Recombinant Tarsius syrichta NADH-ubiquinone oxidoreductase chain 5 (MT-ND5)
Description
Introduction to Recombinant Tarsius syrichta NADH-Ubiquinone Oxidoreductase Chain 5 (MT-ND5)
Recombinant Tarsius syrichta MT-ND5 is a mitochondrial protein subunit of Complex I (NADH:ubiquinone oxidoreductase), critical for electron transport and proton pumping in oxidative phosphorylation . Native MT-ND5 is encoded by mitochondrial DNA but is often studied via recombinant production for structural, functional, and therapeutic research. The Tarsius syrichta (Philippine tarsier) variant has garnered attention due to its unique genomic features, including mitochondrial DNA insertions into the nuclear genome .
Production and Biochemical Characteristics
Recombinant MT-ND5 from Tarsius syrichta is typically expressed in heterologous systems (e.g., E. coli, yeast) with tags (e.g., His-tag) for purification. Key parameters include:
The protein’s amino acid sequence (UniProt ID: Q36151) includes conserved motifs for NADH dehydrogenase activity and interactions with ubiquinone .
4.2. Functional Studies
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Proton Pumping: MT-ND5 contributes to the proton-pumping efficiency of Complex I, with mutations linked to mitochondrial disorders .
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ELISA Applications: Recombinant MT-ND5 is used in immunoassays to detect protein levels in mitochondrial research .
Evolutionary and Genomic Context
The Tarsius syrichta genome uniquely harbors nuclear mitochondrial DNA (NUMT), including a contiguous MT-ND5 sequence . This integration:
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Mechanism: Likely arose via recombination of fragmented mitochondrial DNA insertions.
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Significance: Provides a model to study differential evolution of mitochondrial genes in nuclear vs. mitochondrial environments .
Challenges and Future Directions
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them when placing your order, and we will prepare accordingly.
Important: 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 charges may 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
Q&A
What experimental strategies ensure proper folding and enzymatic activity of recombinant MT-ND5?
Recombinant MT-ND5 requires precise expression and purification protocols to maintain structural integrity. Researchers should employ E. coli-based expression systems with codon optimization for eukaryotic mitochondrial proteins, as demonstrated in rat ortholog studies . Key steps include:
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Cloning the MT-ND5 gene into pET vectors with N-terminal His-tags for affinity chromatography
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Optimizing induction conditions (e.g., 0.5 mM IPTG at 16°C for 18 hours) to minimize inclusion body formation
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Verifying protein conformation via circular dichroism spectroscopy and ATPase activity assays
How can researchers resolve discrepancies in MT-ND5 functional assays across studies?
Contradictions in enzymatic activity measurements (e.g., NADH dehydrogenase rates) often stem from:
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Species-specific post-translational modifications absent in prokaryotic expression systems
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Variations in electron transport chain reconstitution methods
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Mitochondrial membrane potential differences in assay setups
A standardized protocol should include:
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Parallel assays using native tarsier mitochondrial extracts for baseline comparison
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Controlled ubiquinone analog concentrations (10-100 µM range)
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Temperature calibration matching T. syrichta physiology (28-32°C)
What phylogenetic considerations impact comparative studies of MT-ND5?
The Philippine tarsier's MT-ND5 exhibits unique adaptations reflecting insular evolution . For valid cross-species comparisons:
Researchers should:
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Use maximum likelihood phylogenetic models accounting for accelerated evolution in primate mitochondrial genes
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Perform functional complementation assays in ND5-deficient cell lines
Which controls are essential when investigating MT-ND5 mutations?
Robust experimental design requires:
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Isogenic controls: Wild-type vs. mutated MT-ND5 in identical expression systems
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Electron transport chain activity normalization: Citrate synthase activity as internal reference
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Orthologous validation: Compare results with rat ND5 paralogs under identical assay conditions
Critical negative controls:
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Empty vector-transfected mitochondria
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Rotenone-treated samples (Complex I inhibitor)
How does MT-ND5 structural biology inform mutagenesis studies?
The protein’s transmembrane topology dictates rational mutagenesis approaches:
| Domain | Functional Role | Target Residues |
|---|---|---|
| N-terminal β-strand | Ubiquinone binding | Q42, K45 |
| Matrix loop | Proton channel gating | H78, D81 |
| C-terminal helix | Iron-sulfur cluster coordination | C133, C137 |
Advanced methodologies:
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Molecular dynamics simulations of lipid bilayer-embedded structures
What metrics validate successful MT-ND5 integration into artificial proteoliposomes?
Quantitative benchmarks include:
Essential controls:
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Liposomes without MT-ND5
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ATP synthase-coupled systems to verify electrochemical gradient utilization
How do researchers address species-specific codon usage biases in heterologous expression?
T. syrichta MT-ND5 contains 7 rare E. coli codons in critical structural regions . Mitigation strategies:
| Codon | Frequency in E. coli | Optimization Method |
|---|---|---|
| AGG (Arg) | 0.14% | TRNA supplementation plasmids |
| CTA (Leu) | 0.51% | Site-directed mutagenesis to CTG |
| CCC (Pro) | 0.38% | Co-expression with rare codon augmenter strains |
Validation requires:
What advanced techniques characterize MT-ND5’s role in reactive oxygen species (ROS) regulation?
Cutting-edge approaches combine:
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Site-specific spin labeling for EPR spectroscopy of conformational changes
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FLIM-FRET to quantify protein-protein interactions with complex I subunits
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H2O2 biosensor integration in mitochondrial-targeted reporter cell lines
Critical data interpretation considerations:
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Differentiate mitochondrial ROS (mROS) from cytoplasmic sources
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Normalize to mitochondrial membrane potential (TMRE staining controls)
How should researchers design longitudinal stability studies of recombinant MT-ND5?
Accelerated degradation protocols must simulate physiological conditions:
| Stress Factor | Test Condition | Stability Threshold |
|---|---|---|
| Thermal | 37°C, 48 hours | <5% activity loss |
| Oxidative | 100 µM H2O2, 1 hour | >80% structural integrity |
| pH Variation | pH 6.0-8.0 range | <15% conformational change |
Analytical requirements:
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Synchrotron radiation CD for secondary structure analysis
What computational models predict MT-ND5 interactions with novel inhibitors?
Combined quantum mechanics/molecular mechanics (QM/MM) approaches:
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Docking simulations: 50 ns MD runs with explicit membrane models
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Binding energy calculations: MM/GBSA vs. MMPBSA validation
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Pharmacophore mapping: Prioritize residues within 4Å of ubiquinone-binding pocket
Validation pipeline:
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In vitro IC50 determinations using purified complex I
Researchers must cross-validate predictions with:
