Recombinant Macropus robustus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Mitochondrial Genome and MT-ND3
MT-ND3 is encoded by the mitochondrial genome and serves as a critical component of Complex I (NADH:ubiquinone oxidoreductase) in the mitochondrial respiratory chain. The MT-ND3 gene is found in the mitochondrial DNA (mtDNA) of most eukaryotic organisms, including marsupials like Macropus robustus. In mitochondrial genomes, MT-ND3 is one of several genes encoding subunits of the NADH dehydrogenase complex, which is essential for oxidative phosphorylation and ATP production . The organization of MT-ND3 within the mitochondrial genome is highly conserved across species, though variations exist in terms of exact positioning and neighboring genes.
Functional Significance in Energy Metabolism
As a component of Complex I, MT-ND3 participates in the first step of the electron transport chain, where it helps transfer electrons from NADH to ubiquinone. This process is fundamental to cellular respiration and energy production. Complex I functions as a proton pump, using the energy from electron transfer to move protons across the inner mitochondrial membrane, thereby generating the electrochemical gradient necessary for ATP synthesis . MT-ND3, despite its relatively small size, plays a crucial role in maintaining the structural integrity and functional efficiency of this complex molecular machinery.
Gene and Protein Nomenclature
The MT-ND3 gene and protein are known by several synonyms in scientific literature and databases:
| Synonym | Description |
|---|---|
| MT-ND3 | Mitochondrially encoded NADH dehydrogenase 3 |
| MTND3 | Alternative abbreviation |
| NADH3 | Simplified name referring to its role in NADH dehydrogenase complex |
| ND3 | Commonly used short form |
| NADH-ubiquinone oxidoreductase chain 3 | Descriptive name indicating function |
| NADH dehydrogenase subunit 3 | Alternative descriptive name |
This variety of names reflects the historical development of mitochondrial genomics and the different naming conventions used in various research contexts .
Expression and Purification Methods
The recombinant Macropus robustus MT-ND3 protein is produced using Escherichia coli as an expression system . The protein is expressed with an N-terminal His-tag, which facilitates purification through affinity chromatography techniques. The resulting recombinant protein represents the full-length MT-ND3 (amino acids 1-116) with the additional His-tag sequence . This expression system allows for efficient production of the protein in quantities suitable for research applications while maintaining the structural integrity necessary for functional studies.
Physical and Chemical Properties
The recombinant MT-ND3 protein is supplied as a lyophilized powder with a purity greater than 90% as determined by SDS-PAGE . The lyophilized format enhances stability during shipping and storage while maintaining the protein's native conformation. For research applications, the protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance stability during storage, it is recommended to add glycerol to a final concentration of 5-50% after reconstitution .
Biochemical and Biophysical Assays
The purified recombinant MT-ND3 protein can be employed in various biochemical assays to study its interactions with other subunits of Complex I or with inhibitors and substrates . These studies contribute to our understanding of the mechanism of electron transport and proton pumping in mitochondria. Specific applications include binding assays, enzyme kinetics studies, and investigations of protein-protein interactions within the respiratory complex.
Comparative Genomics and Evolutionary Studies
MT-ND3 is a conserved component of mitochondrial genomes across various species. Comparative studies of MT-ND3 from different organisms can provide insights into the evolution of mitochondrial function and the adaptation of energy metabolism to different environmental conditions . The availability of recombinant Macropus robustus MT-ND3 facilitates such comparative analyses, particularly in the context of marsupial evolution and adaptation.
Immunological Research
The recombinant protein can serve as an antigen for the production of specific antibodies against MT-ND3. These antibodies can then be used in various immunological techniques such as Western blotting, immunohistochemistry, and immunoprecipitation to study the expression, localization, and regulation of MT-ND3 in different tissues and under various physiological or pathological conditions.
Gene Structure and Organization
In the mitochondrial genome, MT-ND3 is typically located in a specific position relative to other mitochondrial genes. While the exact organization can vary between species, studies of mitochondrial genomes show that MT-ND3 is often situated between tRNA genes and other protein-coding genes . The complete mitochondrial genome sequencing projects have revealed interesting variations in the organization and expression of MT-ND3 across different taxonomic groups.
Transcription and Translation Mechanisms
The transcription of MT-ND3, like other mitochondrial genes, is controlled by the mitochondrial transcription machinery. The resulting mRNA is translated on mitochondrial ribosomes to produce the MT-ND3 protein. In some species, including the turtle Pelomedusa subrufa, MT-ND3 uses ATA as the initiation codon and has a truncated termination codon (T--) . Additionally, some species exhibit unusual features such as extra nucleotides in the ND3 gene that may require post-transcriptional editing for proper expression . These variations in transcription and translation mechanisms highlight the diversity of mitochondrial gene expression strategies across different species.
Unusual Features in Various Species
Studies of MT-ND3 across different species have revealed several unusual features that may affect protein function. For example, in the African side-necked turtle (Pelomedusa subrufa), researchers found that MT-ND3 had extra nucleotides that potentially affected its open reading frame, suggesting the possibility of post-transcriptional editing mechanisms . Such findings highlight the diversity of mitochondrial gene expression strategies across different taxonomic groups and suggest that similar variations might exist in the expression and function of MT-ND3 in Macropus robustus.
Product Specs
Note: We will prioritize shipping the format that is currently in stock. However, if you have specific requirements for the format, please indicate them in your order notes. We will accommodate your needs to the best of our ability.
Note: All of 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 the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is MT-ND3 and what is its functional role in mitochondrial complex I?
MT-ND3 (Mitochondrially encoded NADH-ubiquinone oxidoreductase chain 3) is one of seven mitochondrially-encoded subunits of complex I, the largest complex of the mitochondrial respiratory chain. Located in the mitochondrial inner membrane, complex I catalyzes electron transfer from NADH to ubiquinone . The mitochondrially encoded subunits, including MT-ND3, are the most hydrophobic components and form the core of the transmembrane region .
MT-ND3 plays a critical but not fully understood role in electron transport, proton pumping, or ubiquinone binding. Experimental evidence suggests that MT-ND3 mutations cause disproportionately greater reductions in enzyme activity compared to reductions in assembled complex I, indicating its importance in catalytic function rather than just structural integrity .
Why is Macropus robustus MT-ND3 of interest for comparative mitochondrial research?
Studying MT-ND3 from Macropus robustus (Wallaroo) provides valuable evolutionary insights into mitochondrial protein conservation and function. As demonstrated by phylogenetic analyses, marsupials diverged from eutherian mammals approximately 130 million years before present (MYBP) .
The complete mitochondrial genome of Macropus robustus (16,896 nucleotides) has been sequenced, providing an important reference point for comparative mitochondrial genomics . Marsupial mitochondria exhibit several unique features, including RNA editing and tRNA gene rearrangements, making them valuable for studying the evolution of mitochondrial function across different mammalian lineages.
What expression systems are appropriate for producing recombinant MT-ND3?
Several expression systems can be employed for MT-ND3 production, each with distinct advantages:
| Expression System | Advantages | Considerations | Applications |
|---|---|---|---|
| E. coli | High yield, cost-effective, rapid expression | May require codon optimization, inclusion body formation common | Structural studies, antibody production |
| Insect cells | Better post-translational modifications, improved folding | More expensive, longer production time | Functional studies, complex reconstitution |
| Mammalian cells | Native-like modifications, proper folding | Lowest yield, highest cost | Interaction studies, therapeutic applications |
| Cell-free systems | Avoids toxicity issues, direct incorporation into lipid systems | Limited scale, expensive | Difficult-to-express variants, rapid screening |
For hydrophobic membrane proteins like MT-ND3, E. coli expression typically requires fusion tags (e.g., His-tag) and specialized solubilization methods . Protein purification often employs affinity chromatography followed by reconstitution into lipid systems to maintain native-like structure.
How can researchers assess the functional integrity of recombinant MT-ND3 protein?
Assessment of recombinant MT-ND3 requires multiple complementary approaches:
Structural integrity:
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Circular dichroism spectroscopy to evaluate secondary structure
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Limited proteolysis to examine folding quality
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Thermal stability assays to assess protein stability
Complex I assembly:
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Blue native PAGE to visualize assembled complex I
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Immunoblotting with appropriate antibodies (e.g., MT-ND3 (E8O4E) Rabbit mAb)
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Co-immunoprecipitation with other complex I subunits
Functional assays:
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NADH:ubiquinone oxidoreductase activity measurements
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Oxygen consumption rate in reconstituted systems
When examining complex I activity, it's important to note that MT-ND3 mutations often affect enzyme activity more severely than they impact complex I assembly, indicating MT-ND3's crucial role in catalytic function .
What techniques can be used to detect and quantify MT-ND3 mutations and heteroplasmy?
Several molecular techniques have been developed for the precise detection and quantification of MT-ND3 mutations:
ARMS-qPCR (Amplification Refractory Mutation System):
This technique uses mutation-specific primers to quantitatively determine mutation rates in MT-ND3. For example, researchers studying the T10158C mutation designed primers with one mismatch at the 3' terminal side to distinguish between wild-type and mutant sequences . The mutation rate can be calculated using the following formula:
Mutation rate (%) = (2^(Ct WT - Ct MT) / (1 + 2^(Ct WT - Ct MT))) × 100
where Ct WT and Ct MT are the threshold cycle values from qPCR using wild-type and mutant-specific primers, respectively .
Sanger sequencing and NGS:
These methods can identify novel mutations and quantify heteroplasmy levels. For NGS approaches, the mutant load can be determined by counting the number of mtDNA reads containing the variant compared to wild-type sequences .
PCR-RFLP (Restriction Fragment Length Polymorphism):
This technique can be used when mutations create or eliminate restriction enzyme recognition sites.
What are the known pathogenic mutations in MT-ND3 and their biochemical consequences?
Several disease-causing mutations have been identified in the MT-ND3 gene, primarily in humans:
These mutations typically cause isolated complex I deficiency, leading to reduced ATP synthesis and increased oxidative stress. The pathogenic variants often affect highly conserved domains of MT-ND3, highlighting functional regions critical for complex I activity .
Interestingly, several mutations appear to be de novo, with no mutation detected in maternal relatives, suggesting that mitochondrial DNA disease may be more prevalent in pediatric populations than previously predicted .
How does the MT-ND3 polymorphism rs2853826 (10398A>G) influence disease risk and mitochondrial function?
The MT-ND3 variant rs2853826 (10398A>G) has significant implications for mitochondrial function and disease:
Disease associations:
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Linked to multiple disease phenotypes according to MitoMap database
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Associated with neurodegenerative conditions including Alzheimer's disease
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Correlated with type 2 diabetes mellitus and increased ROS production
Molecular effects:
Gene network associations:
Gene expression data has revealed that 10398A>G affects networks involved in mitochondrial respiratory chain and Complex I function. This polymorphism provides evidence linking mitochondrial SNPs with mitochondrial heteroplasmy and offers insights into pathomechanisms driven by this pleiotropic disease-associated locus .
What therapeutic approaches have been developed for MT-ND3 mutations?
Recent research has demonstrated promising therapeutic strategies for MT-ND3 mutations:
Allotopic expression:
This approach involves nuclear expression of mitochondrially-encoded genes through:
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Codon optimization for cytoplasmic translation
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Addition of mitochondrial targeting sequences
Nuclear expression of codon-optimized MT-ND3 has been shown to partially restore protein levels, complex I function, and ATP production in patient-derived cells with m.10197G>C and m.10191T>C mutations .
Direct mRNA delivery:
Researchers have developed a mitochondrial delivery system using MITO-Porters to transport therapeutic wild-type MT-ND3 mRNA into diseased mitochondria . Key modifications include:
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Changing the non-standard start codon (ATA) to ATG
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Optimizing the mRNA for mitochondrial translation
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Addition of polyA modifications to improve stability
This approach decreased the mutation rate of mRNA (ND3) in mitochondria and improved mitochondrial respiration in cells derived from Leigh syndrome patients .
What experimental considerations are important when designing studies using recombinant MT-ND3?
When working with recombinant MT-ND3, researchers should consider:
Membrane protein challenges:
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Hydrophobicity may lead to aggregation during expression and purification
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Specialized detergents or lipid systems are required for proper folding
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Storage buffer optimization is critical (e.g., Tris-based buffer with 50% glycerol at -20°C)
Species-specific differences:
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Variations in MT-ND3 sequence between species may affect antibody recognition
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Different codon usage preferences necessitate optimization for the expression system
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Post-translational modifications may vary between species
Functional context:
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MT-ND3 functions within the larger complex I structure
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Isolated protein may not retain native conformation or activity
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Consider reconstitution with other complex I components for functional studies
Mutation analysis:
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Design primers carefully for mutation detection (see ARMS-PCR methodology)
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Consider heteroplasmy effects on experimental outcomes
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Include appropriate controls for both wild-type and mutant sequences
How can MT-ND3 be used as a model for studying mitochondrial complex I assembly and function?
MT-ND3 provides an excellent model for investigating complex I biology for several reasons:
Central role in complex I:
MT-ND3 is one of the core hydrophobic subunits forming the transmembrane domain of complex I. Mutations in MT-ND3 often cause disproportionately greater reductions in enzyme activity than in complex assembly, suggesting its critical role in electron transport or proton pumping functions .
Conservation across species:
The high degree of conservation of MT-ND3 across diverse species facilitates comparative studies. Analyses of MT-ND3 from species like Macropus robustus provide evolutionary insights into mitochondrial function .
Disease relevance:
MT-ND3 mutations cause well-characterized mitochondrial disorders, particularly Leigh syndrome and dystonia, making it relevant for translational research .
Experimental approaches:
Researchers can use techniques such as:
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Site-directed mutagenesis to study specific residues
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Cybrid cell models to examine mutation effects in controlled nuclear backgrounds
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Reconstitution experiments to study MT-ND3's role in complex I assembly
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Cross-linking studies to identify interaction partners within complex I
What gaps remain in our understanding of MT-ND3 structure and function?
Despite significant progress, several knowledge gaps persist in MT-ND3 research:
Structural dynamics:
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How MT-ND3 contributes to conformational changes during electron transfer
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The precise role of specific conserved residues in catalytic function
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Interaction interfaces with other complex I subunits
Species-specific functions:
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Functional differences between MT-ND3 from various species, including marsupials like Macropus robustus
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Adaptive changes in MT-ND3 related to metabolic requirements across species
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Evolution of MT-ND3 in the context of the complete mitochondrial genome
Therapeutic development:
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Optimization of allotopic expression for clinical applications
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Development of small molecules that might compensate for MT-ND3 mutations
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Tissue-specific effects of MT-ND3 mutations and therapeutic approaches
Regulatory mechanisms:
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Factors affecting MT-ND3 expression and incorporation into complex I
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Post-translational modifications that might regulate MT-ND3 function
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Interactions between nuclear and mitochondrial genetic systems in MT-ND3 expression
Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, biochemistry, molecular genetics, and therapeutic development.
