Recombinant Neorickettsia sennetsu ATP synthase subunit c (atpE)

What is Neorickettsia sennetsu ATP synthase subunit c (atpE)?

Neorickettsia sennetsu ATP synthase subunit c (atpE) is a protein component of the F-type ATP synthase complex in the obligate intracellular bacterium Neorickettsia sennetsu. It functions as part of the F0 sector of ATP synthase, which forms a proton channel across the bacterial membrane. This protein has several alternative designations including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein. The gene encoding this protein is named atpE with the ordered locus name NSE_0397. The full amino acid sequence of the protein is MELEGLKFLGIGLSVVGMLGAAIGVSNIFSMMLNGIARNPESEEKLKKYVYAGAALTEAMGLFSFVLALLLIFVA, with an expression region of 1-75 amino acids .

How should Recombinant Neorickettsia sennetsu ATP synthase subunit c (atpE) be stored and handled in laboratory settings?

For optimal stability and activity, Recombinant Neorickettsia sennetsu ATP synthase subunit c (atpE) should be stored at -20°C in its shipped buffer (typically Tris-based buffer with 50% glycerol). For extended storage periods, conservation at -20°C or -80°C is recommended. It is crucial to avoid repeated freezing and thawing cycles as these can compromise protein integrity and activity. For ongoing experiments, working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw damage. Before experimental use, allow the protein to equilibrate to room temperature gradually and mix gently without vortexing to prevent denaturation .

What is the biological significance of ATP synthase subunit c in Neorickettsia sennetsu?

ATP synthase subunit c plays a critical role in the bacterial energy metabolism as part of the F0 portion of the ATP synthase complex. In Neorickettsia sennetsu, an obligate intracellular pathogen with a reduced genome, energy production mechanisms are essential for survival within host cells. The atpE protein forms part of the membrane-embedded proton channel that couples proton flow to ATP synthesis. Given that N. sennetsu is the causative agent of Sennetsu neorickettsiosis, a mononucleosis-like disease contracted from consuming raw fish, understanding the energy production mechanisms of this organism provides insights into its pathogenesis and potential therapeutic targets . The protein's highly conserved structure across bacterial species suggests its fundamental importance to cellular bioenergetics, making it a valuable comparative model for studying ATP synthesis mechanisms.

What is the relationship between Neorickettsia sennetsu and human disease?

Neorickettsia sennetsu is the etiologic agent of Sennetsu neorickettsiosis, a largely forgotten infectious mononucleosis-like disease that has been documented primarily in Japan and Malaysia. The infection is believed to be contracted through the consumption of raw fish, which serves as an intermediate host in the pathogen's life cycle. Clinical studies in the Lao PDR, where raw fish consumption is common, revealed that among 91 patients with undifferentiated fever, one patient's buffy coat tested positive for N. sennetsu through 16S rRNA amplification, sequencing, and real-time PCR targeting N. sennetsu genes. Furthermore, serological surveys showed a high prevalence (17%) of IgG antibodies against N. sennetsu among Lao blood donors and patients with fever, hepatitis, or jaundice, compared to much lower rates in Thailand (4%) and Malaysia (0%). The detection of N. sennetsu DNA in a fish (Anabas testudineus) provided the first molecular evidence supporting the fish-borne transmission hypothesis .

What methodologies are most effective for studying the structure-function relationship of Recombinant Neorickettsia sennetsu ATP synthase subunit c (atpE)?

To effectively investigate the structure-function relationship of Recombinant Neorickettsia sennetsu ATP synthase subunit c (atpE), researchers should employ a multi-faceted approach:

• Structural Analysis: X-ray crystallography or cryo-electron microscopy can elucidate the protein's tertiary structure and its arrangement within the F0 complex. Homology modeling based on structurally characterized homologs provides a complementary approach when crystallization proves challenging.

• Mutagenesis Studies: Site-directed mutagenesis of conserved residues, particularly those in the transmembrane regions, can reveal functional domains. For example, substitutions in the glycine-rich regions might affect proton translocation efficiency.

• Reconstitution Assays: Purified atpE can be reconstituted into proteoliposomes to assess its function in membrane permeability and proton transport, similar to methods used for studying other N. sennetsu membrane proteins such as P51, which demonstrated porin activity in proteoliposome swelling assays .

• Protein-Protein Interaction Analysis: Co-immunoprecipitation or crosslinking studies can identify interactions between atpE and other components of the ATP synthase complex or with host cell factors during infection.

• ATP Synthesis Measurement: ATP production can be quantified in reconstituted systems using luciferase-based assays to assess the functional impact of structural modifications.

This integrated approach enables researchers to correlate structural features with functional properties, providing insights into the molecular mechanisms of ATP synthesis in this intracellular pathogen.

How can surface-exposed protein analysis techniques be applied to study Neorickettsia sennetsu ATP synthase components?

Surface-exposed protein analysis of Neorickettsia sennetsu ATP synthase components can be conducted using methodologies demonstrated effective for other N. sennetsu proteins:

• Biotin Surface Labeling: Live bacteria can be labeled with membrane-impermeable biotin reagents (NHS-SS-biotin) that selectively tag surface-exposed proteins. This approach has successfully identified 42 of the 936 (4.5%) N. sennetsu open reading frames as encoding surface-exposed proteins .

• Streptavidin-Affinity Chromatography: Following biotin labeling, surface proteins can be isolated through streptavidin-affinity chromatography, which enriches for biotinylated proteins with minimal contamination from internal bacterial components .

• LC/MS/MS Analysis: Liquid chromatography-tandem mass spectrometry provides high-resolution identification and quantification of the isolated proteins. This technique has previously revealed both expected membrane proteins and hypothetical proteins of unknown function in N. sennetsu .

• Immunofluorescence Confirmation: Surface exposure can be verified through immunofluorescence microscopy using specific antibodies, which can also reveal the distribution pattern of proteins on the bacterial surface. Previous studies with N. sennetsu surface proteins showed distinctive labeling patterns, such as the rosary-like circumferential distribution of P51 and the polar/punctate distribution of Nsp3 .

• Functional Assays: For ATP synthase components, functional characterization can include membrane potential measurements and ATP synthesis assays to correlate surface exposure with biological activity.

These techniques, when applied to ATP synthase components including atpE, can provide insights into their topological arrangement, accessibility to host immune responses, and potential roles beyond ATP synthesis.

What experimental approaches can determine if Neorickettsia sennetsu ATP synthase subunit c (atpE) exhibits porin-like activity similar to other surface proteins?

To investigate potential porin-like activity of Neorickettsia sennetsu ATP synthase subunit c (atpE), researchers can employ the following experimental approaches:

• Proteoliposome Swelling Assays: Purified native or recombinant atpE can be reconstituted into proteoliposomes loaded with isotonic solutes. Exposure to hypotonic solutions containing test substrates will cause proteoliposome swelling if the protein forms channels permeable to these substrates. This technique successfully demonstrated that N. sennetsu P51 exhibits significant porin activity, allowing diffusion of L-glutamine, monosaccharides (arabinose, glucose), and the tetrasaccharide stachyose .

• Electrophysiological Measurements: Planar lipid bilayer recordings can quantify channel conductance and ion selectivity by measuring current flow across membranes containing reconstituted atpE.

• Inhibition Studies: Specific antibodies against atpE can be tested for their ability to block potential porin activity, similar to how anti-P51 antibodies inhibited the porin function of P51 .

• Substrate Specificity Analysis: A range of potential substrates (ions, amino acids, sugars) can be systematically tested to characterize the selectivity profile of any channel activity.

• Comparative Structure Analysis: Computational structural comparisons between atpE and confirmed porins like P51 can identify shared motifs associated with channel formation.

The table below summarizes the previously observed substrate permeation through N. sennetsu P51 porin as a reference for comparison with atpE studies:

These experimental approaches would help determine whether atpE possesses channel-forming capabilities distinct from its primary role in the ATP synthase complex.

How might the functional characterization of Neorickettsia sennetsu ATP synthase subunit c contribute to understanding the organism's adaptation to intracellular parasitism?

The functional characterization of Neorickettsia sennetsu ATP synthase subunit c provides critical insights into how this obligate intracellular bacterium has adapted to its parasitic lifestyle:

• Energy Metabolism Adaptation: Detailed analysis of atpE functionality can reveal how N. sennetsu has optimized its ATP synthesis machinery for the nutrient-rich yet specialized intracellular environment of monocytes and macrophages. Comparative studies with free-living bacteria could highlight modifications that reflect adaptation to intracellular life.

• Host-Pathogen Interface: Given that some bacterial membrane proteins like P51 have been shown to be surface-exposed in N. sennetsu, determining whether atpE is similarly exposed would indicate its potential role in host-pathogen interactions. Surface exposure would suggest functions beyond energy production, potentially including host cell recognition or immune evasion .

• Metabolic Dependency: Characterizing the substrate specificity and regulation of the ATP synthase complex can elucidate how N. sennetsu has evolved dependencies on host-derived metabolites, helping explain its obligate intracellular nature.

• Evolutionary Constraints: Analysis of sequence conservation in atpE across Neorickettsia species compared to other bacterial taxa can identify signatures of selective pressure unique to intracellular pathogens.

• Therapeutic Target Potential: Understanding the unique structural and functional features of N. sennetsu atpE could guide the development of targeted antimicrobial strategies against this and related intracellular pathogens.

This research is particularly relevant given the fish-borne transmission route of N. sennetsu and its ability to cause disease in humans. The high seroprevalence (17%) observed in populations in Lao PDR indicates that N. sennetsu infection may be more common than previously recognized, highlighting the importance of understanding its basic biology and pathogenic mechanisms .

What are the challenges and solutions in designing experiments to study protein-protein interactions involving Neorickettsia sennetsu ATP synthase subunit c in its native environment?

Studying protein-protein interactions involving Neorickettsia sennetsu ATP synthase subunit c in its native environment presents several significant challenges with corresponding methodological solutions:

Challenges:

• Obligate Intracellular Nature: N. sennetsu cannot be cultivated outside host cells, limiting access to native bacterial proteins.

• Low Bacterial Yields: Obtaining sufficient quantities of bacteria for protein interaction studies is difficult due to the intracellular growth requirements.

• Complex Membrane Integration: As a membrane protein, atpE exists in a lipid environment that is difficult to preserve during isolation.

• Transient Interactions: Some protein-protein interactions may be dynamic or condition-dependent.

• Cross-reactivity with Host Proteins: Distinguishing bacterial protein interactions from host protein contamination can be problematic.

Solutions and Methodologies:

• In situ Crosslinking: Chemical crosslinkers that penetrate host cells can stabilize protein-protein interactions before bacterial isolation. Formaldehyde or photoactivatable crosslinkers can capture interactions in the native environment.

• Proximity Labeling: Techniques like BioID or APEX2, where atpE is fused to a proximity-dependent labeling enzyme, can identify neighboring proteins in the native context through biotinylation of proximal proteins.

• Split Fluorescent/Luminescent Reporters: Protein complementation assays using split GFP or luciferase can detect interactions directly in infected cells.

• Co-immunoprecipitation with Verification: Using antibodies against atpE for immunoprecipitation, followed by mass spectrometry identification, with careful controls for host protein contamination.

• Cryo-Electron Tomography: This technique can visualize macromolecular complexes in situ at near-atomic resolution, potentially revealing the structural arrangement of the ATP synthase complex in intact bacteria.

• Heterologous Expression Systems: For initial screening, bacterial two-hybrid systems can identify potential interactions that can later be verified in more native contexts.

These approaches can help overcome the inherent difficulties in studying protein interactions in obligate intracellular bacteria while providing valuable insights into the structural and functional organization of the ATP synthase complex and its potential interactions with host factors during infection.

What are the optimal expression systems and purification strategies for obtaining functional Recombinant Neorickettsia sennetsu ATP synthase subunit c?

The expression and purification of functional Recombinant Neorickettsia sennetsu ATP synthase subunit c requires specialized approaches due to its hydrophobic nature and membrane integration. Based on successful strategies employed for similar bacterial membrane proteins, the following methodologies are recommended:

Expression Systems:

• E. coli C41(DE3) or C43(DE3): These strains are specifically engineered for toxic and membrane protein expression and provide better yields for hydrophobic proteins like atpE.

• Cell-free Expression Systems: These bypass toxicity issues and allow direct incorporation into nanodiscs or liposomes during synthesis.

• Baculovirus-Insect Cell System: For cases where proper folding requires eukaryotic machinery, this system offers advantages for membrane protein expression.

Optimization Strategies:

• Codon Optimization: Adapting the atpE gene sequence to the expression host's codon usage can significantly improve expression levels.

• Fusion Tags: N-terminal tags like MBP (maltose-binding protein) or SUMO can enhance solubility, while C-terminal His6 or Strep tags facilitate purification.

• Induction Conditions: Low temperature induction (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM) often improves the yield of correctly folded membrane proteins.

Purification Protocol:

• Membrane Isolation: Differential centrifugation followed by membrane fractionation using sucrose gradients.

• Solubilization: Mild detergents like n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), or digitonin are preferable for maintaining protein structure.

• Affinity Chromatography: IMAC (immobilized metal affinity chromatography) for His-tagged constructs, followed by size-exclusion chromatography.

• Detergent Exchange: Gradual exchange to a stabilizing detergent suitable for downstream applications.

• Reconstitution: For functional studies, reconstitution into proteoliposomes with E. coli or synthetic lipids that mimic the bacterial membrane composition.

This methodological framework has been successfully applied to other membrane proteins from Neorickettsia, including the purification of native P51 and Nsp3 under non-denaturing conditions, which subsequently retained functional activity when reconstituted into proteoliposomes .

How can researchers effectively design antibodies against Neorickettsia sennetsu ATP synthase subunit c for immunological studies?

Designing effective antibodies against Neorickettsia sennetsu ATP synthase subunit c requires careful consideration of epitope accessibility, specificity, and functional applications. Based on successful approaches used for other N. sennetsu proteins such as Nsp3, the following strategy is recommended:

• Epitope Selection:

  • Analyze the atpE sequence using predictive algorithms (e.g., DNAStar Protean) to identify antigenic regions

  • Target hydrophilic, surface-exposed segments rather than transmembrane domains

  • Select peptides of 12-20 amino acids with high predicted antigenicity and surface probability

  • For membrane proteins like atpE, N- or C-terminal regions often make optimal epitopes

• Peptide Design Strategy:

  • Add a C-terminal cysteine to peptides for conjugation to carrier proteins (similar to the Nsp3 peptide KKDTKLRTKVPASNC used successfully)

  • Ensure the selected peptide has minimal homology with host proteins to reduce cross-reactivity

  • Consider using multiple peptides targeting different regions to increase success probability

• Antibody Production Protocol:

  • Conjugate selected peptides to carrier proteins like KLH or BSA

  • Immunize rabbits using a standard 56-day protocol with multiple booster injections

  • Collect serum and enrich immunoglobulins through ammonium sulfate precipitation

  • Purify specific antibodies using affinity chromatography with immobilized peptide

• Validation Methods:

  • Western blot against recombinant atpE and native protein from N. sennetsu lysates

  • Immunofluorescence microscopy to confirm specificity and determine localization pattern

  • ELISA titration to determine antibody sensitivity

  • Preabsorption controls with immunizing peptide to confirm specificity

• Functional Testing:

  • Assess whether antibodies inhibit ATP synthase activity in functional assays

  • Determine if antibodies affect bacterial viability or host cell infection

This comprehensive approach has been demonstrated effective for generating specific antibodies against N. sennetsu surface proteins, enabling both localization studies and functional characterization through inhibition assays .

What bioinformatic approaches can reveal evolutionary insights about Neorickettsia sennetsu ATP synthase subunit c compared to homologs in other species?

Studying the evolutionary characteristics of Neorickettsia sennetsu ATP synthase subunit c through bioinformatic approaches can provide valuable insights into its adaptation to an intracellular lifestyle. The following comprehensive bioinformatic pipeline is recommended:

• Sequence-Based Analyses:

  • Homology Identification: BLAST and HMMer searches against diverse bacterial genomes to identify homologs across taxonomic groups

  • Multiple Sequence Alignment: MUSCLE or MAFFT alignment of atpE sequences from free-living bacteria, facultative intracellular pathogens, and obligate intracellular bacteria

  • Conservation Analysis: Calculate position-specific conservation scores using methods like Jensen-Shannon divergence to identify functionally constrained residues

  • Selection Pressure Analysis: Calculate dN/dS ratios using PAML or HyPhy to detect sites under positive or purifying selection

• Phylogenetic Analysis:

  • Tree Construction: Maximum likelihood (RAxML/IQ-TREE) or Bayesian inference methods to reconstruct evolutionary relationships

  • Ancestral Sequence Reconstruction: Estimate ancestral sequences to trace the evolution of specific residues

  • Molecular Clock Analysis: Estimate divergence times to correlate with ecological transitions

• Structural Bioinformatics:

  • Homology Modeling: Generate 3D structural models based on crystallized homologs

  • Molecular Dynamics Simulations: Assess structural stability and conformational flexibility in different lipid environments

  • Protein-Protein Interaction Prediction: Identify potential interaction interfaces with other ATP synthase components

• Comparative Genomics:

  • Synteny Analysis: Examine conservation of gene order around atpE across species

  • Co-evolution Analysis: Identify co-evolving residues within the ATP synthase complex

  • Horizontal Gene Transfer Detection: Search for signatures of potential genetic exchange

• Integrative Analysis:

  • Correlation with Host Range: Analyze if atpE sequence features correlate with host specificity

  • Metabolic Pathway Integration: Connect atpE evolution with changes in other energy metabolism genes

  • Pathogenicity Correlation: Assess whether specific variants associate with virulence

This multi-faceted bioinformatic approach can reveal how N. sennetsu atpE has evolved compared to homologs in other bacterial species, potentially identifying adaptations specific to its intracellular lifestyle and providing context for experimental findings. The analysis is particularly valuable given N. sennetsu's role as a human pathogen transmitted through consumption of raw fish, with significant seroprevalence detected in certain populations .

How might understanding Neorickettsia sennetsu ATP synthase subunit c structure and function contribute to novel therapeutic approaches?

Understanding the structure and function of Neorickettsia sennetsu ATP synthase subunit c offers several promising avenues for therapeutic development:

• Target-Based Drug Design: The detailed structural characterization of atpE can enable the rational design of small molecule inhibitors that specifically disrupt ATP synthesis in N. sennetsu. This approach has precedent in other bacterial systems, where ATP synthase inhibitors like bedaquiline have proven effective against Mycobacterium tuberculosis. The unique features of N. sennetsu atpE could allow for selective targeting without affecting host ATP synthesis.

• Epitope-Based Vaccine Development: If atpE is confirmed to be surface-exposed like other N. sennetsu proteins such as P51 and Nsp3, it could serve as an antigenic target for vaccine development. Surface-exposed portions of the protein could be incorporated into subunit vaccines designed to elicit protective antibody responses against N. sennetsu infection. This approach is particularly relevant given the fish-borne transmission route and the significant seroprevalence (17%) detected in populations in Lao PDR .

• Disruption of Protein-Protein Interactions: Identifying critical interactions between atpE and other components of the ATP synthase complex could lead to the development of peptide mimetics or small molecules that disrupt these interfaces, thereby compromising bacterial energy production.

• Allosteric Modulation: Characterizing regulatory mechanisms of ATP synthase activity could reveal allosteric sites that could be targeted to indirectly inhibit enzyme function, potentially offering greater selectivity than active site inhibitors.

• Targeted Delivery Systems: Knowledge of unique structural features of N. sennetsu atpE could inform the development of targeted delivery systems that selectively deliver antimicrobial agents to infected monocytes and macrophages.

The therapeutic potential is particularly significant considering that Sennetsu neorickettsiosis is likely underdiagnosed due to its mononucleosis-like presentation. The disease's association with raw fish consumption makes it a potential public health concern in regions where such dietary practices are common . Development of specific therapeutics would address an underserved disease and potentially provide insights applicable to related rickettsial pathogens.

What experimental approaches would be most effective for studying the role of ATP synthase subunit c in Neorickettsia sennetsu pathogenesis?

Investigating the role of ATP synthase subunit c in Neorickettsia sennetsu pathogenesis requires specialized approaches that address both the protein's function in bacterial energy metabolism and its potential contributions to host-pathogen interactions:

• Cell Culture Infection Models:

  • Conditional Expression Systems: Develop inducible knockdown or overexpression systems for atpE to assess its impact on bacterial replication in host cells

  • Metabolic Flux Analysis: Measure changes in ATP production and energy utilization during different stages of infection

  • Live Cell Imaging: Track ATP synthase dynamics during the infection cycle using fluorescently tagged components

• Host Response Studies:

  • Transcriptomics/Proteomics: Compare host cell responses to wild-type N. sennetsu versus strains with altered atpE expression

  • Immunomodulation Assessment: Determine if atpE or peptides derived from it affect host immune signaling pathways

  • Inflammasome Activation: Investigate if atpE components contribute to inflammasome activation in infected macrophages

• In Vivo Models:

  • Animal Infection Models: Develop suitable models that recapitulate the mononucleosis-like symptoms of Sennetsu neorickettsiosis

  • Bacterial Dissemination Tracking: Monitor the spread of bacteria with modified atpE throughout host tissues

  • Immune Protection Studies: Test if immunization with atpE components provides protection against challenge infection

• Structural-Functional Correlation:

  • Site-Directed Mutagenesis: Create point mutations in key residues to correlate structural features with pathogenic potential

  • Atomic Force Microscopy: Measure biophysical properties of bacterial membranes with altered atpE expression

  • Cross-linking Studies: Identify interactions between atpE and host cell components during infection

• Translational Approaches:

  • Patient Sample Analysis: Compare atpE expression and antibody responses in patients with different disease severities

  • Ex Vivo Infection: Study infection dynamics in primary human monocytes and macrophages

  • Biomarker Discovery: Evaluate if anti-atpE antibodies could serve as diagnostic biomarkers

This multi-faceted approach would provide comprehensive insights into how ATP synthase subunit c contributes to N. sennetsu pathogenesis, potentially revealing dual roles in energy production and host-pathogen interactions. The findings would be particularly valuable given the evidence for fish-borne transmission of N. sennetsu and its potential public health significance in regions where raw fish consumption is common .

What are the key considerations for designing comparative studies between Neorickettsia sennetsu ATP synthase and related components in other intracellular pathogens?

Designing robust comparative studies between Neorickettsia sennetsu ATP synthase and related components in other intracellular pathogens requires careful consideration of multiple factors to ensure meaningful and interpretable results:

• Phylogenetic Context Selection:

  • Include closely related Neorickettsia species (N. helminthoeca, N. risticii) to identify genus-specific adaptations

  • Select representative obligate intracellular bacteria from diverse lineages (Rickettsia, Ehrlichia, Anaplasma, Chlamydia) to detect convergent evolution

  • Include facultative intracellular pathogens (Salmonella, Listeria) as intermediates in the adaptation spectrum

  • Use free-living bacteria as outgroups to establish ancestral states

• Standardized Experimental Design:

  • Employ identical purification protocols across species to minimize methodology-based variations

  • Develop universal antibodies targeting conserved epitopes for consistent detection

  • Establish normalized activity assays that account for different optimal conditions

  • Use consistent host cell systems for infection studies

• Multi-omics Integration:

  • Combine structural analyses, functional assessments, and genomic contexts

  • Correlate ATP synthase variations with global metabolic adaptations

  • Link proteomic profiles with transcriptional regulation patterns

  • Integrate membrane composition data to account for lipid environment differences

• Specialized Analytical Techniques:

  • Structural Comparisons: Use cryo-EM to resolve ATP synthase structures across species in native membranes

  • Functional Assays: Develop reconstitution systems with defined lipid compositions

  • Evolutionary Rate Analysis: Calculate relative evolutionary rates of different ATP synthase components

  • Host Interaction Mapping: Compare interactomes of ATP synthase components across species

• Interpretative Framework Development:

  • Establish criteria to distinguish adaptation from genetic drift

  • Create models linking ATP synthase modifications to ecological niches

  • Develop metrics for quantifying the degree of host dependence

  • Formulate hypotheses regarding the sequential steps in adaptation to intracellular life

This comprehensive approach would enable researchers to distinguish species-specific adaptations from convergent evolutionary patterns, providing insights into how ATP synthase components have been modified during the transition to obligate intracellular lifestyles. Such comparative studies are particularly valuable for understanding N. sennetsu, which causes a distinct mononucleosis-like disease transmitted through consumption of raw fish, with significant seroprevalence in certain geographical regions .

What are common technical challenges in working with Recombinant Neorickettsia sennetsu ATP synthase subunit c and how can they be overcome?

Working with Recombinant Neorickettsia sennetsu ATP synthase subunit c presents several technical challenges common to membrane proteins, particularly from obligate intracellular pathogens. Here are the major challenges and their solutions:

Challenge 1: Low Expression Yields

• Problem: Hydrophobic membrane proteins often express poorly in heterologous systems.

• Solutions:

  • Try specialized E. coli strains (C41/C43) designed for toxic/membrane protein expression

  • Optimize codon usage for the expression host

  • Use fusion partners (MBP, SUMO, Trx) to enhance solubility

  • Consider cell-free expression systems that bypass toxicity issues

  • Lower induction temperature (16-18°C) and IPTG concentration (0.1-0.3 mM)

  • Test insect cell or mammalian expression systems for improved folding

Challenge 2: Protein Aggregation

• Problem: atpE may form inclusion bodies or aggregate during purification.

• Solutions:

  • Screen multiple detergents (DDM, LMNG, digitonin) for optimal solubilization

  • Add stabilizing agents (glycerol, specific lipids) to all buffers

  • Use on-column refolding during affinity purification

  • Consider native nanodiscs or amphipols for maintaining structure

  • Implement gentle purification protocols with minimal temperature fluctuations

Challenge 3: Loss of Activity/Native Structure

• Problem: Purified protein may lose functional conformation during isolation.

• Solutions:

  • Validate structural integrity using circular dichroism spectroscopy

  • Develop functional assays compatible with detergent-solubilized protein

  • Reconstitute into proteoliposomes with lipid compositions mimicking bacterial membranes

  • Use hydrogen-deuterium exchange mass spectrometry to verify proper folding

  • Implement quality control checkpoints throughout the purification process

Challenge 4: Antibody Cross-Reactivity

• Problem: Antibodies may cross-react with host ATP synthase components.

• Solutions:

  • Design peptide antigens unique to N. sennetsu atpE

  • Perform thorough validation with appropriate negative controls

  • Pre-absorb antibodies against host cell lysates

  • Use epitope-tagging strategies when possible

  • Employ multiple antibodies targeting different epitopes for confirmation

Challenge 5: Reconstitution for Functional Studies

• Problem: Achieving proper orientation and density in artificial membranes.

• Solutions:

  • Optimize protein-to-lipid ratios through systematic screening

  • Test different reconstitution methods (detergent removal via dialysis, Bio-Beads, or cyclodextrin)

  • Include known ATP synthase partners to stabilize the complex

  • Verify incorporation and orientation using protease protection assays

  • Use fluorescent labeling to track reconstitution efficiency

These approaches have been successfully applied to other membrane proteins from Neorickettsia sennetsu, such as P51 and Nsp3, enabling their purification under non-denaturing conditions and subsequent functional characterization in proteoliposome systems .

How can researchers optimize assays to measure the functional activity of Neorickettsia sennetsu ATP synthase subunit c?

Optimizing functional assays for Neorickettsia sennetsu ATP synthase subunit c requires careful consideration of its native environment and biophysical properties. The following comprehensive approach addresses both reconstitution strategies and activity measurement techniques:

1. Proteoliposome Reconstitution Optimization:

• Lipid Composition Selection:

  • Test bacterial lipid extracts versus synthetic lipid mixtures (POPC/POPE/POPG)

  • Include cardiolipin, which is critical for ATP synthase function

  • Optimize cholesterol content to match N. sennetsu membrane properties

  • Screen lipid-to-protein ratios (typically between 50:1 to 200:1 by weight)

• Reconstitution Method Refinement:

  • Compare detergent removal techniques (Bio-Beads, dialysis, cyclodextrin)

  • Optimize protein orientation using pH gradients during reconstitution

  • Verify incorporation efficiency using fluorescence quenching

  • Assess homogeneity using dynamic light scattering and electron microscopy

2. ATP Synthesis Activity Measurement:

• Proton Gradient Establishment:

  • Create artificial pH gradients using acid-base transitions

  • Generate electrochemical gradients using valinomycin/K+ systems

  • Quantify gradient formation using pH-sensitive fluorescent dyes

  • Optimize buffer compositions to maintain stable gradients

• ATP Production Quantification:

  • Implement luciferase-based ATP detection systems

  • Use coupled enzyme assays (hexokinase/glucose-6-phosphate dehydrogenase)

  • Develop 32P-ADP incorporation assays for direct measurement

  • Compare initial reaction rates under varying substrate concentrations

3. Proton Translocation Assays:

• Fluorescence-Based Methods:

  • Use ACMA (9-amino-6-chloro-2-methoxyacridine) quenching to monitor proton pumping

  • Implement pyranine-based internal pH measurements

  • Calibrate fluorescence signals with known proton fluxes

  • Optimize fluorophore concentrations to minimize artifacts

• Electrical Measurements:

  • Develop solid-supported membrane electrophysiology protocols

  • Measure capacitive currents during ATP hydrolysis

  • Optimize electrode configurations for maximum signal-to-noise ratio

  • Implement internal controls with ionophores

4. Specific Subunit c Function Assessment:

• Oligomycin Sensitivity Tests:

  • Determine IC50 values for specific ATP synthase inhibitors

  • Compare inhibition profiles with those of other bacterial ATP synthases

  • Map resistance mutations to structure-function relationships

  • Develop competition assays with labeled inhibitors

• Rotational Catalysis Analysis:

  • Implement single-molecule FRET to detect conformational changes

  • Develop gold nanorod attachment strategies for visualization of rotation

  • Optimize imaging conditions for sustained recording

  • Correlate rotational events with ATP synthesis/hydrolysis

These optimized assays will enable comprehensive functional characterization of N. sennetsu ATP synthase subunit c, building upon methods that have proven successful for other membrane proteins from this organism, such as the P51 porin function assessment in proteoliposome swelling assays .