Recombinant Shigella boydii serotype 4 NADH-quinone oxidoreductase subunit K (nuoK)
Product Specs
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Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid forms is 6 months at -20°C/-80°C. Lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: sbo:SBO_2312
Q&A
What is the structural composition of nuoK from Shigella boydii serotype 4?
The NADH-quinone oxidoreductase subunit K (nuoK) from Shigella boydii serotype 4 is a hydrophobic protein containing three transmembrane segments (TM1-3) . According to UniProt data (Q31YI2), the amino acid sequence is: MIPLQHGLILSAILFVLGLTGLVIRRNLLFMLIGLEIMINA SALAFVVAGSYWGQTDGQVMYILAISLAAA EASIGLALLLQLHRRRQNLNIDSVSEMRG . The protein has an expression region spanning positions 1-100 and is encoded by the nuoK gene (SBO_2312) . The transmembrane organization is critical for its function as part of the NDH-1 complex, with specific residues located in strategic positions across its membrane-spanning domains .
What are the recommended storage conditions for recombinant nuoK protein?
For optimal stability, recombinant Shigella boydii serotype 4 NADH-quinone oxidoreductase subunit K should be stored at -20°C, and for extended storage, conservation at -20°C or -80°C is recommended . The protein is typically provided in a Tris-based buffer with 50% glycerol that has been optimized for this specific protein . Repeated freezing and thawing cycles should be avoided to prevent protein degradation and loss of functional activity . For working solutions, storing aliquots at 4°C for up to one week is acceptable . These storage recommendations help maintain protein integrity for experimental applications.
What experimental design approaches are most effective for studying nuoK function?
When investigating nuoK function, researchers should consider implementing a Solomon 4-Group Design experimental approach, which effectively addresses the potential influence of pretesting on experimental outcomes . This design incorporates four groups: two experimental and two control groups, with pretesting applied to only one experimental and one control group . This approach is particularly valuable when studying the effects of specific mutations on nuoK function, as it controls for potential testing effects.
The experimental design should include:
| Group | Pretest | Treatment | Posttest |
|---|---|---|---|
| 1 | Yes | Mutation | Yes |
| 2 | Yes | No mutation (control) | Yes |
| 3 | No | Mutation | Yes |
| 4 | No | No mutation (control) | Yes |
How do mutations in conserved residues affect the energy transduction properties of nuoK?
Research has demonstrated that specific conserved residues in nuoK are critical for its energy transduction function. Two glutamic acid residues located in adjacent transmembrane helices play particularly important roles . Mutation of the highly conserved carboxyl residue (K)Glu-36 in TM2 to alanine results in complete loss of NDH-1 activities, indicating this residue is essential for function . In contrast, mutation of the second conserved carboxyl residue (K)Glu-72 in TM3 only moderately reduces activities, suggesting a supportive but non-essential role .
Experimental relocation of these conserved residues provides further insights:
| Mutation Type | Position Change | Functional Effect |
|---|---|---|
| (K)Glu-36 shift | To positions 32, 38, 39, 40 | Largely retained energy transducing activities |
| (K)Glu-36 to Ala | Position 36 | Complete loss of NDH-1 activities |
| (K)Glu-72 to Ala | Position 72 | Moderate reduction in activities |
| Double mutation of Arg residues | Loop-1 | Drastic effect on energy transducing activities |
The functional resilience when (K)Glu-36 is shifted to nearby positions (32, 38, 39, and 40) indicates that these locations share a similar environment within the protein structure, likely positioned in the same helix phase immediately before or after a helix turn . These findings highlight the importance of not just the residue identity but also its precise positioning within the three-dimensional structure of the protein.
What methodological approaches can be used to study the proton translocation mechanism of nuoK?
Investigating the proton translocation mechanism of nuoK requires a multi-faceted methodological approach. Site-directed mutagenesis targeting the cytosolic loop between TM1 and TM2 (containing residues (K)Arg-25, (K)Arg-26, and (K)Asn-27) has proven effective in elucidating the importance of this region for energy transduction . These residues should be systematically mutated individually and in combination to assess their contributions to the proton translocation pathway.
For comprehensive analysis, researchers should implement:
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Site-directed mutagenesis targeting conserved residues
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Proton pumping assays using pH-sensitive fluorescent dyes
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Membrane potential measurements using voltage-sensitive probes
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Structural analysis through X-ray crystallography or cryo-EM
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Molecular dynamics simulations to visualize conformational changes during the catalytic cycle
When designing experiments to study the relocation of key residues, it's important to consider their position relative to the membrane plane and their interactions with other subunits of the NDH-1 complex . The experimental data should be integrated with computational models to develop a comprehensive understanding of the proton translocation mechanism.
What considerations are important when analyzing data from transmembrane protein studies?
When analyzing data from transmembrane protein studies such as those involving nuoK, researchers must account for several confounding factors that can affect experimental outcomes. The hydrophobic nature of transmembrane proteins presents unique challenges for expression, purification, and functional characterization .
Critical considerations include:
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Detergent effects on protein conformation and activity
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Lipid environment influences on protein function
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Potential artifacts from fusion tags or expression systems
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Maintaining native-like membrane environment during functional assays
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Distinguishing between direct and indirect effects of mutations
Data analysis should employ appropriate controls to account for these factors. For example, when studying the effects of relocating conserved residues like (K)Glu-36 along TM2, researchers should consider how each position might interact differently with the surrounding lipid environment or neighboring protein subunits . Computational tools such as molecular dynamics simulations can help interpret experimental data by providing insights into how mutations might affect protein dynamics within a membrane context.
How does understanding nuoK function contribute to our knowledge of Shigella pathogenicity?
Shigella boydii is a significant human pathogen that causes diarrhea and bacillary dysentery . Understanding the function of nuoK contributes to our knowledge of Shigella pathogenicity by elucidating essential aspects of bacterial energy metabolism that support pathogen survival and virulence.
The NDH-1 complex containing nuoK plays a crucial role in bacterial respiration and energy generation, which is essential for:
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Bacterial growth and proliferation during infection
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Adaptation to changing host environments
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Resistance to host-imposed stress conditions
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Maintenance of membrane potential necessary for various virulence mechanisms
Research on nuoK function may reveal vulnerabilities in Shigella's energy metabolism that could be targeted for therapeutic intervention. Since respiratory chain components like NDH-1 are often essential for pathogen survival but differ significantly from human mitochondrial complexes, they represent promising targets for pathogen-specific inhibition .
What techniques can be used to evaluate nuoK as a potential antimicrobial target?
Evaluating nuoK as a potential antimicrobial target requires a systematic approach combining structural biology, biochemistry, and in vivo infection models. The following methodological framework can guide this evaluation:
| Evaluation Phase | Techniques | Expected Outcomes |
|---|---|---|
| Target validation | Gene knockout/knockdown | Viability assessment, growth kinetics |
| Druggability assessment | Structural analysis (X-ray, cryo-EM) | Identification of potential binding pockets |
| Inhibitor screening | High-throughput biochemical assays | Discovery of lead compounds |
| Mechanism studies | Site-directed mutagenesis, molecular dynamics | Understanding of inhibition mechanism |
| Specificity testing | Comparative studies with human homologs | Safety assessment |
| In vivo efficacy | Animal infection models | Therapeutic potential evaluation |
Researchers should focus on the unique features of nuoK, particularly the conserved residues (K)Glu-36, (K)Glu-72, and the cytosolic loop containing (K)Arg-25, (K)Arg-26, and (K)Asn-27, which have been demonstrated to be critical for function . These regions represent potential binding sites for inhibitors that could specifically target bacterial NDH-1 without affecting human mitochondrial complexes.
How can researchers determine the precise role of nuoK in the proton translocation pathway of NDH-1?
Determining the precise role of nuoK in the proton translocation pathway requires a combination of structural analysis, functional studies, and computational modeling. Studies have shown that relocating the conserved residue (K)Glu-36 to positions 32, 38, 39, and 40 largely preserves energy transducing activities, indicating these positions are functionally equivalent . This suggests these residues are located in the same helix phase, positioned immediately before and after a helix turn .
A comprehensive approach should include:
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High-resolution structural determination using cryo-EM or X-ray crystallography
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Hydrogen-deuterium exchange mass spectrometry to identify regions exposed to solvent
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Cysteine-scanning mutagenesis to map functionally important residues
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Cross-linking experiments to determine proximity relationships with other subunits
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Computational modeling of proton transfer pathways
The results from these complementary approaches should be integrated to develop a mechanistic model of how nuoK participates in proton translocation. Particular attention should be paid to the conserved glutamic acid residues and the cytosolic loop between TM1 and TM2, which have been implicated in the energy transduction mechanism .
What are the technical challenges in expressing and purifying functional recombinant nuoK for structural studies?
Expressing and purifying functional recombinant nuoK presents significant technical challenges due to its hydrophobic nature and the presence of multiple transmembrane segments . Researchers must overcome several obstacles to obtain sufficient quantities of properly folded protein for structural and functional studies.
Key challenges and solutions include:
When working with recombinant nuoK, researchers should consider using the baculovirus expression system, which has been successfully employed for this protein . The storage buffer typically contains Tris-based buffer with 50% glycerol optimized for protein stability . For structural studies, it's crucial to verify that the recombinant protein retains its native conformation and functional properties through activity assays before proceeding with structural analysis.
