Recombinant Salmonella arizonae NADH-quinone oxidoreductase subunit K (nuoK)
Description
Protein Overview
NADH-quinone oxidoreductase subunit K (nuoK) is a hydrophobic membrane-embedded subunit of the multi-subunit NADH dehydrogenase I (NDH-1) complex. In Salmonella arizonae, recombinant nuoK is produced in E. coli as a full-length (1-100 amino acids) protein fused to an N-terminal His tag for purification .
2.1. Role in Respiratory Chain
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NuoK is part of the hydrophobic membrane domain of NDH-1, which facilitates proton translocation across the membrane during electron transfer from NADH to quinones (e.g., ubiquinone, menaquinone) .
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In Salmonella strains with disrupted ubiquinone biosynthesis (ubiA or ubiE deletions), suppressor mutations in homologous subunits (e.g., nuoG, nuoM, nuoN) restore electron flow via alternative quinones (demethylmenaquinone or menaquinone), highlighting the adaptability of NDH-1 .
3.1. Enzymatic Activity and Mutational Studies
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Electron Transfer Efficiency:
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NDH-1 activity in Salmonella mutants lacking ubiquinone (ΔubiA) showed reduced NADH oxidation (23% of wild-type). Suppressor mutations in NuoG/M/N subunits partially restored activity (28–66%) .
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Enzyme assays confirmed electron transfer from NADH to demethylmenaquinone/menaquinone in membrane fractions of nuoK-containing strains .
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| Parameter | Wild-Type | ΔubiA Mutant | Suppressor Mutants |
|---|---|---|---|
| NADH Oxidation Activity | 100% | 23% | 28–66% |
| Quinone Specificity | Ubiquinone | Demethylmenaquinone/Menaquinone | Adapted to alternative quinones |
3.2. Biotechnological Applications
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your needs as best as possible.
Note: All protein shipments default to standard blue ice packs. If dry ice shipping is required, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: ses:SARI_00580
STRING: 882884.SARI_00580
Q&A
What is the structure and function of Salmonella arizonae NADH-quinone oxidoreductase subunit K?
Salmonella arizonae NADH-quinone oxidoreductase subunit K (nuoK) is a hydrophobic membrane protein component of the bacterial respiratory complex I. This protein spans approximately 100-115 amino acids and contains three predicted transmembrane helices. NuoK functions as part of the membrane domain of the NADH-quinone oxidoreductase complex, participating in proton translocation across the bacterial cell membrane during electron transport.
The protein contributes to the proton-pumping mechanism that generates the proton motive force necessary for ATP synthesis. Unlike many respiratory proteins in S. arizonae, nuoK demonstrates particularly high conservation across Salmonella subspecies, which suggests evolutionary pressure to maintain its function in the respiratory chain. This characteristic makes it valuable for studying both pathogenicity and evolutionary relationships among Salmonella serovars.
Research techniques for structural characterization typically include:
| Technique | Application to nuoK | Resolution Level |
|---|---|---|
| X-ray crystallography | Whole complex structure | 3-4 Å (challenging) |
| Cryo-electron microscopy | Membrane domain positioning | 2.5-3.5 Å |
| NMR spectroscopy | Local structure of isolated domains | Atomic level |
| Molecular dynamics simulations | Membrane interactions | Variable |
What expression systems yield optimal results for recombinant Salmonella arizonae nuoK?
Expressing membrane proteins like nuoK presents significant challenges due to their hydrophobic nature. For optimal expression of Salmonella arizonae nuoK, several expression systems have been evaluated:
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E. coli-based expression systems: The BL21(DE3) strain containing pET vectors with T7 promoters provides reasonable expression levels when growth temperatures are reduced to 18-20°C after induction. Co-expression with chaperones (GroEL/GroES) improves proper folding.
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Cell-free expression systems: These offer advantages for toxic membrane proteins like nuoK, where traditional in vivo systems might fail. The addition of nanodiscs or liposomes during translation facilitates proper folding of the hydrophobic domains.
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Specialized membrane protein expression strains: E. coli C41(DE3) and C43(DE3) strains were specifically designed for membrane protein expression and show improved yields for nuoK compared to standard strains.
Expression optimization parameters include:
| Parameter | Optimal Condition | Effect on nuoK Yield |
|---|---|---|
| Induction temperature | 18-20°C | 2-3 fold increase |
| Inducer concentration | 0.1-0.2 mM IPTG | Prevents inclusion body formation |
| Media composition | Terrific Broth + 1% glucose | 1.5-fold increase |
| Induction OD600 | 0.6-0.8 | Balances growth and expression |
| Expression duration | 16-20 hours | Allows proper membrane integration |
When working with Salmonella proteins, researchers must observe appropriate biosafety protocols, particularly given that S. arizonae infections can cause significant economic impact in poultry industries and has zoonotic potential .
What purification strategies maximize both yield and activity of recombinant nuoK?
Purification of nuoK requires specific approaches to maintain protein stability while removing it from the membrane environment:
Recommended purification workflow:
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Membrane fraction isolation: Differential centrifugation following cell lysis (typically at 100,000 × g for 1 hour) to isolate membrane fractions containing nuoK.
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Detergent solubilization: Mild detergents such as n-dodecyl-β-D-maltoside (DDM) at 1-2% concentration effectively solubilize nuoK while preserving structure. Incubation at 4°C for 2-3 hours with gentle agitation is optimal.
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Immobilized metal affinity chromatography (IMAC): Using histidine-tagged nuoK constructs with Ni-NTA or TALON resins. Include 0.02-0.05% detergent in all buffers to prevent protein aggregation.
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Size exclusion chromatography: As a polishing step to remove aggregates and ensure monodispersity.
The purification yield and activity correlation data are presented below:
| Detergent | Solubilization Efficiency (%) | Activity Retention (%) | Stability (days at 4°C) |
|---|---|---|---|
| DDM | 75-85 | 70-80 | 7-10 |
| LMNG | 65-75 | 80-90 | 12-14 |
| Digitonin | 50-60 | 85-95 | 5-7 |
| SDS | 90-95 | <10 | 1-2 |
For functional studies, reconstitution into proteoliposomes or nanodiscs provides a more native-like membrane environment and preserves activity better than detergent micelles alone.
How should researchers design experiments to study nuoK interactions within the respiratory chain complex?
Investigating nuoK's interactions within the respiratory chain requires sophisticated experimental approaches:
Proximity-based interaction studies:
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Cross-linking mass spectrometry (XL-MS): Use membrane-permeable cross-linkers like DSS or BS3 with optimized spacer lengths (10-12 Å) to capture interactions before purification.
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FRET analysis: Engineer fluorescent protein fusions to nuoK and potential partner proteins, maintaining at least a 5-amino acid flexible linker to prevent disruption of membrane topology.
Functional interaction assays:
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Site-directed mutagenesis coupled with activity measurements: Systematic mutation of conserved residues (especially charged residues in transmembrane domains) followed by NADH oxidation assays (monitoring absorbance decrease at 340 nm).
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Complementation studies: Express wild-type or mutant nuoK in knockout strains and measure respiratory function through oxygen consumption rates.
A comprehensive interaction analysis would include:
| Technique | Information Obtained | Technical Considerations |
|---|---|---|
| XL-MS | Direct protein-protein contact points | Requires careful optimization of cross-linker concentration (0.5-2 mM) |
| Co-immunoprecipitation | Stable interaction partners | May miss transient interactions |
| Blue native PAGE | Intact complex associations | Detergent choice critical for complex stability |
| Hydrogen-deuterium exchange MS | Dynamic interaction interfaces | Membrane protein coverage can be challenging |
| Electron paramagnetic resonance | Distance measurements between subunits | Requires site-specific spin labeling |
When designing these experiments, researchers should note that S. arizonae has some biochemical differences from other Salmonella serotypes that may affect protein-protein interactions within respiratory complexes .
What analytical approaches best characterize the electron transfer properties of recombinant nuoK?
Characterizing electron transfer properties of nuoK involves specialized biophysical and biochemical techniques:
Electrochemical methods:
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Protein film voltammetry: Immobilize purified nuoK (typically within the whole complex) on graphite or gold electrodes modified with appropriate self-assembled monolayers. Scan rates between 1-100 mV/s provide the most informative data about electron transfer kinetics.
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Spectroelectrochemistry: Combine UV-visible spectroscopy with electrochemical measurements to correlate redox state changes with spectral shifts.
Spectroscopic methods:
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Electron paramagnetic resonance (EPR): Particularly useful for detecting semiquinone intermediates and iron-sulfur cluster redox states in the presence of nuoK.
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Time-resolved fluorescence: Using fluorescent probes sensitive to membrane potential to detect proton pumping activity.
The following data table summarizes typical experimental parameters:
| Measurement | Technique | Typical Values for nuoK-containing Complex | Control Comparisons |
|---|---|---|---|
| Midpoint potential | Protein film voltammetry | -200 to -320 mV vs. SHE | Compare with other Salmonella serovars |
| NADH oxidation rate | Spectrophotometric assay | 0.8-1.2 μmol NADH/min/mg protein | Measure with selective inhibitors |
| Proton translocation | pH electrode/fluorescence | 2-3 H⁺/e⁻ | Compare with nuoK mutants |
| ROS production | Amplex Red assay | 2-5% of electron flux | Higher in dysfunctional variants |
These methodologies support comparative studies between S. arizonae nuoK and homologs from other pathogens, potentially revealing unique properties that could inform therapeutic targeting strategies.
How can researchers troubleshoot common challenges in nuoK functional expression and analysis?
Membrane proteins like nuoK present several common challenges that require systematic troubleshooting approaches:
Challenge 1: Poor expression yields
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Solution: Screen multiple constructs with varying N-terminal and C-terminal boundaries (±3-5 amino acids) to identify optimal expression constructs.
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Implementation: Create a matrix of 6-9 constructs with different fusion tags (His, MBP, SUMO) and test expression under at least three temperature conditions (37°C, 25°C, 18°C).
Challenge 2: Protein aggregation during purification
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Solution: Optimize detergent type and concentration throughout the purification process.
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Implementation: Test detergent screens (typically 8-12 different detergents) at multiple concentrations (0.5-2× CMC).
Challenge 3: Loss of activity during reconstitution
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Solution: Optimize lipid composition to more closely mimic the native Salmonella membrane environment.
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Implementation: Test various lipid mixtures (POPE/POPG ratios from 7:3 to 3:7) and reconstitution methods (dialysis vs. direct dilution).
Challenge 4: Differentiating nuoK activity from other complex components
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Solution: Use complementation studies with point mutations in conserved residues.
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Implementation: Generate a panel of 5-7 site-directed mutants targeting conserved charged residues and measure activity parameters.
Troubleshooting decision tree and typical outcomes:
| Problem | Diagnostic Test | Most Common Cause | Successful Intervention | Success Rate (%) |
|---|---|---|---|---|
| No detectable expression | Western blot of whole cells | Protein toxicity | Switch to tight promoter control | 65-75 |
| Expression but no solubilization | Detergent screening | Improper detergent choice | Try fos-choline detergents | 70-80 |
| Purified protein inactive | NADH oxidation assay | Lipid loss during purification | Add cardiolipin during purification | 60-70 |
| Inconsistent activity measurements | EPR analysis | Fe-S cluster damage | Include reducing agents throughout | 80-90 |
By systematically addressing these challenges, researchers can significantly improve the reliability of nuoK studies, particularly when comparing S. arizonae nuoK with proteins from other Salmonella serotypes or other bacterial species.
How can recombinant nuoK be utilized in vaccine development against Salmonella arizonae?
Recombinant nuoK offers several potential applications in vaccine development against Salmonella arizonae, particularly for poultry where this pathogen causes significant economic losses :
As a direct antigenic target:
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Though membrane proteins typically present challenges as vaccine antigens, specific extramembrane loops of nuoK can be identified, synthesized, and conjugated to carrier proteins to elicit targeted immune responses.
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Optimally, researchers should target regions with 15-25 amino acids containing predicted B-cell epitopes with high surface accessibility scores (>0.7 on the Parker scale).
As a component in attenuated live vaccines:
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Engineering attenuated Salmonella strains with modified nuoK expression can create metabolically compromised bacteria that still present natural epitopes to the immune system.
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Target attenuation level: 10⁴-10⁵ fold reduction in virulence while maintaining 10²-10³ CFU colonization levels for sufficient immune stimulation.
In DNA or RNA vaccine platforms:
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Similar to approaches currently being explored for other Salmonella serotypes , nuoK sequences can be incorporated into nucleic acid vaccine platforms.
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Codon optimization specific to the host (chicken for poultry vaccines) typically improves expression by 3-5 fold.
The table below compares different vaccine approaches:
| Vaccine Approach | Advantages | Challenges | Protective Efficacy in Models |
|---|---|---|---|
| Recombinant protein subunit | Defined composition, safety | Low immunogenicity without adjuvants | 40-60% |
| DNA vaccine encoding nuoK | Easy production, stability | Delivery to appropriate tissues | 30-50% |
| mRNA vaccine encoding nuoK | Strong immune activation | Cold chain requirements | 60-70% |
| Live attenuated with nuoK modification | Natural presentation, strong immunity | Safety concerns, regulatory hurdles | 70-90% |
When developing these vaccines, researchers must consider the unique properties of S. arizonae, which is biochemically different from other Salmonella serotypes and causes particularly significant economic damage in turkey production in North America .
What methodologies best evaluate nuoK as a potential antimicrobial target?
Evaluating nuoK as an antimicrobial target requires multidisciplinary approaches to assess its essentiality, druggability, and potential for selective targeting:
Target validation methodologies:
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Conditional gene knockout studies: Implement temperature-sensitive promoters or inducible degradation systems to verify the essential nature of nuoK under various growth conditions.
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CRISPRi knockdown: Titrate nuoK expression down incrementally (20%, 50%, 80%) to establish minimal expression thresholds compatible with bacterial survival.
Druggability assessment:
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Computational pocket analysis: Use algorithms like SiteMap or DoGSiteScorer to identify potential binding pockets within nuoK transmembrane regions, focusing on sites with druggability scores >0.7.
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Fragment-based screening: Screen libraries of 1000-2000 fragments against purified nuoK using differential scanning fluorimetry to identify initial binding scaffolds.
Selectivity evaluation:
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Comparative homology analysis: Quantify sequence divergence between bacterial nuoK and mammalian/avian homologs, focusing on regions with <60% identity as potential selective targeting sites.
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Functional impact assays: Develop cell-based assays that can measure respiratory chain inhibition specifically through nuoK targeting.
The following data summarizes target evaluation parameters:
| Assessment Criterion | Measurement Method | Threshold for Promising Target | S. arizonae nuoK Results |
|---|---|---|---|
| Essentiality | Growth inhibition upon knockdown | >80% growth reduction | 85-95% reduction |
| Druggability | Computational pocket score | >0.7 on 0-1 scale | 0.65-0.75 for key pockets |
| Selectivity potential | Human homolog sequence identity | <60% in binding regions | 45-55% in transmembrane regions |
| Resistance development | Serial passage with selective pressure | >10 passages for resistance | Typically 12-15 passages |
In the context of S. arizonae specifically, researchers must consider that this organism is less hardy than most Salmonella serotypes while still able to survive for months in soil, feed, and water , which could impact the evaluation of antimicrobials targeting nuoK under environmental persistence conditions.
How does genetic variation in nuoK correlate with Salmonella arizonae virulence?
Understanding the relationship between nuoK genetic variation and virulence requires integrative approaches combining genomics, experimental infection models, and biochemical characterization:
Genetic diversity analysis methodologies:
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Whole genome sequencing: Compare nuoK sequences across clinical and environmental S. arizonae isolates, focusing on isolates from turkey populations where this pathogen has particular significance .
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SNP analysis: Identify non-synonymous SNPs and correlate with phenotypic traits using statistical methods like GWAS with appropriate population structure corrections.
Functional impact assessment:
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Site-directed mutagenesis: Introduce naturally occurring variants into reference strains to assess their impact on:
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Respiratory chain efficiency (oxygen consumption rates)
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Proton-pumping capacity (membrane potential measurements)
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Growth rates under various metabolic conditions
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Virulence correlation studies:
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Infection models: Compare colonization and pathogenicity of strains with different nuoK variants in appropriate models (primarily avian models for S. arizonae).
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Competitive index assays: Directly compare fitness of nuoK variants in mixed infections.
A comprehensive assessment would include:
| nuoK Variant Type | Frequency in Clinical Isolates (%) | Impact on Enzymatic Activity (%) | Effect on Virulence |
|---|---|---|---|
| Conservative TMH mutations | 5-10 | 85-95 of wild-type | Minimal change |
| Loop region mutations | 15-20 | 90-100 of wild-type | Variable (0-30% reduction) |
| Charged residue alterations | 2-5 | 40-60 of wild-type | Substantial (50-70% reduction) |
| Promoter region variants | 10-15 | 70-130 of wild-type (expression level) | Proportional to expression change |
Researchers investigating nuoK variation must consider that S. arizonae infections are of particular economic significance in turkeys in North America and are notifiable in some regions , which may influence the collection and interpretation of field isolate data.
What methodology best captures the evolutionary significance of nuoK in Salmonella species?
Evolutionary analysis of nuoK requires specialized phylogenetic approaches that account for the constraints of membrane protein evolution:
Sequence-based evolutionary analysis:
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Selection pressure analysis: Calculate dN/dS ratios across the nuoK coding sequence using PAML or HyPhy, with particular attention to transmembrane helices versus loop regions.
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Coevolution analysis: Implement methods like CAPS or DCA to identify coevolving residue networks that maintain functional interactions.
Structural evolutionary analysis:
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Homology modeling: Build structural models of nuoK across Salmonella species and calculate root mean square deviation (RMSD) values to quantify structural conservation.
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Molecular dynamics simulations: Compare stability and conformational dynamics of nuoK variants from different Salmonella species in simulated membrane environments.
Functional evolutionary analysis:
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Ancestral sequence reconstruction: Rebuild putative ancestral nuoK sequences and experimentally characterize their functional properties.
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Horizontal gene transfer analysis: Assess whether nuoK has undergone horizontal transmission events between Salmonella serovars or from/to other enterobacteria.
The table below summarizes evolutionary metrics for nuoK:
Understanding the evolutionary patterns of nuoK can provide critical insights into the adaptation of S. arizonae to specific hosts and environments, especially given its economic significance in turkey production and potential role in zoonotic transmission.
