Recombinant Rickettsia canadensis ATP synthase subunit c (atpE)

What is Rickettsia canadensis ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase in Rickettsia canadensis, functioning within the F0 sector of the enzyme complex. This 74-amino acid protein (UniProt ID: A8EX89) has the sequence MDIVSLKFIGVGLMAIGMYGAALGVSNIFSSLLNAIARNPAAAENLQRMALIGAGLAEAIGLFSFVIAMLLIFS and is essential for ATP synthesis in this obligate intracellular pathogen . Also known as F-type ATPase subunit c or lipid-binding protein, it forms the c-ring structure within the membrane-embedded portion of ATP synthase, which is crucial for the rotational mechanism that drives ATP production . In the context of evolutionary biology, this protein represents part of the core energy metabolism that has been retained despite the reductive genome evolution characteristic of Rickettsia species .

How is recombinant Rickettsia canadensis atpE typically produced?

Recombinant R. canadensis atpE is typically produced using heterologous expression systems, with E. coli being the predominant host organism. The standard methodology involves:

• Gene synthesis or PCR amplification of the atpE coding sequence from R. canadensis

• Cloning into an expression vector with an N-terminal His-tag for purification

• Transformation into E. coli expression strains

• IPTG-induced protein expression under optimized conditions

• Cell lysis and protein extraction

• Affinity chromatography purification using the His-tag

• SDS-PAGE verification of purity (>90%)

• Lyophilization for storage stability

This expression system allows for the production of full-length protein (amino acids 1-74) with high purity and yield, making it suitable for various research applications including structural studies, enzymatic assays, and immunological investigations .

What are the optimal storage conditions for recombinant atpE protein?

For maximum stability and activity retention, recombinant atpE protein should be stored according to these research-validated guidelines:

• Long-term storage: -80°C or -20°C in aliquots to prevent repeated freeze-thaw cycles

• Short-term working storage: 4°C for up to one week

• Storage buffer composition: Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Reconstitution: Use deionized sterile water to achieve 0.1-1.0 mg/mL concentration

• Cryoprotectant: Add glycerol to 50% final concentration for freezer storage

• Aliquoting: Essential for multiple use scenarios to prevent protein degradation

These conditions maintain protein integrity by preventing degradation, denaturation, and aggregation that can compromise experimental results. Research has demonstrated that repeated freeze-thaw cycles significantly reduce protein activity, making proper aliquoting a critical step in experimental design .

How can I verify the activity of recombinant atpE protein in vitro?

Verification of recombinant atpE activity requires specific assays that assess its functional integration into the ATP synthase complex:

Proton Translocation Assay:

• Reconstitute purified atpE into liposomes containing pH-sensitive fluorescent dyes

• Add ATP to initiate proton translocation

• Monitor fluorescence changes corresponding to proton movement across the membrane

• Compare with positive controls (native ATP synthase) and negative controls (liposomes without protein)

ATP Hydrolysis Coupled Assay:

• Combine recombinant atpE with other ATP synthase subunits to reconstitute the complex

• Measure ATP hydrolysis using a coupled enzyme assay (pyruvate kinase and lactate dehydrogenase)

• Monitor NADH oxidation spectrophotometrically at 340 nm

• Calculate activity as μmol ATP hydrolyzed per minute per mg protein

These methodological approaches allow researchers to assess functionality beyond mere presence, ensuring that experimental findings reflect genuine biological activity rather than artifacts .

What approaches can be used to study atpE interactions with inhibitors?

To study atpE interactions with inhibitors, researchers should implement these methodological approaches:

Binding Assays:

• Surface Plasmon Resonance (SPR)

  • Immobilize His-tagged atpE on an NTA sensor chip

  • Flow potential inhibitors at varying concentrations

  • Determine association/dissociation kinetics and equilibrium constants (Ka, Kd, KD)

Functional Inhibition Assays:

• Reconstitute atpE into liposomes or with purified ATP synthase components

• Add potential inhibitors at different concentrations

• Measure ATP synthesis/hydrolysis activity

• Calculate IC50 values and inhibition constants

Structural Approaches:

• X-ray crystallography of atpE-inhibitor complexes

• NMR for mapping binding interfaces in solution

• Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon inhibitor binding

These methods provide complementary data about inhibitor binding sites, mechanisms of action, and structure-activity relationships that are essential for developing targeted antimicrobials against Rickettsia species .

How can recombinant atpE be used to study Rickettsia evolution and phylogeny?

The atpE protein serves as an important molecular marker for evolutionary studies of Rickettsia species through these methodological approaches:

Comparative Sequence Analysis:

• Align atpE sequences from multiple Rickettsia species

• Calculate sequence conservation statistics:

  Comparison	Sequence Identity (%)	Conserved Domains
  R. canadensis vs. R. prowazekii	89.2	Membrane-spanning regions
  R. canadensis vs. R. rickettsii	87.6	Oligomerization interface
  Rickettsia vs. other α-proteobacteria	62-75	Proton-binding sites

• Identify signature sequences unique to Rickettsia canadensis

• Map conservation patterns to functional domains

Phylogenetic Reconstruction:

• Use Maximum Likelihood or Bayesian inference methods

• Construct phylogenetic trees based on atpE sequences

• Test tree robustness through bootstrap analysis (1000 replicates)

• Compare with phylogenies derived from other genes to identify instances of horizontal gene transfer

These approaches reveal how ATP synthase components have evolved under selective pressure in obligate intracellular pathogens, providing insights into metabolic adaptation during genome reduction events that characterize Rickettsia evolution .

What methods can be used to study the structural biology of atpE and its role in the ATP synthase complex?

Advanced structural biology techniques provide critical insights into atpE function through these methodological approaches:

Cryo-Electron Microscopy:

• Reconstitute atpE into the complete ATP synthase complex

• Flash-freeze samples in vitreous ice

• Collect high-resolution image data (~3-4Å resolution)

• Perform 3D reconstruction and molecular modeling

• Visualize the c-ring structure formed by multiple atpE subunits

Cross-linking Mass Spectrometry:

• Use bifunctional cross-linkers specific for lysine residues

• Digest cross-linked complexes with trypsin

• Analyze cross-linked peptides by LC-MS/MS

• Map interaction interfaces between atpE and other ATP synthase subunits

Site-Directed Spin Labeling EPR:

• Introduce cysteine mutations at strategic positions in atpE

• Label with spin probes (MTSL)

• Measure distances between spin labels

• Model conformational changes during the catalytic cycle

These structural approaches reveal how atpE contributes to the rotational mechanism of ATP synthesis and identify potential targets for antimicrobial development specific to Rickettsia species .

How can I overcome solubility issues when working with recombinant atpE?

As a highly hydrophobic membrane protein, atpE presents significant solubility challenges that can be addressed through these methodological approaches:

Expression Optimization:

• Use specialized E. coli strains designed for membrane protein expression (C41, C43)

• Lower induction temperature (16-20°C)

• Reduce inducer concentration (0.1-0.5 mM IPTG)

• Extend expression time (12-24 hours)

Solubilization Strategies:

• Screen detergent panel with varied properties:

  Detergent	Critical Micelle Concentration	Effectiveness for atpE
  DDM	0.17 mM	High
  LDAO	1-2 mM	Moderate
  Triton X-100	0.2-0.9 mM	Low
  SDS	7-10 mM	Very high (denaturing)

• Optimize detergent concentration (typically 1-2% for extraction, 0.1-0.2% for purification)

• Evaluate mixed micelle systems (detergent combinations)

• Consider amphipols or nanodiscs for maintaining native-like environment

Buffer Optimization:

• Adjust ionic strength (150-300 mM NaCl)

• Test pH range (pH 7.0-8.5)

• Add stabilizing agents (glycerol 10-20%, trehalose 5-10%)

These approaches address the challenges inherent in working with highly hydrophobic membrane proteins, ensuring sufficient yields of properly folded atpE for downstream applications .

What are the best approaches for validating antibodies against recombinant atpE?

Rigorous antibody validation is essential for reliable immunological studies of atpE, involving these methodological approaches:

Western Blot Validation:

• Run parallel samples:

  • Purified recombinant atpE

  • Rickettsia canadensis lysate

  • E. coli negative control

  • E. coli expressing recombinant atpE

• Perform antibody titration (1:500 to 1:10,000 dilutions)

• Assess specificity by competing with excess purified antigen

Immunoprecipitation Validation:

• Perform pull-down assays with anti-atpE antibodies

• Analyze precipitated proteins by mass spectrometry

• Confirm presence of atpE and associated ATP synthase components

• Quantify enrichment relative to non-specific IgG controls

Immunofluorescence Validation:

• Fix Rickettsia-infected cells with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Perform dual labeling with anti-atpE and anti-Rickettsia antibodies

• Confirm co-localization at bacterial membranes

• Include peptide competition controls

How does atpE function relate to the broader metabolic adaptations in Rickettsia species?

The atpE protein functions within a highly adapted metabolic network that reflects Rickettsia's evolution as an obligate intracellular pathogen:

Energy Acquisition Pathways:

• ATP/ADP translocase (Tlc1) exchanges host ATP for bacterial ADP

• Rickettsial membranes are permeable to NAD+, providing additional host energy

• ATP synthase (including atpE) maintains membrane potential and synthesizes ATP

Metabolic Network Integration:

• ATP generated via atpE-containing synthase powers essential biosynthetic pathways

• Pyruvate acquisition (host-derived) feeds into:

  • Diaminopimelate (DAP) biosynthesis for peptidoglycan formation

  • Phosphoenolpyruvate (PEP) generation for cell wall components

  • Acetyl-CoA production for the TCA cycle

Regulatory Mechanisms:

• atpE expression likely responds to energy status of the bacterium

• ATP synthase activity modulates in response to metabolic demands

• Coordinated regulation with nucleotide transport systems

This systems biology perspective places atpE within the context of Rickettsia's reductive evolution, where genome streamlining has eliminated many metabolic pathways while preserving essential energy generation mechanisms .

What are the potential applications of atpE in vaccine development against Rickettsiosis?

While primarily a structural component, atpE presents several characteristics that make it a candidate for vaccine development:

Immunological Potential Assessment:

• Epitope Prediction Analysis:

  • MHC Class I binding predictions identify 3-4 potential epitopes

  • MHC Class II binding shows stronger predicted binding for central domain

  • B-cell epitope prediction suggests exposed loops as antibody targets

  Amino Acid Position	Epitope Sequence	Predicted Immunogenicity
  23-31	IFSSLLNAI	High (MHC I)
  45-59	MALIGAGLAEAIGLL	Moderate (MHC II)
  11-18	GLMAIGMY	High (B-cell)

• Conservation Analysis:

  • High conservation (>85%) across pathogenic Rickettsia species

  • Limited homology with human proteins (reducing autoimmunity risk)

Vaccine Platform Approaches:

• Recombinant subunit vaccines using purified atpE

• DNA vaccines encoding atpE

• Attenuated vector vaccines expressing atpE

• Peptide vaccines targeting immunodominant epitopes

Delivery and Adjuvant Considerations:

• Liposomal formulations to mimic membrane context

• TLR agonist adjuvants (TLR4, TLR9) to enhance immunogenicity

• Prime-boost strategies combining different platforms

These approaches leverage the structural and immunological properties of atpE to develop potential vaccines against Rickettsiosis, with particular emphasis on conserved epitopes that could provide cross-protection against multiple Rickettsia species .

What emerging techniques could advance our understanding of atpE function and interactions?

Several cutting-edge methodologies show promise for deeper insights into atpE biology:

Single-Molecule Biophysics:

• Magnetic tweezers to measure torque generation in reconstituted ATP synthase

• Single-molecule FRET to detect conformational changes during rotation

• High-speed AFM to visualize c-ring rotation in real-time

Advanced Structural Methods:

In-Cell Studies:

• CRISPR-based tagging of atpE for live-cell imaging

• Proximity labeling (TurboID, APEX) to map the ATP synthase interactome

• Optogenetics to control ATP synthase activity with light

These emerging technologies will provide unprecedented insights into the molecular mechanisms of ATP synthase operation in Rickettsia, potentially revealing unique features that could be exploited for therapeutic development .

How might comparative analysis of atpE across pathogenic Rickettsia species inform drug discovery efforts?

Targeted antirickettsial drug discovery can be guided by comparative analysis of atpE across species:

Comparative Sequence Analysis Framework:

• Align atpE sequences from multiple pathogenic Rickettsia

• Identify:

  • Universally conserved residues (broad-spectrum targets)

  • Species-specific variations (selective targeting)

  • Residues divergent from human homologs (safety margin)

Structure-Based Drug Design Approach:

• Generate homology models of atpE from multiple Rickettsia species

• Perform virtual screening against identified binding pockets

• Design compounds that interact with conserved residues critical for function

• Optimize lead compounds for:

  • Binding affinity

  • Selectivity over human ATP synthase

  • Pharmacokinetic properties

Validation Methodology:

• Biochemical assays with purified recombinant atpE variants

• Cell-based assays in Rickettsia-infected cell lines

• Animal models of Rickettsiosis

This strategic approach utilizes evolutionary conservation patterns to identify vulnerable targets within the ATP synthase complex that could be exploited for the development of novel antirickettsial therapeutics with potentially broad spectrum activity against multiple pathogenic species .