Recombinant Salmonella choleraesuis ATP synthase subunit b (atpF)

What is the function of ATP synthase subunit b in Salmonella choleraesuis?

ATP synthase subunit b (atpF) functions as a critical structural component of the F₀ sector of the ATP synthase complex in Salmonella choleraesuis. This protein serves as a peripheral stalk that connects the membrane-embedded F₀ sector to the catalytic F₁ sector, thereby facilitating the mechanical coupling necessary for ATP synthesis. The protein's structure includes a transmembrane domain that anchors it in the bacterial cell membrane and a helical domain that extends into the cytoplasm to interact with the F₁ sector. This structural arrangement enables the protein to participate in the rotational mechanism that couples proton translocation to ATP synthesis, essential for bacterial energy metabolism under various environmental conditions .

How is recombinant Salmonella choleraesuis ATP synthase subunit b typically produced for research purposes?

Recombinant Salmonella choleraesuis ATP synthase subunit b is typically produced using prokaryotic expression systems, most commonly E. coli, transformed with a plasmid containing the atpF gene sequence from Salmonella choleraesuis (strain SC-B67). The expressed protein is then purified using affinity chromatography methods appropriate to the tag system employed during the production process. The specific tag type is determined during the production process based on experimental requirements. After purification, the protein is typically stored in a Tris-based buffer containing 50% glycerol, which has been optimized for protein stability. For research applications, the protein is typically provided at a concentration sufficient for experimental use, with standard quantities around 50 μg, though other quantities can be produced as needed for specific research applications .

What are the recommended storage conditions for maintaining the stability of recombinant atpF protein?

For optimal stability of recombinant Salmonella choleraesuis ATP synthase subunit b, short-term storage should be maintained at -20°C in the provided Tris-based buffer with 50% glycerol. For extended storage periods, it is recommended to store the protein at either -20°C or -80°C, with the latter providing better long-term stability by minimizing protein degradation and denaturation. It is crucial to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity through structural damage and aggregation. If the protein will be used frequently over a short period, working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw damage. Proper storage conditions are essential for maintaining the native conformation and biological activity of the protein for reliable experimental results .

How does the structure of Salmonella choleraesuis ATP synthase subunit b compare to homologous proteins in other bacterial pathogens?

In methodological terms, researchers investigating these structural relationships should employ multiple sequence alignment with CLUSTAL or MUSCLE algorithms followed by homology modeling using software like SWISS-MODEL or Phyre2. Specific attention should be paid to the dimerization interface residues that are critical for proper assembly and function. Surface electrostatic potential mapping can reveal differences in charge distribution that may influence interaction with other ATP synthase components or potential inhibitors. These comparative structural analyses provide valuable insights for developing species-specific inhibitors that could serve as narrow-spectrum antimicrobial agents targeting the ATP synthase complex .

What experimental approaches are most effective for investigating interactions between ATP synthase subunit b and other components of the ATP synthase complex?

Investigation of interactions between ATP synthase subunit b and other components of the ATP synthase complex requires a multi-technique approach. Crosslinking mass spectrometry (XL-MS) provides valuable spatial constraints by identifying amino acid residues in close proximity between interacting proteins. For this method, researchers should use membrane-permeable crosslinkers like DSS or BS3 followed by proteolytic digestion, enrichment of crosslinked peptides, and LC-MS/MS analysis. Co-immunoprecipitation coupled with Western blot analysis can verify protein-protein interactions, though care must be taken with membrane proteins to use appropriate detergents that maintain native structure.

For more refined analysis, surface plasmon resonance (SPR) or microscale thermophoresis (MST) can quantify binding affinities between purified subunit b and other ATP synthase components. Researchers should also consider hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of subunit b that undergo conformational changes upon interaction with partner proteins. Fluorescence resonance energy transfer (FRET) can be employed to study these interactions in living bacterial cells when combined with fluorescent protein tagging strategies. For validating interaction sites, researchers should implement site-directed mutagenesis of predicted interface residues followed by functional assays measuring ATP synthesis or proton translocation efficiency .

What methodological approaches are recommended for investigating the role of ATP synthase subunit b in bacterial pathogenesis?

Investigating the role of ATP synthase subunit b in bacterial pathogenesis requires a comprehensive approach combining genetic manipulation, infection models, and systems biology. Researchers should first develop targeted gene knockout or conditional expression systems for atpF in Salmonella choleraesuis using CRISPR-Cas9 or allelic exchange methodologies. These mutant strains should then be characterized for growth kinetics in various media conditions that mimic host environments, including acidic pH, limited nutrients, and oxidative stress.

In vitro infection models using relevant cell lines (e.g., intestinal epithelial cells, macrophages) can assess the impact of atpF manipulation on bacterial adhesion, invasion, and intracellular survival. These experiments should include time-course studies to capture dynamic changes in host-pathogen interactions. For in vivo relevance, researchers should utilize appropriate animal models of salmonellosis to measure colonization efficiency, dissemination patterns, and disease severity.

Transcriptomic and proteomic analyses comparing wild-type and atpF-modified strains under infection-relevant conditions can reveal broader metabolic and virulence factor changes resulting from ATP synthase disruption. Metabolomic profiling focusing on energy metabolism intermediates can provide insights into how ATP synthase dysfunction affects bacterial metabolism during infection. Additionally, researchers should investigate whether ATP synthase components, including subunit b, are recognized by the host immune system using serum antibody profiling from infected hosts .

What techniques are most suitable for analyzing the structure-function relationship of recombinant ATP synthase subunit b?

For comprehensive structure-function analysis of recombinant ATP synthase subunit b, researchers should implement a combination of biophysical techniques, computational methods, and functional assays. Circular dichroism (CD) spectroscopy provides essential information about secondary structure content and thermal stability of the purified protein. Nuclear Magnetic Resonance (NMR) spectroscopy is particularly valuable for studying the solution structure of specific domains, especially the hydrophilic regions extending into the cytoplasm.

For regions that prove difficult to analyze by experimental methods, researchers should employ molecular dynamics (MD) simulations to predict conformational flexibility and identify potential functional motifs. These computational predictions should then be validated through site-directed mutagenesis targeting conserved residues, followed by functional reconstitution assays measuring ATP synthesis activity.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map solvent-accessible regions and conformational changes under different physiological conditions. For studying membrane insertion and orientation, researchers should utilize oriented CD spectroscopy or solid-state NMR with isotopically labeled protein. Protein crosslinking coupled with mass spectrometry (XL-MS) provides valuable information about the spatial relationship between different regions of the protein and its interaction partners within the ATP synthase complex. Collectively, these approaches can generate a comprehensive understanding of how specific structural elements contribute to the function of ATP synthase subunit b in energy transduction .

How can researchers effectively measure the functional activity of recombinant ATP synthase subunit b in reconstituted systems?

Measuring the functional activity of recombinant ATP synthase subunit b requires reconstitution into systems that approximate its native environment. The most direct approach involves reconstituting the complete ATP synthase complex into liposomes, incorporating purified recombinant subunit b along with other isolated ATP synthase components. Researchers should establish a proton gradient across the liposome membrane using techniques such as acid-base transition or valinomycin-mediated potassium diffusion, then measure ATP synthesis rates using luciferin-luciferase assays that provide real-time luminescence readouts.

For analyzing specific aspects of subunit b function, researchers can develop partial reconstitution systems. The interaction between subunit b and the F₁ sector can be measured using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding kinetics and thermodynamic parameters. The dimerization properties of subunit b can be analyzed using analytical ultracentrifugation or size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS).

To assess the impact of specific mutations or post-translational modifications, researchers should compare the functional parameters of wild-type versus modified proteins in these reconstituted systems. Fluorescence-based assays using environment-sensitive probes attached at specific positions can provide insights into conformational changes during the catalytic cycle. Each functional assay should include appropriate controls, including negative controls lacking essential components and positive controls with well-characterized bacterial ATP synthase complexes .

What computational methods are recommended for predicting post-translational modifications and their impact on ATP synthase subunit b function?

For predicting post-translational modifications (PTMs) in ATP synthase subunit b, researchers should implement a multi-algorithm approach combining both general and bacteria-specific PTM prediction tools. NetPhos, PhosphoSitePlus, and GPS can identify potential phosphorylation sites, while bacteria-specific tools like NetBacPhos focus on bacterial phosphorylation patterns. For analyzing other modifications, researchers should use CSS-Palm for palmitoylation, UbPred for ubiquitination, and NetOGlyc and NetNGlyc for glycosylation, though the latter modifications are less common in bacterial proteins.

After identifying potential PTM sites, molecular dynamics simulations should be employed to predict how these modifications might alter protein structure, flexibility, and interaction interfaces. These simulations should compare the native protein versus models with simulated PTMs, focusing on regions involved in dimer formation and interactions with other ATP synthase components.

To validate computational predictions, researchers should use site-directed mutagenesis to create phosphomimetic mutations (e.g., serine to aspartate) or non-modifiable variants (e.g., serine to alanine) at predicted PTM sites. These mutants should then be analyzed for altered stability, interactions, and function in reconstitution assays. Mass spectrometry approaches, including targeted multiple reaction monitoring (MRM), can detect and quantify actual PTMs under different physiological conditions. Researchers should also investigate whether predicted PTMs are differentially present during various growth conditions or infection stages, potentially indicating regulatory roles in ATP synthase function or assembly .

What are the optimized protocols for using recombinant ATP synthase subunit b in structural studies?

For structural studies of recombinant ATP synthase subunit b, researchers must carefully optimize sample preparation to account for the protein's membrane association and structural complexity. Beginning with expression and purification, high yields of correctly folded protein can be achieved using bacterial expression systems with specialized vectors containing fusion partners that enhance solubility (such as MBP or SUMO). For membrane domain studies, expression in cell-free systems supplemented with detergent micelles or nanodiscs often produces better results than traditional in vivo expression.

For X-ray crystallography, researchers should screen multiple detergents (including DDM, LDAO, and CHAPS) during purification to identify conditions that maintain native structure while promoting crystal formation. Lipidic cubic phase (LCP) crystallization has proven particularly effective for membrane proteins like ATP synthase components. If crystallization proves challenging, cryo-electron microscopy offers an alternative approach, especially for visualizing subunit b in the context of larger ATP synthase subcomplexes.

For NMR studies, researchers should initially focus on the soluble domain using truncated constructs, then gradually incorporate the membrane domain using appropriate membrane mimetics. Specific isotope labeling strategies (¹⁵N, ¹³C, ²H) are essential for reducing spectral complexity. For the transmembrane domain, solid-state NMR using oriented bicelle samples can provide valuable structural constraints. Importantly, all structural studies should include functional validation to ensure that the observed structures represent physiologically relevant conformations .

How can researchers design effective antibodies against Salmonella choleraesuis ATP synthase subunit b for immunological studies?

Designing effective antibodies against Salmonella choleraesuis ATP synthase subunit b requires careful epitope selection and validation strategies. Researchers should begin with computational epitope prediction using tools such as BepiPred, Ellipro, or ABCpred, focusing on regions with high surface accessibility, hydrophilicity, and predicted antigenicity. Researchers should prioritize epitopes unique to Salmonella choleraesuis to minimize cross-reactivity with homologous proteins from other species, particularly in the cytoplasmic domain which shows more sequence divergence than the transmembrane region.

For antibody production, researchers can use two complementary approaches: (1) peptide immunization using synthesized peptides corresponding to predicted epitopes conjugated to carrier proteins like KLH; and (2) recombinant protein immunization using purified soluble domains of ATP synthase subunit b. Multiple host species (rabbit, mouse, chicken) should be considered to maximize epitope recognition diversity.

Post-production validation is critical and should include ELISA, Western blotting, and immunofluorescence microscopy using both recombinant protein and native Salmonella lysates. Cross-reactivity testing against related bacterial species is essential for confirming specificity. For advanced applications, researchers should further characterize antibody affinity using surface plasmon resonance (SPR) and epitope binning to select complementary antibodies that recognize different regions of the protein. These characterized antibodies can then be effectively deployed in various immunological applications, including tracking ATP synthase localization during infection processes .

What methodological approaches should be used to investigate potential inhibitors targeting ATP synthase subunit b for antimicrobial development?

Investigating potential inhibitors targeting ATP synthase subunit b requires a systematic approach spanning from in silico screening to in vivo validation. Researchers should begin with computational structure-based drug design, utilizing homology models of Salmonella choleraesuis ATP synthase subunit b to identify potential binding pockets, particularly at interfaces critical for protein-protein interactions within the ATP synthase complex. Virtual screening of compound libraries should prioritize molecules predicted to disrupt these interactions rather than simply binding to the protein.

For biochemical screening, researchers should develop assay systems that specifically measure subunit b function. These could include FRET-based assays measuring conformational changes or disruption of protein-protein interactions, surface plasmon resonance (SPR) to quantify binding affinities, and thermal shift assays to detect compound-induced stability changes. Compounds showing activity in these primary screens should progress to functional assays using reconstituted ATP synthase complexes in liposomes, measuring inhibition of ATP synthesis or proton translocation.

Promising candidate inhibitors should be tested against whole bacterial cells to assess antimicrobial activity, membrane permeability, and target engagement within living bacteria. Target validation can be performed using resistant mutant generation and whole-genome sequencing to confirm that resistance mutations map to the atpF gene. Researchers should also evaluate potential cytotoxicity against mammalian cells using standard cell viability assays and mitochondrial function tests, as mammalian cells also contain ATP synthase complexes. Structure-activity relationship studies should guide compound optimization, focusing on enhancing bacterial selectivity while maintaining antimicrobial efficacy .

How can researchers effectively analyze the expression patterns of ATP synthase subunit b under different environmental conditions?

To comprehensively analyze ATP synthase subunit b expression patterns under varying environmental conditions, researchers should implement a multi-omics approach. At the transcriptional level, quantitative RT-PCR offers precise measurement of atpF gene expression, while RNA-seq provides a broader perspective by simultaneously monitoring the entire ATP synthase operon and related energy metabolism genes. For high-throughput analysis across multiple conditions, researchers can develop reporter strains with the atpF promoter fused to fluorescent proteins or luciferase.

At the protein level, targeted quantitative proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) mass spectrometry provides absolute quantification of ATP synthase subunit b. These methods should employ isotopically labeled peptide standards derived from unique regions of the protein. Western blotting with validated antibodies offers a more accessible approach for relative quantification across conditions.

Table: Biochemical Properties of Recombinant Salmonella choleraesuis ATP synthase subunit b

What are the key research findings regarding ATP synthase as a potential antimicrobial target in Salmonella species?

Recent research has established ATP synthase as a promising antimicrobial target in Salmonella species, with several key findings supporting this approach. Studies have demonstrated that ATP synthase activity is essential for Salmonella survival during infection, particularly when bacteria transition between host environments with varying pH and nutrient availability. Unlike some other bacteria, Salmonella lacks alternative energy generation pathways that could fully compensate for ATP synthase inhibition, creating a metabolic vulnerability that can be therapeutically exploited.

Structural analysis has revealed several unique features in Salmonella ATP synthase compared to human mitochondrial ATP synthase, particularly in the membrane-embedded F₀ sector where subunit b resides. These differences provide potential binding sites for selective inhibitors that could target bacterial ATP synthase while sparing the human homolog. Molecular dynamics simulations have identified specific interaction interfaces between subunit b and other ATP synthase components that could be disrupted by small molecules or peptide mimetics.

Experimental validation using genetic approaches has confirmed that attenuation of ATP synthase function reduces Salmonella virulence in animal models. Specifically, mutations affecting ATP synthase assembly or function lead to decreased colonization ability and reduced survival within macrophages. Preliminary screening efforts have identified several chemical scaffolds that selectively inhibit bacterial ATP synthase activity, with some compounds showing promising in vitro activity against multidrug-resistant Salmonella isolates. These findings collectively support ongoing efforts to develop ATP synthase inhibitors as a novel class of antimicrobials with potential efficacy against Salmonella infections resistant to conventional antibiotics .

How does the amino acid sequence of ATP synthase subunit b contribute to its functional properties in ATP synthesis?

The amino acid sequence of ATP synthase subunit b contains several distinct domains that directly contribute to its functional properties in ATP synthesis. The N-terminal region (approximately residues 1-30) is rich in hydrophobic amino acids forming a transmembrane alpha-helix that anchors the protein in the bacterial membrane. This precise membrane positioning is critical for proper assembly of the entire ATP synthase complex and for establishing the correct spatial relationship between the F₀ and F₁ sectors.

The central portion of the protein (approximately residues 31-110) forms an extended alpha-helical structure that participates in dimerization with another subunit b molecule. This dimerization is facilitated by a specific pattern of hydrophobic residues creating a coiled-coil interaction. Mutational studies have demonstrated that disruption of this dimerization interface severely compromises ATP synthase assembly and function. The precise length of this region serves as a molecular ruler that determines the distance between the membrane sector and the catalytic domain.