not directly affect pathogenicity in mammals, as seen in R. rickettsii .
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Functional Redundancy: Other subunits (e.g., a, b) or compensatory pathways might mitigate atpE defects in ATP synthesis, masking its role in viability .
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Assay Limitations: qPCR-based growth rate measurements in Rickettsia may not capture subtle metabolic impacts of atpE mutations .
Resolution Strategy:
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Isolate atpE Function: Use recombinant atpE (His-tagged, expressed in E. coli) in in vitro proton transport assays to deconvolute its role from other subunits .
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Cross-Species Complementation: Express R. peacockii atpE in R. rickettsii mutants to test rescue of virulence defects .
What Methods Are Effective for Studying the Proton Transport Function of Recombinant atpE?
To characterize atpE’s role in proton translocation:
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Reconstitution into Liposomes: Purify recombinant atpE (74 aa, His-tagged) and reconstitute into phospholipid vesicles. Measure proton leakage using ACMA fluorescence quenching assays .
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Site-Directed Mutagenesis: Target conserved residues (e.g., Glycine residues in transmembrane helices) critical for proton channel formation. Analyze mutant phenotypes in proton transport assays .
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Cryo-EM Structural Studies: Compare R. peacockii atpE (1-74 aa) to mammalian subunit c (e.g., mitochondrial isoforms) to identify structural motifs affecting proton coupling .
Key Challenges:
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Low solubility of atpE in E. coli necessitates optimized refolding protocols .
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Membrane protein crystallization for structural studies remains technically demanding .
How Do Transposons Influence the Evolution of atpE in Rickettsia?
The ISRpe1 transposon in R. peacockii drives genomic instability and atpE evolution:
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Genomic Reorganization: Recombination between ISRpe1 copies causes deletions, disrupting synteny with R. rickettsii .
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Gene Decay: Transposon insertion or excision may lead to atpE truncations or loss of critical functional domains (e.g., lipid-binding regions) .
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Horizontal Gene Transfer: Plasmid-borne genes (e.g., glycosylation islands from Pseudomonas) may compensate for atpE defects, enabling survival despite reduced ATP synthesis .
Table 2: Transposon-Driven Effects on R. peacockii atpE
What Are Best Practices for Producing and Handling Recombinant atpE?
To optimize recombinant atpE (His-tagged, 1-74 aa) production and stability :
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Expression Conditions: Grow E. coli at 18–25°C with IPTG induction to minimize inclusion body formation.
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Purification: Use Ni-NTA affinity chromatography followed by size-exclusion chromatography (SEC) to achieve >90% purity .
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Storage: Aliquot in Tris/PBS buffer with 50% glycerol at -20°C to prevent aggregation. Avoid repeated freeze-thaw cycles .
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Functional Validation: Confirm proton transport activity via liposome assays before use in structural or biochemical studies .
Troubleshooting:
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Low Yield: Optimize codon usage for Rickettsia genes in E. coli.
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Protein Aggregation: Use detergents (e.g., DPC) during purification to maintain membrane protein solubility .
How Can Researchers Study the Structural and Functional Evolution of atpE Across Rickettsia Species?
To trace atpE evolution and functional divergence:
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Phylogenetic Trees: Align atpE sequences from Rickettsia and outgroups (e.g., Francisella) to identify lineage-specific mutations .
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Homology Modeling: Predict 3D structures of R. peacockii atpE (using E. coli F0 ATP synthase as a template) to identify conserved/variable residues .
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Comparative Proton Transport: Measure proton pumping rates of recombinant atpE from non-pathogenic vs. pathogenic Rickettsia in liposome assays .
Key Insight:
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R. peacockii atpE’s shorter length (74 aa vs. 76 aa in mammals) may reduce proton channel stability, linking to reduced virulence .
What Approaches Reveal atpE’s Role in Membrane Interactions and Lipid Binding?
To study atpE’s interaction with lipid bilayers:
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Lipid Composition Assays: Test atpE’s binding affinity to cardiolipin (CL), phosphatidylethanolamine (PE), or other lipids using surface plasmon resonance .
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Membrane Permeability: Measure dye leakage from liposomes containing atpE to assess pore formation or lipid perturbation .
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Cryo-EM with Nanodiscs: Reconstitute atpE into lipid nanodiscs to capture native-like conformations for structural studies .
Table 3: Lipid Binding Assay Parameters
How Can atpE Mutations Be Linked to Reduced Virulence in Rickettsia?
To establish causality between atpE defects and virulence loss:
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Gene Replacement: Replace R. rickettsii atpE with R. peacockii atpE and assess virulence in tick/mammalian models .
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Metabolic Profiling: Compare ATP levels, PMF, and redox states (e.g., NAD+/NADH ratios) in Rickettsia with atpE mutations vs. wild-type .
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Host Cell Survival: Measure Rickettsia replication in Vero E6 or ISE6 cells using qPCR to quantify growth rates .
Example: R. peacockii atpE mutations may reduce ATP synthesis efficiency, limiting replication in host cells .
What Are the Challenges in Studying atpE’s Tissue-Specific Roles in Rickettsia?
While Rickettsia lack mitochondrial targeting peptides, comparative studies with mammalian subunit c isoforms (P1, P2, P3) offer insights:
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Expression Profiling: Use qRT-PCR to quantify atpE expression in Rickettsia isolated from ticks vs. mammalian hosts .
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Proteomic Analysis: Identify post-translational modifications (e.g., phosphorylation) that regulate atpE activity in different environments .
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Host Cell Interaction: Co-culture Rickettsia with tick (e.g., ISE6) vs. mammalian (e.g., Vero E6) cells to assess atpE-dependent pH regulation .
Key Challenge: Rickettsia’s obligate intracellular lifestyle complicates in vivo modulation of atpE expression.
How Does atpE’s Evolutionary History Inform Its Functional Studies?
AtpE’s evolution reflects trade-offs between ATP synthesis and pathogenicity:
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Horizontal Gene Transfer: Plasmid-encoded genes in R. peacockii (e.g., glycosylation genes from Pseudomonas) may compensate for atpE defects, enabling survival despite reduced ATP synthesis .
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Gene Loss Events: Deletions in R. peacockii atpE and other virulence factors (e.g., RickA, OmpA) suggest convergent evolution toward commensalism .
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Comparative Genomics: Align atpE sequences from Rickettsia, Francisella, and E. coli to identify conserved residues critical for proton transport .
Table 4: Evolutionary Trade-Offs in Rickettsia atpE
