Recombinant Salmonella newport ATP synthase subunit c (atpE)

What is the Salmonella Newport ATP synthase subunit c (atpE) protein and what is its function?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase in Salmonella Newport. As part of the ATP synthase complex, it plays an essential role in cellular energy production through oxidative phosphorylation. The protein functions as a lipid-binding component within the membrane-embedded F0 portion of the ATP synthase, contributing to the proton channel that drives ATP synthesis . The protein consists of 79 amino acids and has the amino acid sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The atpE gene is also known by several alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, and lipid-binding protein .

How is recombinant S. Newport atpE protein typically expressed and purified for research applications?

Recombinant S. Newport ATP synthase subunit c (atpE) is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The protein can be expressed as a full-length construct (amino acids 1-79) based on the sequence from Salmonella Newport strain SL254 (UniProt ID: B4SYD6) . After expression, the protein is purified to greater than 90% purity as determined by SDS-PAGE analysis . The purified protein is typically provided in a lyophilized form in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For research applications, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage stability .

What are the optimal storage and handling conditions for recombinant S. Newport atpE protein?

For optimal stability and activity retention, recombinant S. Newport atpE protein should be stored at -20°C or -80°C immediately upon receipt . Since the protein is typically provided in lyophilized form, proper reconstitution is essential. After reconstitution, it is recommended to add glycerol to a final concentration of 50% for long-term storage . Working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw cycles, which are detrimental to protein stability and function . Prior to opening the vial, it should be briefly centrifuged to bring contents to the bottom . Researchers should avoid repeated freeze-thaw cycles as they significantly reduce protein activity and structural integrity . When planning experiments, it's advisable to prepare multiple small-volume aliquots during initial reconstitution to ensure consistent protein quality across experiments.

How does S. Newport atpE compare structurally and functionally to atpE proteins from other Salmonella serotypes?

Comparative genomic analysis indicates that S. Newport Lineages II and III diverged early in the serotype evolution and have evolved largely independently, with evidence of genetic flow and homologous recombination events around certain genes, including the mutS region near which functional genes like atpE may have been affected . The atpE region may therefore serve as one of the potential loci that could delineate sublineages within the phylogenetic tree of S. Newport and could potentially be used as a biomarker for trace-back investigations during outbreaks .

How can recombinant S. Newport atpE protein be used in structural biology studies and what challenges might researchers encounter?

Recombinant S. Newport atpE protein presents valuable opportunities for structural biology studies, but researchers must navigate several technical challenges. As a membrane protein component, atpE is hydrophobic in nature, which can complicate structural determination using techniques like X-ray crystallography or cryo-electron microscopy. For successful structural studies, researchers should consider using the His-tagged version for initial purification, followed by tag removal if needed for structural integrity .

The lyophilized form of the protein requires careful reconstitution, ideally in detergent-containing buffers that mimic the native lipid environment . When designing structural biology experiments, researchers should account for the protein's small size (79 amino acids) and consider studying it within the context of the larger ATP synthase complex or with stabilizing fusion partners. For NMR studies, the reconstitution buffer should be optimized to minimize interference while maintaining protein stability. Given the protein's high purity (>90% by SDS-PAGE) , researchers can expect relatively homogeneous preparations suitable for structural investigations, provided they maintain strict temperature control and minimize freeze-thaw cycles that could lead to conformational heterogeneity.

What role does the atpE gene play in the evolutionary history and phylogenetic analysis of S. Newport lineages?

The atpE gene and surrounding genomic regions have significant implications for understanding S. Newport evolution and lineage differentiation. Whole genome sequencing and comparative genomic analysis of S. Newport strains have identified over 140,000 informative SNPs, revealing four distinct sublineages with clear geographic structure . Analysis indicates that strains from Asia are divergent from those originating in the Americas .

Evolutionary history studies have shown that S. Newport Lineages II and III diverged early in the serotype's evolution and have evolved largely independently, with the atpE gene potentially being subject to selection pressures related to its function in energy metabolism . The region around the mutS gene, which could affect neighboring functional genes like atpE, shows evidence of genetic flow and homologous recombination events between lineages . This suggests that certain functional genes may have undergone adaptive evolution to different environmental niches.

Researchers conducting phylogenetic studies should consider atpE alongside other genetic markers that can differentiate between lineages, particularly loci such as those between invH and mutS genes, the ste fimbrial operon, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated proteins (cas) . When using whole genome sequencing for population structure analysis, computational approaches like those described for hierarchical-based population mining can be particularly effective in identifying lineage-specific patterns .

How can researchers use recombinant S. Newport atpE in immunological studies to develop detection methods or vaccines?

Recombinant S. Newport atpE presents significant potential for immunological research applications. Because the protein is expressed in E. coli with a His-tag and purified to >90% purity , it provides a reliable antigen for raising antibodies or developing detection systems. For immunological studies, researchers should reconstitute the lyophilized protein under non-denaturing conditions to preserve conformational epitopes .

When developing antibody-based detection methods, researchers should consider:

• Immunization protocols optimized for small membrane proteins

• Adjuvant selection to enhance immune response while minimizing protein denaturation

• ELISA-based detection systems utilizing the recombinant protein as a standard

• Cross-reactivity testing against atpE from other Salmonella serotypes

For potential vaccine development, researchers must evaluate whether atpE contains conserved epitopes across S. Newport lineages. Whole genome sequencing studies indicate that S. Newport has distinct lineages (I, II, and III) with different geographical distributions , which may impact antigenic variation. Immunoinformatics approaches can be applied to analyze the 79-amino acid sequence (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) for potential epitopes conserved across clinically relevant strains.

The high purity preparation enables precise dosing in immunization studies, while the lyophilized format allows for long-term stability during vaccine formulation development . Researchers should validate any immunological tools developed with the recombinant protein against native protein expressed in actual S. Newport isolates representing different lineages to ensure clinical relevance.

What insights can functional studies of atpE provide about antimicrobial resistance mechanisms in S. Newport?

Functional studies of S. Newport atpE can provide critical insights into antimicrobial resistance mechanisms, particularly those related to membrane integrity and energy metabolism. Some S. Newport strains, like SL254, have shown resistance to multiple antibiotics including ampicillin, chloramphenicol, gentamicin, streptomycin, ceftriaxone, sulfonamides, and tetracycline , suggesting complex resistance mechanisms that may involve membrane-associated proteins like atpE.

ATP synthase components, including subunit c, can be targeted by certain antimicrobials that disrupt bacterial energy metabolism. Researchers investigating this aspect should design functional assays that:

• Measure ATP synthesis activity in the presence of various antibiotics

• Evaluate proton flux across membranes containing recombinant atpE

• Assess potential structural changes in atpE upon exposure to membrane-active antimicrobials

• Compare activity of atpE from resistant versus susceptible strains

The recombinant protein's high purity (>90%) enables precise structure-function studies using techniques like circular dichroism or fluorescence spectroscopy to detect conformational changes upon antimicrobial binding. When conducting these studies, researchers should reconstitute the protein in membrane-mimetic environments such as liposomes or nanodiscs to maintain native-like function .

Comparative studies across different S. Newport lineages may reveal lineage-specific adaptations in atpE that correlate with antimicrobial resistance profiles, particularly given the evidence that S. Newport Lineages II and III have evolved independently and may have different resistance mechanisms .

What are the recommended protocols for functional characterization of recombinant S. Newport atpE protein?

For comprehensive functional characterization of recombinant S. Newport atpE protein, researchers should implement multi-faceted approaches focusing on both biochemical and biophysical properties. When working with the lyophilized protein, initial reconstitution should follow manufacturer recommendations using deionized sterile water to a concentration of a 0.1-1.0 mg/mL with 5-50% glycerol added for stability .

Table 1: Recommended Functional Characterization Methods for S. Newport atpE

For optimal results, all functional assays should be performed with freshly reconstituted protein or samples stored at 4°C for no more than one week . Temperature, pH, and ionic strength should be carefully controlled, with optimal conditions typically being 25-30°C, pH 7.0-8.0, and physiological salt concentrations. When reconstituting the protein into membranes, a gradual detergent removal approach using dialysis or Bio-Beads is recommended to ensure proper protein folding and orientation.

How can whole genome sequencing and bioinformatics approaches be applied to study S. Newport atpE in the context of bacterial evolution?

Whole genome sequencing (WGS) and advanced bioinformatics provide powerful tools for studying S. Newport atpE in evolutionary contexts. Based on current methodologies, researchers should consider the following comprehensive approach:

First, implement high-quality genome sequencing using Illumina paired-end technology as described in population mining studies of Salmonella serovars . After sequencing, conduct quality control and genome assembly using platforms such as ProkEvo for population-based analysis . For phylogenetic analysis, identify core genome regions through progressive MAUVE alignment with extraction of locally collinear blocks (LCBs) longer than 500 bp that are present across all genomes .

For evolutionary analysis specifically focusing on atpE:

• Extract and align atpE sequences from multiple S. Newport isolates representing different lineages

• Identify single nucleotide polymorphisms (SNPs) and calculate nucleotide diversity

• Conduct selection analysis using dN/dS ratios to identify evolutionary pressures

• Perform recombination analysis using methods like ClonalFrame to detect potential horizontal gene transfer events

When examining atpE in the context of S. Newport lineages, researchers should analyze surrounding genomic regions including loci between invH and mutS genes, which have shown evidence of genetic flow and homologous recombination between lineages . For comprehensive population structure analysis, implement hierarchical-based approaches that combine database-derived loci identification with agnostic searches for lineage-differentiating markers .

Visualization of results should incorporate core-genome phylogenetic trees with mapped gene presence/absence patterns and SNP distributions . This approach provides a multi-dimensional view of atpE evolution within the broader context of S. Newport population dynamics and can reveal potential functional adaptations related to geographical distribution or host specificity.

What quality control measures should be implemented when working with recombinant S. Newport atpE protein for experimental reproducibility?

Ensuring experimental reproducibility with recombinant S. Newport atpE protein requires rigorous quality control measures throughout the research workflow. Based on the properties of this specific protein, researchers should implement the following comprehensive quality control protocol:

Table 2: Quality Control Measures for S. Newport atpE Protein Research

To minimize batch-to-batch variation, researchers should maintain detailed records of protein lot numbers, reconstitution dates, and storage conditions . Working aliquots should be stored at 4°C for no more than one week to preserve activity . For long-term experimental planning, stability studies should be conducted with regular testing intervals to establish reliable activity retention profiles under various storage conditions.

When designing experiments, include appropriate positive and negative controls, and implement blinding procedures where feasible. All buffer components should be analytical grade, and water should be molecular biology grade to prevent contaminants that might affect protein behavior. Implementing these systematic quality control measures will significantly enhance reproducibility across different research groups working with this recombinant protein.

How can researchers effectively use recombinant S. Newport atpE protein in structural and functional studies of ATP synthase inhibitors?

Recombinant S. Newport atpE protein offers a valuable tool for developing and characterizing ATP synthase inhibitors, which may have potential as novel antimicrobials. For effective use in inhibitor studies, researchers should implement a systematic approach combining structural and functional methodologies.

Begin by reconstituting the lyophilized protein according to recommended protocols in buffers optimized for your specific assay system. For membrane protein inhibitor studies, reconstitution into liposomes or nanodiscs provides a more native-like environment than detergent micelles. The protein's high purity (>90% by SDS-PAGE) ensures that observed inhibitory effects are specific to atpE interactions.

For inhibitor binding studies:

• Implement biophysical techniques such as isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to determine binding constants

• Use fluorescence-based approaches with environment-sensitive probes to detect conformational changes upon inhibitor binding

• Consider hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map inhibitor binding sites within the protein structure

For functional inhibition assays:

• Establish proton translocation assays using pH-sensitive fluorophores to measure inhibition of c-subunit function

• Develop reconstituted ATP synthase systems incorporating recombinant atpE to assess effects on ATP synthesis

• Use membrane potential measurements to evaluate inhibitor effects on proton gradient maintenance

When designing inhibitor screening campaigns, create a diverse compound library targeting different aspects of atpE function, such as c-ring assembly, proton binding sites, or interface regions with other ATP synthase components. The availability of multiple S. Newport lineages provides an opportunity to test inhibitor efficacy across genetic variants, potentially identifying broad-spectrum candidates.

For data analysis, implement dose-response modeling to determine IC50 values and inhibition mechanisms. Correlation of functional data with structural insights will accelerate rational inhibitor optimization and may reveal novel antimicrobial strategies targeting this essential component of bacterial energy metabolism.

What are the potential applications of recombinant S. Newport atpE protein in vaccine development and diagnostic tools?

Recombinant S. Newport atpE protein has significant potential in both vaccine development and diagnostic applications, leveraging its conserved structure and immunogenic properties. For vaccine applications, the protein's high purity (>90%) provides a well-defined antigen that can be formulated with appropriate adjuvants to stimulate protective immunity. Researchers developing atpE-based vaccines should consider both humoral and cell-mediated immune responses, as membrane proteins often contain T-cell epitopes that may contribute to protection.

The small size (79 amino acids) of atpE may limit its immunogenicity when used alone, suggesting potential strategies such as:

• Conjugation to carrier proteins to enhance immune recognition

• Incorporation into virus-like particles or nanoparticles

• Multivalent vaccine approaches combining atpE with other Salmonella antigens

• DNA vaccine strategies encoding the atpE gene

For diagnostic applications, the recombinant protein serves as an excellent capture antigen for developing serological assays to detect S. Newport infections. The His-tagged version facilitates oriented immobilization on sensor surfaces, improving detection sensitivity. When developing such assays, researchers should evaluate cross-reactivity with antibodies against atpE from other Salmonella serotypes and related bacteria.

Notably, phylogenetic analysis showing distinct S. Newport lineages with geographical distribution patterns suggests that diagnostic tools may need to account for sequence variations in atpE across lineages. This could involve either targeting highly conserved epitopes or developing lineage-specific detection systems for improved specificity in epidemiological investigations. The recombinant protein's stability in lyophilized form further supports its use in field-deployable diagnostic platforms that require minimal cold chain maintenance.

How might S. Newport atpE protein be used in the study of host-pathogen interactions during Salmonella infection?

Recombinant S. Newport atpE protein offers valuable opportunities for investigating host-pathogen interactions during Salmonella infection. As a membrane protein component of ATP synthase, atpE may play roles beyond energy metabolism that influence host responses or bacterial adaptation within host environments.

Researchers can utilize the recombinant protein to:

• Examine interactions with host innate immune receptors such as pattern recognition receptors

• Investigate potential modulation of host cell ATP metabolism during intracellular infection

• Study adaptive immune responses to conserved bacterial ATP synthase components

• Evaluate whether atpE undergoes structural modifications in response to host environments

For cellular interaction studies, the reconstituted protein can be labeled with fluorescent tags while maintaining its native conformation . When investigating host responses, researchers should compare atpE from different S. Newport lineages to identify any lineage-specific interaction patterns that may correlate with virulence or host adaptation.

The evolutionary divergence observed between S. Newport lineages II and III suggests potential differences in host adaptation strategies that may be reflected in structural or functional modifications of ATP synthase components. Comparative studies using recombinant atpE from representative strains of each lineage could reveal adaptation mechanisms specific to different host environments or geographical regions.

For comprehensive host-pathogen interaction studies, researchers should complement in vitro protein-based approaches with genomic analyses examining atpE expression patterns during infection, potentially identifying environmental triggers that modulate ATP synthase activity as part of bacterial adaptation to host stresses.