SOD Human, 15N
Description
Introduction to SOD Human, 15N
SOD Human, 15N refers to isotopically labeled human superoxide dismutase (SOD1), a copper-zinc metalloenzyme critical for neutralizing superoxide radicals in oxidative stress mitigation. The 15N isotope is incorporated into SOD1 to enable advanced nuclear magnetic resonance (NMR) studies, which probe structural dynamics, metal binding, and interactions with molecular partners like the copper chaperone for SOD1 (CCS). This labeling strategy enhances resolution in NMR spectra, allowing precise characterization of conformational changes and ligand binding events .
Production and Validation of 15N-Labeled SOD1
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Cloning and Expression: A monomeric human SOD1 variant (T2M4SOD1) was engineered for stability and overexpressed in E. coli. High yields of uniformly 15N-labeled SOD1 were achieved using optimized growth media containing 15NH₄Cl as the nitrogen source .
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Folding and Activity: 15N-labeled T2M4SOD1 retained native folding, confirmed by ¹H-NMR spectroscopy, and exhibited enzymatic activity comparable to wild-type SOD1 in in-gel assays .
Metal Binding and Protein Interactions
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Copper-Zinc Coordination: 15N relaxation analysis revealed that apo-SOD1 (metal-free) exhibits higher flexibility (τₘ = 10.3 ns) compared to its holo form (τₘ = 18.6 ns when bound to CCS-D2) .
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CCS Chaperone Binding: Titration of 15N-labeled apo-SOD1 with CCS-D2 showed broadened NMR peaks, indicating complex formation. Affinity increased with stoichiometric ratios, requiring >2 equivalents of CCS-D2 for full binding .
Histidine Residue Analysis
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Active Site Probes: 15N NMR identified His side-chain environments in Fe-containing SOD analogs. Paramagnetic shifts revealed hydrogen-bonding patterns between His69 and coordinated solvent molecules, critical for catalytic activity .
Applications in Drug Screening
15N-labeled SOD1 enabled NMR-based binding studies with small molecules:
| Compound Tested | Binding Observation (via ¹H/¹⁵F NMR) | Citation |
|---|---|---|
| 5-Fluorouridine (FUrd) | Weak binding (line broadening) | |
| Uridine | No significant changes | |
| Trifluridine | Minimal spectral perturbations |
Key Research Findings
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Hyperpolarization Potential: While not directly tested on SOD Human, 15N₃-azide tags in related biomolecules achieved hyperpolarization lifetimes (T₁) up to 9.8 minutes, highlighting 15N’s utility for long-lived signal enhancement in metabolic imaging .
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Disease Relevance: Altered 15N isotopic abundance in proteins correlates with metabolic disorders, though direct links to SOD1 activity remain underexplored .
Comparative Dynamics with Prokaryotic SOD
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Flexibility Differences: Bacillus subtilis SOD (BsSOD) lacking Cu-binding His residues showed higher flexibility (average ¹H-¹⁵N NOE = 0.87) compared to human SOD1, underscoring evolutionary adaptations in metal coordination .
Future Directions
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Targeted Hyperpolarization: Expanding 15N-DNP (dynamic nuclear polarization) techniques to SOD Human could enhance real-time tracking of enzymatic activity in vivo.
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Therapeutic Development: High-resolution 15N NMR data may guide the design of SOD1-stabilizing drugs for neurodegenerative diseases like ALS.
Product Specs
Human Cu/Zn Superoxide Dismutase (SOD) is an enzyme that catalyzes the breakdown of superoxide radicals into hydrogen peroxide and molecular oxygen. This process is crucial for protecting cells from the harmful effects of superoxide radicals. SOD contains copper and zinc ions and is one of three isozymes responsible for eliminating free superoxide radicals in the body. The protein encoded by the SOD1 gene neutralizes superoxide molecules, preventing cellular damage caused by their uncontrolled levels.
Recombinant Human Superoxide Dismutase, 15N is a protein produced in E. coli. It is a single, non-glycosylated polypeptide chain composed of 153 amino acids, with a molecular weight of 15.8 kDa.
The product is lyophilized from a 0.2 μm filtered solution at a concentration of 1 mg/ml in PBS containing 0.1 mM CuCl2 and 0.2 mM ZnCl2.
The purity of the product is determined to be greater than 95.0% by SDS-PAGE analysis.
ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGD NTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSV ISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ.
Q&A
Why is 15N^{15}\text{N}15N-labeling used in SOD1 studies, and how does it enhance NMR-based research?
-labeling enables precise tracking of nitrogen atoms in SOD1 via NMR spectroscopy, critical for studying ligand interactions, conformational dynamics, and aggregation pathways. This isotopic labeling resolves chemical shift ambiguities in -NMR by providing distinct - correlation spectra, particularly for aromatic residues like Trp32, which are pivotal in binding studies . For example, in monomeric T2M4SOD1 (a six-mutation variant designed for NMR), -labeling facilitates detection of weak interactions with pyrimidine ligands (e.g., 5-fluorouridine) through line broadening or chemical shift perturbations .
Table 1: Advantages of -Labeling in SOD1 Research
How is 15N^{15}\text{N}15N-T2M4SOD1 expressed and purified for NMR studies?
The monomeric variant T2M4SOD1 is engineered with six mutations (C6A, C111S, and four surface charges) to disrupt dimerization while retaining enzymatic activity . Expression in E. coli uses:
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Minimal M9 media with as the sole nitrogen source.
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IPTG induction (0.2 mM) at mid-log phase (OD ~0.6–0.8) for 16–24 hours .
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Purification via Ni-NTA affinity chromatography, followed by dialysis to remove imidazole.
Key Optimization: Yields exceed 15 mg/L, achieved by omitting zinc/copper during growth and using glucose as the carbon source .
What challenges arise in interpreting NMR shifts for SOD1-ligand interactions, and how are they addressed?
Challenges:
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Spectral overlap: Residues in flexible regions (e.g., electrostatic loop) exhibit broadened peaks, complicating assignment .
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Intermediate exchange dynamics: Weak binding (e.g., 5-fluorouridine) causes line broadening rather than distinct chemical shift changes, masking precise interaction sites .
Solutions:
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Molecular docking and MD simulations: Predicts hydrogen bonding (e.g., Asp96-uracil) and validates NMR-observed interactions .
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Targeted mutagenesis: Trp32 mutants test binding specificity; -NMR detects fluorinated ligands with high sensitivity .
How can expression yields of 15N^{15}\text{N}15N-T2M4SOD1 be maximized?
Critical Parameters:
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Media composition: M9 media with (4 g glucose/L) supports high biomass without isotope dilution .
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IPTG concentration: 0.2 mM induces optimal expression without toxicity .
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Temperature control: Growth at 25°C reduces inclusion body formation compared to 37°C .
Table 2: Expression Optimization Data
| Parameter | Optimal Value | Yield (mg/L) | Reference |
|---|---|---|---|
| IPTG | 0.2 mM | >15 | |
| Temperature | 25°C | 12–15 | |
| Induction | Overnight | 15–20 |
How does 15N^{15}\text{N}15N-T2M4SOD1 aid in studying ALS-related SOD1 aggregation?
Monomeric T2M4SOD1 mimics immature SOD1, which is prone to misfolding and aggregation in ALS . NMR studies reveal:
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Conformational heterogeneity: Transient states (e.g., non-native oligomers) detected via - NMR .
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Disulfide bond dynamics: Cys57–Cys146 bond cleavage promotes aggregation, monitored via - HSQC .
Clinical Relevance: Insights into how familial (fALS) and sporadic (sALS) SOD1 variants share aggregation pathways .
How do NMR-based SOD1 binding studies compare to X-ray crystallography?
Advantages of NMR:
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Solution-state dynamics: Captures transient interactions (e.g., Trp32-uracil) missed in static crystal structures .
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Weak binding detection: Identifies low-affinity ligands via line broadening, complementary to X-ray’s high-resolution structural data .
Limitations:
What advanced data analysis methods resolve spectral ambiguities in SOD1-ligand NMR?
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Line-shape analysis: Quantifies binding kinetics (e.g., , ) from -NMR line broadening .
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Molecular docking: Predicts binding poses (e.g., uracil-Asp96 interactions) validated via MD simulations .
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Relaxation dispersion: Detects microsecond-timescale conformational changes linked to aggregation .
Software: CSP, TALOS-N, and HADDOCK for structure prediction .
What are the implications of 15N^{15}\text{N}15N-SOD1 studies for neuroprotective drug discovery?
-T2M4SOD1 screens identify small molecules (e.g., uracil analogs) that stabilize monomeric SOD1, preventing aggregation. This approach:
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Guides medicinal chemistry: Prioritizes ligands with strong hydrogen bonding (e.g., Asp96 interactions) .
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Validates structure-activity relationships: Correlates SOD activity (e.g., carboxyfullerenes) with neuroprotection .
Future Directions: Targeting SOD1’s electrostatic loop to disrupt non-native oligomerization .
How does 15N^{15}\text{N}15N-labeling in SOD1 differ from 13C^{13}\text{C}13C-labeling in carbohydrates?
While both isotopes enable NMR studies, -labeling in SOD1 focuses on protein-ligand interactions, whereas -labeling in carbohydrates (e.g., hyaluronan) resolves saccharide conformation . Key distinctions:
| Isotope | Application | Key Challenge |
|---|---|---|
| Protein dynamics, binding sites | Low natural abundance (0.36%) | |
| Carbohydrate structure | Overlap in -NMR signals |
Product Science Overview
Superoxide dismutase (SOD) is a crucial enzyme that plays a significant role in protecting cells from oxidative stress by catalyzing the dismutation of superoxide radicals into oxygen and hydrogen peroxide. The human recombinant form of this enzyme, labeled with nitrogen-15 (^15N), is particularly valuable for scientific research due to its enhanced stability and traceability in various experimental settings.
Superoxide dismutase exists in several isoforms, with the most common being the copper-zinc (CuZn-SOD), manganese (Mn-SOD), and iron (Fe-SOD) variants. The human recombinant SOD, often produced in Escherichia coli, retains the essential structural and functional characteristics of the native enzyme. The ^15N labeling allows for detailed nuclear magnetic resonance (NMR) studies, providing insights into the enzyme’s structure, dynamics, and interactions at the atomic level .
The primary function of SOD is to catalyze the conversion of superoxide radicals (O_2^•−) into molecular oxygen (O_2) and hydrogen peroxide (H_2O_2). This reaction is vital for mitigating the harmful effects of reactive oxygen species (ROS) in biological systems. The enzyme’s active site typically contains metal ions, such as copper and zinc, which facilitate the redox reactions necessary for dismutation .
The recombinant form of SOD, especially when labeled with ^15N, is extensively used in various research fields, including biochemistry, molecular biology, and medicine. Some key applications include:
- Structural Studies: ^15N-labeled SOD is instrumental in NMR spectroscopy, allowing researchers to study the enzyme’s structure and dynamics in detail.
- Oxidative Stress Research: SOD is a critical tool for investigating the mechanisms of oxidative stress and its role in diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases.
- Therapeutic Potential: Due to its antioxidative properties, SOD has potential therapeutic applications in treating conditions associated with oxidative damage, such as inflammation, ischemia-reperfusion injury, and aging .
The production of recombinant SOD involves cloning the human SOD gene into an expression vector, which is then introduced into a suitable host, such as E. coli. The bacteria are cultured under conditions that promote the expression of the recombinant protein. The ^15N labeling is achieved by growing the bacteria in a medium containing ^15N-labeled compounds. The protein is then purified using techniques such as affinity chromatography and characterized to ensure its activity and stability .
