Recombinant Salmonella paratyphi B ATP synthase subunit b (atpF)

What is ATP synthase subunit b (atpF) and its functional role in Salmonella paratyphi B?

ATP synthase subunit b (atpF) is a critical component of the F0 sector of ATP synthase in Salmonella paratyphi B. This protein functions as part of the stator, connecting the F1 catalytic domain to the membrane-embedded F0 domain. The b subunit forms a dimer that helps prevent rotation of the F1 domain, thereby enabling the rotational catalysis mechanism that produces ATP. In the complete ATP synthase complex, the rotation of the c-ring (driven by proton translocation) is coupled to the rotation of the γ-stalk in the F1 region, where the γ-subunit functions as a shaft inside the α3β3 head, driving the catalysis of ATP production .

The atpF gene in Salmonella paratyphi B encodes a 156-amino acid protein that is highly conserved among bacterial species. The amino acid sequence is:
MNLNATILGQAIAFILFVWFCMKYVWPPLMAAIEKRQKEIADGLASAERAHKDLDLAKASATDQLKKAKAEAQVIIEQANKRRAQILDEAKTEAEQERTKIVAQAQAEIEAERKRAREEL RKQVAILAVAGAEKIIERSVDEAANSDIVDKLVAEL

What expression systems are recommended for recombinant production of Salmonella paratyphi B atpF?

The most widely used expression system for recombinant Salmonella paratyphi B ATP synthase subunit b is the Escherichia coli-based expression system. This approach offers several advantages:

• High yield production with well-established protocols

• Compatibility with various induction systems (IPTG, arabinose, etc.)

• Availability of specialized strains optimized for membrane protein expression

For optimal expression, consider using E. coli strains such as BL21(DE3), C41(DE3), or C43(DE3), which are designed to handle potentially toxic membrane proteins. The recombinant protein is typically expressed with an N-terminal 10xHis-tag to facilitate purification . When designing your expression construct, it's important to optimize codon usage for E. coli and include appropriate regulatory elements.

Experimental evidence suggests that in vitro E. coli expression systems can successfully produce full-length atpF protein (residues 1-156) in quantities sufficient for biochemical and structural studies . Similar approaches have been successfully used for other ATP synthase subunits, such as the c subunit from spinach chloroplast .

What purification strategies yield high-purity recombinant atpF protein?

Purification of recombinant Salmonella paratyphi B ATP synthase subunit b requires a multi-step approach to achieve high purity while maintaining protein structure and function:

Step 1: Cell lysis and membrane fraction isolation

• Use mechanical disruption (sonication or French press) in a buffer containing 20-50 mM Tris/PBS (pH 8.0)

• Add protease inhibitors to prevent degradation

• Separate membrane fraction by ultracentrifugation (100,000g for 1 hour)

Step 2: Detergent solubilization

• Solubilize membrane proteins using mild detergents (DDM, LDAO, or C12E8)

• Incubate with gentle agitation at 4°C for 1-2 hours

Step 3: Affinity chromatography

• Apply solubilized fraction to Ni-NTA column for His-tagged protein

• Use step-wise imidazole gradient for elution (50 mM, 100 mM, 250 mM)

• Monitor elution with SDS-PAGE and Western blot

Step 4: Size exclusion chromatography

• Further purify by gel filtration to remove aggregates and obtain homogeneous protein

• Use buffer containing 0.05% detergent to maintain protein stability

The purified protein can be stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0. For long-term storage, lyophilization is recommended, as the shelf life of lyophilized form is typically 12 months at -20°C/-80°C compared to 6 months for liquid form .

How can I confirm the proper folding and oligomeric state of purified recombinant atpF?

To assess the proper folding and oligomeric state of purified recombinant Salmonella paratyphi B ATP synthase subunit b, several complementary techniques should be employed:

The crystal structure of the dimerization domain (residues 62-122) of ATP synthase subunit b has been solved at 1.55 Å resolution, revealing an α-helical structure with a length of 90 Å . This information can serve as a reference point for validating your recombinant protein structure.

What are the critical regions of atpF for dimerization and functional interactions within the ATP synthase complex?

The ATP synthase subunit b (atpF) contains distinct functional domains that are critical for its role in the ATP synthase complex:

• N-terminal membrane anchor domain (residues 1-30)

  • Contains a transmembrane helix that anchors the protein to the membrane

  • Mutations in this region can disrupt membrane integration and affect ATP synthase assembly

• Dimerization domain (residues 62-122)

  • Forms an extremely elongated α-helical coiled-coil structure

  • Critical for b-b dimer formation with a frictional ratio of 1.60

  • The structure has a maximal dimension of 95 Å and a radius of gyration of 27 Å

  • Crystal structure resolved at 1.55 Å shows a monomeric α-helix with a length of 90 Å

• C-terminal domain (residues 123-156)

  • Interacts with the F1 sector of ATP synthase

  • Essential for connecting the membrane-embedded F0 domain to the catalytic F1 domain

Mutagenesis studies in related systems have shown that specific residues within the dimerization domain form key interactions that stabilize the coiled-coil structure. Disruption of these interactions can lead to destabilization of the b-b dimer and consequent dysfunction of the entire ATP synthase complex. The elongated structure of the b dimer serves as a stator arm that prevents rotation of the F1 domain while allowing rotation of the c-ring and γ-subunit, which is essential for the mechanochemical coupling that drives ATP synthesis.

How can recombinant atpF be used in vaccine development against Salmonella paratyphi B?

Recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) holds potential for vaccine development through several strategies:

• As a component of subunit vaccines

  • Purified recombinant atpF can be formulated with appropriate adjuvants

  • The protein's conserved nature may provide cross-protection against multiple Salmonella serovars

  • Can be combined with other antigens for broader protection

• As part of live attenuated vaccine strains

  • Modified atpF can be used to create attenuated Salmonella strains

  • Attenuation through deletion or mutation of atpF affects bacterial metabolism without compromising immunogenicity

  • Such strains can serve as vectors for delivering heterologous antigens

• As a carrier protein for conjugate vaccines

  • The structural properties of atpF make it suitable as a carrier for polysaccharide antigens

  • Similar approaches have been used with other Salmonella proteins such as flagellin (FliC)

The development of vaccines against Salmonella remains an important public health goal, as current licensed vaccines only target Salmonella Typhi and offer limited cross-protection against other serovars such as Salmonella Paratyphi B . The extensive knowledge of Salmonella virulence mechanisms and the ability to genetically modify this organism make it an excellent candidate for new vaccine approaches.

Experimental approaches using other ATP synthase subunits have shown promise. For example, researchers have developed reagent strains for O:4 OPS and flagellin purification (CVD 1925) and O:9 OPS and FliC purification (CVD 1943) for conjugate vaccine development .

What methods are effective for studying interactions between atpF and other ATP synthase subunits?

To investigate the interactions between recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) and other ATP synthase components, researchers can employ these methodologies:

• Co-immunoprecipitation and pull-down assays

  • Use His-tagged atpF to pull down interacting partners

  • Identify binding partners via mass spectrometry

  • Quantify binding affinities with varying salt and detergent conditions

• Surface Plasmon Resonance (SPR)

  • Immobilize purified atpF on sensor chip

  • Measure real-time kinetics of interactions with other subunits

  • Determine binding constants (KD) for each interaction pair

• Crosslinking coupled with mass spectrometry

  • Use chemical crosslinkers of varying lengths to capture transient interactions

  • Identify crosslinked peptides to map interaction interfaces at amino acid resolution

  • Provides spatial constraints for structural modeling

• Förster Resonance Energy Transfer (FRET)

  • Label atpF and potential binding partners with fluorescent probes

  • Monitor distance-dependent energy transfer

  • Can be used in reconstituted systems or living cells

• Reconstitution experiments

  • Combine purified atpF with other ATP synthase components

  • Assess assembly efficiency and functionality of the reconstituted complex

  • Compare activity with native ATP synthase as a control

These approaches can reveal not only which subunits interact with atpF but also the structural basis and functional consequences of these interactions. Similar methods have been successfully applied to study the reconstitution of the c subunit multimeric ring in chloroplast ATP synthase .

How do mutations in atpF affect ATP synthase assembly and function in Salmonella paratyphi B?

Mutations in ATP synthase subunit b (atpF) can significantly impact the assembly and function of the ATP synthase complex through several mechanisms:

Site-directed mutagenesis of conserved residues in atpF can provide valuable insights into structure-function relationships. For example, mutations affecting the coiled-coil interactions in the dimerization domain may still allow complex assembly but reduce efficiency of energy coupling, resulting in decreased ATP synthesis rates.

The functional impact of atpF mutations can be assessed by:

• ATP synthesis/hydrolysis assays in reconstituted systems

• Proton pumping measurements using pH-sensitive fluorescent dyes

• Growth phenotype analysis under different energy sources

• Structural integrity assessment using electron microscopy

Evolutionary analysis of natural sequence variations in atpF across Salmonella serovars can also reveal functionally critical residues that have been conserved due to selective pressure. Comparative genomic studies of Salmonella Paratyphi A have shown that homologous recombination, which was extensive before the most recent common ancestor (MRCA), has essentially stopped during more recent history, with 99% of SNPs arising by mutation rather than recombination .

What are the optimal conditions for expression and solubilization of recombinant atpF?

The expression and solubilization of recombinant Salmonella paratyphi B ATP synthase subunit b require careful optimization:

Expression Optimization:

Solubilization Protocol:

• Harvest cells and resuspend in buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10% glycerol

• Lyse cells using French press or sonication with protease inhibitors

• Isolate membrane fraction by ultracentrifugation (100,000g, 1 hour, 4°C)

• Solubilize membrane proteins using detergent screening approach:

  • Try DDM (1%), LDAO (1%), or C12E8 (1%)

  • Incubate with gentle agitation at 4°C for 2 hours

  • Remove insoluble material by ultracentrifugation

• Monitor solubilization efficiency by SDS-PAGE and Western blotting

The choice of detergent is critical for maintaining the native structure of atpF, particularly for preserving the dimerization interface. A systematic detergent screening approach is recommended to identify conditions that yield properly folded, functional protein.

Similar approaches have been successfully used for the recombinant production of other ATP synthase subunits, such as the c subunit from spinach chloroplast ATP synthase .

How can I troubleshoot low expression yields or protein aggregation issues with recombinant atpF?

When encountering challenges with recombinant Salmonella paratyphi B ATP synthase subunit b expression or aggregation, consider these troubleshooting strategies:

For Low Expression Yields:

• Codon optimization

  • Analyze the atpF sequence for rare codons in E. coli

  • Synthesize a codon-optimized gene or use specialized strains (Rosetta)

• Expression vector modification

  • Test different promoter strengths (T7, tac, araBAD)

  • Optimize the ribosome binding site sequence and spacing

• Fusion tags approach

  • Try alternative N-terminal tags (MBP, SUMO, TrxA)

  • These can improve translation initiation and protein solubility

• Growth conditions adjustment

  • Reduce expression temperature further (16°C)

  • Test auto-induction media for gentle, gradual protein expression

For Protein Aggregation:

• Detergent optimization

  • Screen additional detergents (CHAPS, digitonin, amphipols)

  • Try detergent mixtures for improved solubilization

• Buffer composition

  • Add stabilizing agents (glycerol 10-20%, trehalose 6%)

  • Test different pH ranges (pH 7.0-8.5)

  • Include specific lipids that may stabilize the native structure

• Refolding strategies

  • If inclusion bodies form, develop a refolding protocol

  • Use gradual dialysis with decreasing denaturant concentrations

• Co-expression approaches

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Co-express with other ATP synthase subunits that interact with atpF

Molecular chaperones like Hsp70 may be particularly beneficial, as recent research has shown that Hsp70 not only acts as a "folding helper" of proteins in mitochondria but also promotes the assembly of ATP synthase . Similar chaperoning mechanisms likely exist in bacterial systems and could be leveraged to improve recombinant atpF production.

What crystallization approaches are most promising for structural studies of recombinant atpF?

Crystallization of membrane proteins like ATP synthase subunit b presents unique challenges due to their hydrophobic surfaces and conformational flexibility. The following approaches have shown promise for structural studies of recombinant atpF:

Construct Design Strategies:

• Domain-based approach

  • Focus on the soluble dimerization domain (residues 62-122)

  • This region has been successfully crystallized at 1.55 Å resolution

  • Consider multiple constructs with varying N- and C-terminal boundaries

• Fusion protein strategy

  • Incorporate crystallization chaperones (T4 lysozyme, BRIL)

  • These can provide crystal contacts and stabilize flexible regions

  • Position fusions to avoid disrupting functional domains

Crystallization Methods:

• Detergent screening

  • Systematic testing of detergents with varying micelle sizes

  • Include detergent mixtures and facial amphiphiles

  • Monitor protein monodispersity by dynamic light scattering

• Lipidic cubic phase (LCP)

  • Embed protein in lipid bilayer environment

  • More native-like environment for membrane proteins

  • Has proven successful for challenging membrane protein targets

• Antibody fragment co-crystallization

  • Generate Fab or nanobody fragments against atpF

  • These can stabilize specific conformations and provide crystal contacts

  • Particularly useful for flexible proteins like atpF

Optimization Techniques:

• Additive screening

  • Include metal ions, small molecules, and lipids

  • These can stabilize specific protein conformations

  • Try amphiphiles that mimic the natural membrane environment

• Surface entropy reduction

  • Identify surface residues with high entropy

  • Mutate clusters of Lys/Glu/Gln to alanine

  • Creates potential new crystal contacts

• Crystallization at lipid-detergent interfaces

  • Use bicelles or nanodiscs to provide a more native-like environment

  • Maintains the oligomeric state of the protein complex

The crystal structure of the dimerization domain (residues 62-122) has been solved, revealing an isolated, monomeric α-helix with a length of 90 Å . This suggests that crystallizing the full-length protein might require stabilizing the dimeric interface, possibly through engineered disulfide bonds or binding partners.

How can recombinant atpF be used to study ATP synthase assembly in vitro?

Recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) provides a valuable tool for investigating the assembly process of ATP synthase through several experimental approaches:

• Reconstitution of subcomplexes

  • Combine purified atpF with other F0 components (a, c subunits)

  • Monitor assembly using native PAGE and electron microscopy

  • Study the kinetics and order of subunit incorporation

• Protein-lipid interactions

  • Reconstitute atpF into liposomes of defined composition

  • Investigate how lipid environment affects insertion and oligomerization

  • Use fluorescence techniques to monitor changes in protein conformation

• Time-resolved assembly studies

  • Develop a cell-free expression system for ATP synthase components

  • Monitor assembly in real-time using fluorescently labeled subunits

  • Identify rate-limiting steps and assembly intermediates

• Chaperone-assisted assembly

  • Investigate the role of molecular chaperones like Hsp70 in atpF folding and assembly

  • Recent research has shown that Hsp70 promotes ATP synthase assembly in mitochondria

  • Test whether similar mechanisms apply to bacterial ATP synthase

• Cross-subunit interactions

  • Map the interaction network using chemical crosslinking

  • Identify critical residues that mediate subunit recognition

  • Create assembly maps based on detected interactions

Similar approaches have been used to study the reconstitution of the multimeric c-ring in chloroplast ATP synthase . These methods can be adapted for studying the b subunit dimer formation and its integration into the larger ATP synthase complex.

The development of recombinant expression systems for ATP synthase subunits enables molecular biology techniques that cannot otherwise be applied to native proteins, making it possible to introduce specific mutations, isotopic labeling, or fusion tags that facilitate detailed mechanistic studies of assembly and function.

How do ATP synthase b subunits compare across different Salmonella serovars?

The ATP synthase subunit b (atpF) exhibits both conservation and variation across different Salmonella serovars, reflecting evolutionary pressures and functional constraints:

Evolutionary analysis of Salmonella genomes has revealed interesting patterns in how genes evolve. For instance, studies of Salmonella paratyphi A have shown that while extensive homologous recombination occurred before the most recent common ancestor (MRCA), it has essentially stopped during more recent history, with 99% of SNPs arising by mutation rather than recombination . It would be interesting to examine whether atpF follows similar evolutionary patterns.

The core genome of Salmonella paratyphi A contains 4,073,403 bp encoding 3,365 intact CDSs . ATP synthase genes are part of this core genome, highlighting their essential nature. Comparative genomics can reveal whether atpF has been subject to purifying selection (conservation of function) or positive selection (adaptation to new conditions) across different Salmonella lineages.

How can structural studies of atpF contribute to understanding ATP synthase evolution?

Structural studies of recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) can provide valuable insights into the evolution of ATP synthase:

• Structure-function conservation

  • Comparing atpF structures across species reveals evolutionarily conserved motifs

  • The dimerization domain (residues 62-122) forms an extremely elongated α-helical coiled-coil structure

  • This structural feature is likely conserved across diverse bacterial species

• Evolutionary adaptation mechanisms

  • Identify species-specific structural features that may represent adaptations

  • Compare these with environmental niches or metabolic requirements

  • Correlate structural variations with differences in ATP synthase efficiency

• Phylogenetic analysis

  • Use structural information to refine molecular phylogenies

  • Identify structurally constrained regions versus variable regions

  • Create structure-guided sequence alignments for more accurate evolutionary trees

• Evolutionary design principles

  • Reveal how nature has solved the mechanical challenge of coupling proton translocation to ATP synthesis

  • The b subunit dimer serves as a stator that prevents rotation of F1 while allowing rotation of the c-ring

  • This mechanical principle is conserved from bacteria to mitochondria and chloroplasts

ATP synthase is classified as an F-type enzyme in chloroplasts and is reversible, similar to archaeal (A-type) ATP synthases, while vacuolar (V-type) ATP-ases function only as proton or ion pumps driven by ATP hydrolysis . Structural studies of atpF can help elucidate how these different types of rotary ATPases evolved from a common ancestor.

The process by which ATP is synthesized is mechanically coupled to the rotation of a ring of c-subunits embedded in the membrane, with the rotation of the c-ring coupled to the rotation of the γ-stalk in the F1 region . Structural studies of atpF can illuminate how this subunit contributes to maintaining the correct spatial relationships required for this mechanical coupling.

How can recombinant atpF contribute to vaccine development against Salmonella infections?

Recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) offers several promising avenues for vaccine development:

• As a target antigen in subunit vaccines

  • atpF could serve as a protective antigen due to its surface exposure

  • The conserved nature across Salmonella serovars may provide broad protection

  • Can be combined with other antigens for multivalent vaccine formulations

• For attenuated live vaccine development

  • Mutations in atpF can create metabolically compromised but immunogenic strains

  • Such strains can colonize host tissues transiently without causing disease

  • The approach has been successful with other ATP synthase components

• As a carrier for polysaccharide conjugate vaccines

  • The protein can be conjugated to O-antigen polysaccharides

  • Similar approaches have been used with other Salmonella proteins

  • Reagent strains for O:4 OPS and O:9 OPS purification have been developed

• In recombinant vector vaccines

  • Engineered Salmonella expressing modified atpF can elicit targeted immune responses

  • Can be used to deliver heterologous antigens from other pathogens

  • Leverages natural Salmonella interaction with host immune system

Current licensed Salmonella vaccines only target Salmonella Typhi and include the orally administered live-attenuated Ty21a vaccine and injectable Vi capsular polysaccharide and conjugate vaccines . The Ty21a vaccine offers some cross-protection against Salmonella Paratyphi B but not Salmonella Paratyphi A, highlighting the need for broader-spectrum vaccines.

Recent advances in attenuated Salmonella strains have included deletions in various genes including guaBA (guanine biosynthesis), clpPX (regulatory protease), htrA (heat-shock protein), and pipA (part of Salmonella pathogenicity island 5) . Similar approaches could be applied to atpF to develop new vaccine candidates.

What biotechnological applications exist for engineered variants of atpF?

Engineered variants of recombinant Salmonella paratyphi B ATP synthase subunit b (atpF) have potential applications beyond basic research:

• Biosensors for antimicrobial discovery

  • Modified atpF can serve as a target for high-throughput screening

  • Compounds that disrupt atpF function could be developed as antimicrobials

  • The assay could utilize FRET or bioluminescence readouts

• Bioenergetic engineering

  • Modified atpF variants could alter ATP synthase efficiency

  • Potential applications in optimizing bacterial production strains

  • Could enhance ATP yield for biotechnology processes

• Nanobiotechnology applications

  • The elongated structure of the b-b dimer (approximately 95 Å) makes it suitable as a molecular scaffold

  • Could be used to position functional domains at defined distances

  • Applications in synthetic biology and molecular machines

• Protein delivery systems

  • Attenuated Salmonella strains expressing modified atpF could deliver therapeutic proteins

  • The natural ability of Salmonella to invade host cells makes it an attractive delivery vector

  • Similar approaches have been used with other bacterial systems

• Template for synthetic ATP synthases

  • Understanding atpF structure-function relationships can guide design of synthetic energy-generating systems

  • Could lead to novel biomimetic energy conversion technologies

  • Potentially applicable in artificial cells or biohybrid systems

The extensive knowledge of Salmonella virulence mechanisms and the ability to genetically modify this organism make it an excellent platform for various biotechnological applications . Attenuated Salmonella strains can serve as ideal tools for the delivery of foreign antigens to create multivalent live carrier vaccines for simultaneous immunization against several unrelated pathogens.