Recombinant Macaca hecki NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)
Description
Functional Role in Mitochondrial Respiration
MT-ND4L is a core subunit of Complex I, facilitating electron transfer from NADH to ubiquinone. Its roles include:
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Electron transport: Participates in the first step of oxidative phosphorylation by transferring electrons to ubiquinone .
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Proton pumping: Supports the generation of the proton gradient across the mitochondrial membrane, essential for ATP synthesis .
Comparative Functional Insights:
| Feature | Macaca hecki MT-ND4L | Human MT-ND4L |
|---|---|---|
| Amino acid length | 98 residues | 98 residues |
| Molecular weight | 10.8 kDa | 10.7 kDa |
| Key mutation sites | Not reported | Val65Ala (LHON-linked) |
| Complex I integration | Transmembrane core | Transmembrane core |
Production and Purification
Recombinant MT-ND4L is typically expressed in E. coli with an N-terminal His-tag for purification .
Research Applications
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Mitochondrial disease studies: Used to model mutations linked to Leber’s hereditary optic neuropathy (LHON) and metabolic disorders .
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Drug discovery: Screens for compounds targeting Complex I dysfunction in neurodegenerative diseases .
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Structural biology: Aids in cryo-EM studies to resolve Complex I architecture .
Limitations and Future Directions
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The specific tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Recombinant Macaca hecki NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It catalyzes electron transfer from NADH through the respiratory chain, utilizing ubiquinone as the electron acceptor.
Q&A
What is the functional role of MT-ND4L in mitochondrial Complex I?
MT-ND4L is a core subunit of mitochondrial Complex I (NADH:ubiquinone oxidoreductase), which catalyzes electron transfer from NADH to ubiquinone during oxidative phosphorylation . This subunit is embedded in the inner mitochondrial membrane and contributes to proton translocation across the membrane, a critical step for ATP synthesis . Methodologically, its role can be studied using blue native PAGE to isolate intact Complex I assemblies, followed by ubiquinone-reduction assays to measure electron transfer kinetics . For example, researchers have observed that mutations in ND4L (e.g., Val65Ala) disrupt proton pumping without affecting baseline electron transfer rates, suggesting its specific role in coupling electron transport to proton translocation .
How is recombinant MT-ND4L expressed and purified for structural studies?
Recombinant MT-ND4L requires mitochondrial codon-optimized expression systems due to its hydrophobic, multi-pass transmembrane structure . A validated protocol involves:
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Cloning the MT-ND4L gene into a plasmid with a mitochondrial targeting sequence (e.g., pMIT-Vector).
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Transfecting mammalian cell lines (e.g., HEK-293) and inducing expression under hypoxia-mimicking conditions (1% O₂).
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Purifying the protein using Ni-NTA affinity chromatography after tagging with a histidine-rich sequence at the C-terminus .
Critical quality checks include circular dichroism to confirm α-helical content (>80%) and liposome reconstitution assays to verify membrane integration .
How do researchers reconcile conflicting data on ND4L’s role in ubiquinone binding vs. proton translocation?
Discrepancies arise from methodological differences in studying isolated Complex I subunits versus intact assemblies. For example:
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Isolated ND4L: Shows no direct ubiquinone-binding activity in surface plasmon resonance assays .
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Intact Complex I: ND4L mutations (e.g., ND4/11778) reduce NAD-linked substrate oxidation by 40% in mitochondrial preparations, implicating it in inter-subunit communication rather than direct catalysis .
To resolve this, cryo-EM studies at <3.0 Å resolution have mapped ND4L’s interaction with ND2 and ND3 subunits, revealing a hydrogen-bond network critical for proton channel stability . Researchers should combine structural biology with functional mutagenesis (e.g., introducing Cys mutations for crosslinking studies) to dissect domain-specific roles.
What strategies address the low yield of recombinant MT-ND4L in in vitro studies?
How does the T10663C (Val65Ala) mutation in MT-ND4L impair mitochondrial function?
This mutation reduces proton-pumping efficiency by 60% while preserving 85% of electron transfer activity, as shown in cysteine-labeling experiments and ATP synthesis assays . Key steps to model this:
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Introduce the T10663C mutation via CRISPR-Cas9 in cybrid cell lines.
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Measure Δψm (mitochondrial membrane potential) using TMRE fluorescence.
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Perform BN-PAGE/Western blot to confirm intact Complex I assembly.
Data from patient-derived fibroblasts show a 30% reduction in maximal respiration (Seahorse XF analysis), highlighting the mutation’s bioenergetic impact .
What controls are essential when using MT-ND4L antibodies in localization studies?
Why do some studies report normal Complex I activity despite ND4L mutations?
Discrepancies arise from tissue-specific isoforms and compensatory mechanisms. For example:
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Skeletal muscle homogenates: Retain 70% Complex I activity due to alternate NADH dehydrogenases (e.g., NDI1) .
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Fibroblasts: Show 50% reduced activity, as seen in spectrophotometric assays monitoring NADH oxidation at 340 nm .
Researchers must standardize tissue sources and use in-gel activity assays to isolate Complex I-specific function.
