Recombinant Bartonella tribocorum ATP synthase subunit delta (atpH)

What is the structural organization of Bartonella tribocorum ATP synthase and where does the delta subunit fit?

The ATP synthase in B. tribocorum, like other bacterial F1Fo ATP synthases, consists of two major domains: the membrane-embedded Fo domain and the catalytic F1 domain. The delta (δ) subunit functions as part of the stator, connecting the α3β3 hexamer of F1 to the membrane-bound Fo domain. This connection is crucial for maintaining the proper orientation of the catalytic sites during rotational catalysis. The δ subunit interacts with both the F1 domain (primarily through the α subunits) and components of the Fo domain, particularly the b subunit . Unlike the ε subunit which has been extensively characterized in some bacteria as having regulatory functions through ATP binding, the δ subunit primarily serves a structural role in maintaining the integrity of the enzyme complex.

How does the atpH gene expression respond to environmental pH conditions in Bartonella?

Expression of ATP synthase components, including atpH, demonstrates pH-dependent regulation in many bacteria. While specific data for B. tribocorum is limited, research on related systems suggests that expression may increase under alkaline conditions where ATP synthesis is favored over ATP hydrolysis. In facultative alkaliphiles like C. thermarum, ATP synthase components show altered expression patterns between pH 7.0-9.5, with optimal expression at pH 9.5 . For B. tribocorum, researchers should consider examining atpH expression across a pH range of 6.5-8.5 to determine if similar regulatory patterns exist. Quantitative PCR and western blot analysis using anti-atpH antibodies can effectively track these expression changes.

What sequence homology exists between B. tribocorum atpH and those of other bacterial species?

Sequence alignment analysis reveals that B. tribocorum atpH shares significant homology with delta subunits from other alpha-proteobacteria. While detailed structural data specific to B. tribocorum is not available in current literature, comparative analysis with characterized homologs suggests conservation of key functional domains. Researchers should note that despite sequence similarities, species-specific variations in certain residues may significantly impact protein-protein interactions within the ATP synthase complex. When designing experiments, these potential variations should be considered, especially when extrapolating functional data from model organisms to B. tribocorum.

What expression systems yield optimal production of soluble recombinant B. tribocorum atpH?

For successful expression of soluble recombinant B. tribocorum atpH, E. coli BL21(DE3) remains the preferred expression system when combined with specific optimization strategies:

The addition of 500 mM NaCl and 10% glycerol to lysis buffers significantly improves solubility. Co-expression with chaperones (GroEL/GroES) may further enhance proper folding of the recombinant protein. When using the BL21(DE3) system, maintaining post-induction temperature below 20°C is critical for reducing inclusion body formation.

What purification strategies yield the highest purity and functional integrity for recombinant atpH?

A multi-step purification approach is recommended for obtaining high-purity functional atpH:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and an imidazole gradient (20-250 mM).

• Intermediate purification: Ion exchange chromatography using Q-Sepharose at pH 8.0 effectively separates atpH from remaining contaminants.

• Polishing step: Size exclusion chromatography using Superdex 75 in a buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5% glycerol.

Throughout purification, maintaining buffer pH between 7.5-8.0 is crucial as the protein stability appears pH-dependent, similar to other ATP synthase components . Adding 1 mM DTT to all buffers helps maintain the native conformation by preventing oxidation of cysteine residues. The purified protein should be assessed using SDS-PAGE and western blotting techniques similar to those described for other ATP synthase components .

What analytical methods are most effective for assessing the structural integrity of purified atpH?

Multiple complementary approaches should be employed to thoroughly characterize the structural integrity of purified atpH:

• Circular dichroism (CD) spectroscopy: Use far-UV CD (190-260 nm) to analyze secondary structure content and thermal stability through melting curves.

• Intrinsic fluorescence spectroscopy: Measure the emission spectrum (310-400 nm) following excitation at 280 nm to assess tertiary structure integrity.

• Limited proteolysis: Digestion with trypsin or chymotrypsin followed by mass spectrometry analysis to identify stable domains and flexible regions.

• Dynamic light scattering: Determine the hydrodynamic radius and polydispersity to assess homogeneity and potential oligomerization.

• Nuclear magnetic resonance (NMR): For detailed structural analysis if isotopically labeled protein can be produced.

These methods, when used in combination, provide comprehensive information about the protein's folding state, stability, and structural features. Similar analytical approaches have been successfully applied to other ATP synthase components, as demonstrated in studies of the ε subunit .

How can researchers effectively study atpH interactions with other ATP synthase subunits?

To investigate interactions between atpH and other ATP synthase components, researchers should employ multiple complementary approaches:

• Pull-down assays: Using His-tagged atpH as bait to capture interacting partners from B. tribocorum lysate, followed by mass spectrometry identification.

• Surface plasmon resonance (SPR): Immobilize purified atpH on a sensor chip and measure binding kinetics with other purified subunits (particularly the b and α subunits).

• Isothermal titration calorimetry (ITC): Determine binding affinities and thermodynamic parameters of interactions.

• Chemical cross-linking coupled with mass spectrometry (XL-MS): Identify specific residues involved in subunit interactions.

• Fluorescence resonance energy transfer (FRET): For monitoring dynamic interactions in reconstituted systems.

Interactions should be studied across a pH range (7.0-8.5) to determine if they exhibit pH dependency similar to that observed with the ε subunit in C. thermarum . When designing these experiments, researchers should consider that subunit interactions might be modulated by nucleotide binding, as demonstrated for other ATP synthase components.

What is known about potential post-translational modifications of B. tribocorum atpH and how can they be characterized?

While specific post-translational modifications (PTMs) of B. tribocorum atpH have not been extensively characterized, related research suggests several possible modifications that warrant investigation:

• Phosphorylation: May regulate assembly or activity of the ATP synthase complex.

• AMPylation: Given the presence of AMPylation mechanisms in Bartonella species through FIC domains , this modification should be investigated as a potential regulatory mechanism.

• Acetylation: Often regulates bacterial metabolic enzymes in response to carbon source availability.

To identify and characterize these PTMs:

• Mass spectrometry: Use high-resolution LC-MS/MS with enrichment strategies specific to each PTM type.

• Phospho-specific or acetylation-specific antibodies: For western blot detection.

• In vitro modification assays: Test whether B. tribocorum kinases, acetylases, or AMPylating enzymes (such as Fic proteins) modify purified atpH .

• Mutational analysis: Create variants where predicted modification sites are mutated to non-modifiable residues to assess functional consequences.

The potential for AMPylation is particularly interesting given the presence of FIC domain proteins in Bartonella that mediate AMPylation of target proteins .

How does pH affect the function and interactions of atpH in the context of the complete ATP synthase complex?

pH-dependent regulation appears to be a significant factor in ATP synthase function, as demonstrated by studies showing pH-dependent ATP binding in the ε subunit of C. thermarum . For B. tribocorum atpH, researchers should investigate:

• pH-dependent conformational changes: Using circular dichroism and fluorescence spectroscopy to monitor structural changes across a pH range of 6.5-9.0.

• Interaction studies: Performing pull-down or co-immunoprecipitation assays at different pH values to determine if atpH-subunit interactions are pH-sensitive.

• Reconstitution experiments: Incorporating purified atpH into partially assembled ATP synthase complexes at varying pH to assess assembly efficiency.

• Activity assays: Measuring ATP synthesis/hydrolysis rates of reconstituted complexes with wild-type versus mutant atpH across a pH spectrum.

These studies are particularly relevant given that Bartonella species must adapt to different pH environments during their infection cycle. The observed pH-dependent ATP binding in C. thermarum (with distinct affinity clusters either side of pH 7.75) suggests that similar pH-sensitive mechanisms might exist in B. tribocorum to regulate ATP synthase function in response to environmental conditions.

What role might atpH play in Bartonella tribocorum adaptation to different host environments?

ATP synthase components, including atpH, likely contribute significantly to B. tribocorum's adaptation to varying host environments. During infection, Bartonella encounters different pH conditions and energy availability states that require metabolic adaptation:

• Erythrocyte infection phase: Within erythrocytes, the bacterium faces a relatively stable pH environment but limited nutrient availability, potentially requiring ATP synthase optimization for energy conservation.

• Endothelial cell infection phase: The pH gradient across endothelial cell membranes may necessitate adjustments in ATP synthase function.

• Stress response: During transitions between hosts or host cell types, ATP synthase regulation via structural components like atpH may help Bartonella respond to changing energy demands.

Studies should focus on comparing atpH expression and ATP synthase assembly during different infection phases. Unlike other bacteria where ATP synthases can operate bidirectionally, some bacterial ATP synthases appear mono-directional (like in C. thermarum) , suggesting specialized adaptations that might also exist in Bartonella species. Researchers should investigate whether atpH contributes to any potential directionality preference in B. tribocorum ATP synthase function.

Can atpH be targeted for therapeutic intervention against Bartonella infections?

The essential nature of ATP synthase for bacterial viability makes atpH a potential therapeutic target, though several factors require consideration:

• Structural uniqueness: Comparative sequence analysis between human and B. tribocorum ATP synthase components can identify regions unique to the bacterial protein that could be targeted with minimal host toxicity.

• Drug accessibility: Since ATP synthase is membrane-associated, drug design must account for penetration of both the bacterial outer membrane and potentially host cell membranes during intracellular infection phases.

• Binding pocket characterization: Computational analysis and structural studies can identify potential binding pockets in atpH that differ from human homologs.

• Small molecule screening: High-throughput screening against purified atpH can identify compounds that disrupt its interaction with other ATP synthase components.

• Peptide inhibitors: Designing peptides that mimic interaction interfaces between atpH and other subunits to competitively inhibit ATP synthase assembly.

When developing therapeutic approaches, researchers should consider that targeting structural components like atpH might be more effective than targeting catalytic sites, as it could avoid resistance mechanisms that often arise with active site mutations.

How does atpH contribute to Bartonella tribocorum energy metabolism during different phases of infection?

The role of atpH in energy metabolism likely varies during the complex infection cycle of B. tribocorum:

• During initial colonization: ATP synthesis may be prioritized to support rapid growth and establishment of infection, with atpH ensuring proper ATP synthase assembly and function.

• During persistent infection: Energy conservation may become more important, potentially involving structural adjustments in the ATP synthase complex that could involve atpH.

• During nutrient limitation: The ATP synthase might need to operate with maximal efficiency, requiring optimal interactions between all components including atpH.

Research approaches should include:

• Gene expression analysis: Comparing atpH expression levels during different infection phases using qRT-PCR.

• Conditional knockdown experiments: Using inducible systems to reduce atpH expression at different infection stages and assess consequences.

• Measurement of ATP/ADP ratios: In wild-type versus atpH-deficient strains under various conditions.

• Metabolomics analysis: To determine shifts in energy metabolism pathways when atpH function is compromised.

These studies should consider the pH-dependent nature of ATP synthase function observed in related systems , as B. tribocorum likely experiences pH fluctuations during its infectious cycle that could influence atpH function.

What are the most effective strategies for creating site-directed mutations in B. tribocorum atpH for structure-function studies?

Site-directed mutagenesis of atpH presents several technical challenges requiring specific methodological approaches:

Based on studies of other ATP synthase components, researchers should prioritize mutations of residues potentially involved in subunit interactions or those conserved among Bartonella species but divergent from host homologs.

How can researchers effectively assess the impact of atpH mutations on ATP synthase assembly and function?

A multi-layered approach is necessary to comprehensively evaluate how atpH mutations affect ATP synthase function:

• In vitro assembly assays:

  • Reconstitution of ATP synthase complexes using purified components with wild-type or mutant atpH.

  • Blue native PAGE to analyze complex formation and stability.

  • Analytical ultracentrifugation to determine complex stoichiometry and integrity.

• Functional assays:

  • ATP synthesis/hydrolysis assays using reconstituted complexes in liposomes.

  • Proton pumping assays using pH-sensitive fluorescent dyes.

  • Membrane potential measurements using potentiometric dyes.

• In vivo assessment:

  • Complementation studies in atpH-deficient strains.

  • Growth rate analysis under different energy source conditions.

  • Competitive fitness assays comparing wild-type and mutant strains.

• Structural analysis:

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics.

  • Cross-linking mass spectrometry to map changes in subunit interactions.

Researchers should assess these parameters across different pH conditions (pH 7.0-8.5) to determine if mutations affect any pH-dependent functional characteristics, similar to those observed in the ATP synthase ε subunit .

What considerations are important when designing experiments to study potential regulatory roles of atpH in ATP synthase activity?

When investigating potential regulatory roles of atpH, researchers should consider:

• Environmental conditions that might trigger regulatory changes:

  • pH variations (pH 6.5-8.5) that might alter protein conformation or interactions.

  • ATP/ADP ratio fluctuations that could influence complex assembly or stability.

  • Ionic strength variations that might affect subunit interactions.

• Potential regulatory mechanisms:

  • Conformational changes in atpH affecting its interaction with other subunits.

  • Post-translational modifications such as phosphorylation or AMPylation .

  • Protein-protein interactions with regulatory factors outside the ATP synthase complex.

• Experimental designs:

  • Use of fluorescently labeled atpH to monitor conformational changes in real-time.

  • Development of conformation-specific antibodies to detect regulatory states.

  • Application of hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility under different conditions.

• Controls and validation:

  • Comparison with other ATP synthase components known to have regulatory roles, such as the ε subunit.

  • Use of atpH variants with mutations in potential regulatory sites.

  • Correlation of in vitro observations with in vivo phenotypes.

These approaches should be informed by the understanding that bacterial ATP synthases often have evolved specialized regulatory mechanisms to respond to their unique environmental niches, as demonstrated by the pH-dependent ATP binding observed in C. thermarum .