Recombinant Peromyscus polionotus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Introduction to Recombinant Peromyscus polionotus MT-ND3
NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) is one of the seven mitochondrial DNA-encoded subunits of Complex I in the mitochondrial respiratory chain. Complex I, also known as NADH:ubiquinone oxidoreductase, serves as the primary entry point for electrons into the respiratory chain and plays a crucial role in cellular energy metabolism . MT-ND3 specifically functions as part of the membrane arm of Complex I, contributing to the proton translocation module that pumps protons across the inner mitochondrial membrane . Recombinant Peromyscus polionotus MT-ND3 refers to the artificially produced version of this protein from the Oldfield mouse (Peromyscus polionotus), expressed using recombinant DNA technology in bacterial systems for research purposes .
The significance of studying MT-ND3 lies in its fundamental role in mitochondrial function and cellular energy production. As part of Complex I, it contributes to oxidative phosphorylation, a process that generates ATP, the primary energy currency of cells . Dysfunction of MT-ND3 or other Complex I components has been implicated in various mitochondrial disorders and neurodegenerative diseases, emphasizing the importance of understanding its structure and function .
Protein Structure and Domains
As a component of Complex I, MT-ND3 contributes to the characteristic L-shaped structure of the complex, specifically residing in the membrane arm that is embedded in the inner mitochondrial membrane . Within the functional architecture of Complex I, MT-ND3 is part of the proton translocation module (P module), which is responsible for pumping protons across the inner membrane .
The transmembrane domains of MT-ND3 are critical for its integration into the lipid bilayer of the inner mitochondrial membrane. These hydrophobic regions form alpha-helical structures that span the membrane, while the connecting loops provide interaction surfaces with other subunits of Complex I .
Biochemical Properties of Recombinant Peromyscus polionotus MT-ND3
The commercially available recombinant Peromyscus polionotus MT-ND3 protein has been expressed in Escherichia coli and purified to greater than 90% purity as determined by SDS-PAGE . The protein is supplied as a lyophilized powder, which enhances its stability during storage and shipping .
For research applications, the recombinant MT-ND3 can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . The addition of glycerol (recommended final concentration of 50%) helps maintain protein stability during long-term storage at -20°C or -80°C .
The storage buffer for the recombinant protein consists of a Tris/PBS-based buffer containing 6% trehalose, with a pH of 8.0 . This formulation helps maintain the protein's integrity and biological activity during storage.
| Property | Description |
|---|---|
| Source | Expressed in E. coli |
| Tag | N-terminal His-tag |
| Protein Length | Full Length (1-115 amino acids) |
| Form | Lyophilized powder |
| Purity | >90% (SDS-PAGE) |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Recommended Storage | -20°C/-80°C (aliquoted) |
| Reconstitution | Deionized sterile water (0.1-1.0 mg/mL) |
Function in the Respiratory Chain
MT-ND3, as a component of Complex I, participates in the first step of the electron transport chain in mitochondrial respiration. Complex I oxidizes NADH, which is generated through the Krebs cycle in the mitochondrial matrix, and uses the two electrons to reduce ubiquinone to ubiquinol . This electron transfer is coupled with proton pumping from the mitochondrial matrix to the intermembrane space, contributing to the establishment of a proton gradient across the inner mitochondrial membrane .
The energy stored in this proton gradient is subsequently utilized by ATP synthase (Complex V) to produce ATP from ADP and inorganic phosphate, completing the oxidative phosphorylation process . As part of the proton translocation module within Complex I, MT-ND3 plays a critical role in this energy-generating process.
Assembly and Stability of Complex I
The assembly of Complex I involves the preassembly of the membrane and matrix arms through independent pathways, which subsequently join to form the complete complex . MT-ND3, along with other mitochondrial DNA-encoded subunits, is part of the membrane arm assembly pathway . The specific interactions between MT-ND3 and other subunits during this assembly process are still being elucidated through ongoing research.
Production and Purification of Recombinant Peromyscus polionotus MT-ND3
The production of recombinant Peromyscus polionotus MT-ND3 involves several steps of genetic engineering and protein expression. The gene encoding the MT-ND3 protein (UniProt ID: Q95921) is cloned into an expression vector, which is then transformed into an E. coli host for protein production .
After expression and purification, the protein undergoes quality control assessments, including SDS-PAGE to confirm its purity and size . The final product is a highly purified (>90%) recombinant protein suitable for various research applications .
Applications and Research Significance
Recombinant Peromyscus polionotus MT-ND3 serves as a valuable tool for researchers studying mitochondrial function, respiratory chain dynamics, and related disorders. The availability of purified recombinant protein enables various experimental approaches, including:
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Structural studies to elucidate the three-dimensional organization of Complex I and the specific role of MT-ND3 within this structure.
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Functional assays to investigate the contribution of MT-ND3 to electron transport and proton pumping activities of Complex I.
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Interaction studies to identify binding partners and regulatory factors that modulate MT-ND3 function.
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Immunological studies using the recombinant protein as an antigen for antibody production or as a standard in quantitative assays.
The research significance of MT-ND3 extends beyond basic mitochondrial biology to potential implications in human health and disease. Mutations in mitochondrial DNA-encoded Complex I subunits, including MT-ND3, have been associated with various mitochondrial disorders . Understanding the structure and function of these proteins can provide insights into the molecular mechanisms underlying such diseases and potentially inform therapeutic strategies.
Potential Role in ROS Generation and Oxidative Stress
Complex I is one of the main sites of reactive oxygen species (ROS) production in mitochondria, where electron leakage leads to the formation of superoxide anions . These ROS can act as signaling molecules, activating various cellular pathways, including protein kinase C, mitogen-activated protein kinase (MAPK), PI3K, Akt, and p38 MAPK, as well as calcium signaling .
Recent evidence suggests that Complex I contributes significantly to ROS generation in intact mitochondria . There are two potential sites on Complex I where oxygen could access electrons: the flavin moiety and the ubiquinone-binding site . As a component of Complex I, MT-ND3 may indirectly influence ROS production and thereby contribute to cellular oxidative stress responses.
Mutations in genes encoding Complex I subunits, including MT-ND3, have been linked to alterations in ROS levels . This connection between Complex I dysfunction and oxidative stress has implications for understanding aging processes and various pathological conditions, including neurodegenerative diseases .
Product Specs
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Target Background
Function: Recombinant Peromyscus polionotus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) 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 an electron acceptor. MT-ND3 is essential for the catalytic activity of Complex I.
Q&A
How is recombinant MT-ND3 protein typically prepared for research applications?
Recombinant Peromyscus polionotus MT-ND3 is commonly expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The protein is typically supplied as:
| Characteristic | Specification |
|---|---|
| Form | Lyophilized powder |
| Purity | >90% as determined by SDS-PAGE |
| Storage buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Tag | N-terminal His-tag |
| Protein length | Full length (1-115aa) |
For optimal use, researchers should reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add glycerol to a final concentration of 5-50% for long-term storage .
What evolutionary significance does MT-ND3 have across species?
While complete cross-species analysis is not fully detailed in the search results, evidence indicates that MT-ND3 contains highly conserved functional domains, particularly in the ND3 loop involved in the active/deactive state transition of Complex I . This conservation suggests critical evolutionary importance for mitochondrial function across species. Naturally occurring mtDNA polymorphisms, including those affecting MT-ND3, can serve as valuable tools for analyzing pathogenic effects in research models .
What methodologies are currently employed for mitochondrial base editing of MT-ND3?
Current research has demonstrated successful in vivo base editing of mouse mitochondrial DNA using DddA-derived cytosine base editors (DdCBE) delivered via adeno-associated viral (AAV) vectors . The protocol involves:
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Design of DdCBE pairs containing TALE domains binding to specific mtDNA regions
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Integration of different DddA toxin split combinations (G1333 or G1397)
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Targeting the complementary cytosine residues corresponding to MT-ND3 positions
In a specific experiment targeting mouse MT-ND3 (mtDNA positions m.9576 G and m.9577 G), researchers designed four DdCBE pairs with different configurations. These targeted a 19bp sequence in mouse MT-ND3 with TALE domains binding to the light strand (positions m.9549–m.9564) and heavy strand (positions m.9584–m.9599) .
Editing efficiency analysis revealed that three of the four pairs (pairs 1, 2, and 3) demonstrated effective editing, with pair 1 showing up to ~43% editing of the target cytosines .
How can researchers effectively quantify MT-ND3 editing efficiency in experimental systems?
Comprehensive assessment of MT-ND3 editing requires multiple complementary approaches:
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Sanger sequencing: Initial detection and qualitative assessment of editing at target cytosines.
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Next-generation sequencing (NGS): Provides detailed quantitative analysis of:
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Editing percentage at each target cytosine
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Proportion of specific amino acid changes (e.g., G40K, G40E, G40*)
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Simultaneous editing patterns across multiple target sites
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Off-target editing across the mitochondrial genome
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Controls: Vehicle-injected controls and catalytically inactive DdCBE controls are essential to distinguish editing-induced changes from natural background heteroplasmy .
A typical experimental workflow involves isolating mtDNA from the tissue of interest, PCR amplification of the target region, followed by both Sanger sequencing for qualitative assessment and NGS for quantitative analysis .
What considerations are important for AAV delivery of MT-ND3 editors to different tissues and age groups?
Research has revealed significant variations in editing efficiency and off-target effects based on delivery approach and subject age:
| Parameter | Adult Mice | Neonatal Mice |
|---|---|---|
| Delivery Route | Tail vein injection | Temporal vein injection |
| Viral Dose | 4×10¹² vg per monomer | 2×10¹² vg per monomer |
| Editing Efficiency (target sites) | 10-20% at 24 weeks post-injection | 20-30% at 3 weeks post-injection |
| Off-target Effects | 7-fold higher with extended treatment (24 weeks vs. 3 weeks) | Highest among all groups studied |
These findings demonstrate that treatment of younger subjects significantly enhances editing efficacy but may increase off-target effects. Researchers should carefully consider this trade-off when designing experimental protocols .
How do MT-ND3 polymorphisms impact mitochondrial function and disease pathogenesis?
Mutations in MT-ND3 can significantly alter Complex I function, with cascading effects on mitochondrial respiration and cellular metabolism. Research has demonstrated that:
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Specific amino acid changes in MT-ND3 (e.g., G40K, G40E, or premature stop codons) affect the conserved ND3 loop involved in Complex I active/deactive transition .
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mtDNA variants, including those affecting MT-ND3, can "interfere with cognitive abilities and differentially modulate mitochondrial oxidative phosphorylation (OXPHOS) and the generation of reactive oxygen species (ROS)" .
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There are potential links between mitochondrial dysfunction and neurodegenerative diseases, with evidence that mitochondria from Alzheimer's disease patients show respiratory chain deficiency and increased amyloid-β accumulation when transferred to mtDNA-depleted cells .
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The age-related decline of respiratory chain function appears more strongly affected by mtDNA point mutations than by deletions, highlighting the potential significance of specific nucleotide changes in genes like MT-ND3 .
What methods can researchers use to minimize off-target effects in MT-ND3 editing experiments?
Off-target effects represent a significant challenge in mitochondrial base editing. Research has shown varying off-target C:G-to-T:A editing frequencies:
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Adult mice treated for 3 weeks: 0.026–0.046% (comparable to controls)
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Adult mice treated for 24 weeks: 0.22-0.30% (~7-fold higher than controls)
To minimize these effects, researchers should consider:
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Editor design optimization: Testing multiple DdCBE pair configurations to identify those with highest on-target specificity.
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Treatment duration calibration: Balancing editing efficiency with off-target accumulation over time.
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Comprehensive controls: Including vehicle-injected and catalytically inactive editor controls to accurately distinguish editor-induced mutations from background.
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Age-appropriate protocols: Recognizing that while neonatal treatment may increase editing efficiency, it also increases off-target effects .
What experimental models are most effective for studying MT-ND3 function and mutations?
Several experimental models have proven valuable for MT-ND3 research:
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In vitro cellular models:
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In vivo models:
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Specialized experimental systems:
Each model system offers distinct advantages depending on the specific research question being addressed.
How should recombinant MT-ND3 protein be handled to maintain optimal activity?
For maximum stability and experimental reproducibility, observe these guidelines for recombinant MT-ND3:
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Storage conditions:
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Store at -20°C/-80°C upon receipt
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Aliquot to minimize freeze-thaw cycles
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Working aliquots can be stored at 4°C for up to one week
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Reconstitution procedure:
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Centrifuge vial briefly before opening
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Reconstitute in deionized sterile water to 0.1-1.0 mg/mL
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Add glycerol to 5-50% final concentration for long-term storage
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Quality assessment:
Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided.
How can MT-ND3 research contribute to understanding neurodegenerative disorders?
MT-ND3 research offers promising insights into neurodegenerative disease mechanisms:
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Mitochondrial dysfunction and oxidative damage are implicated in neurodegenerative diseases such as Alzheimer's disease (AD) .
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mtDNA variants can influence cognitive abilities and modulate oxidative phosphorylation and ROS production, which are key factors in neurodegeneration .
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Conplastic mouse models with variant mtDNA backgrounds represent valuable tools for investigating how specific MT-ND3 polymorphisms might affect Aβ proteostasis and other pathological aspects of neurodegenerative diseases .
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The ability to perform targeted base editing of MT-ND3 in vivo opens new possibilities for creating animal models with specific mitochondrial mutations relevant to human disease .
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The conserved ND3 loop's role in Complex I active/deactive transition suggests that MT-ND3 mutations could significantly impact neuronal energy metabolism, a critical factor in neurodegenerative progression .
What technical challenges remain in MT-ND3 research and what approaches might address them?
Despite significant advances, several challenges persist in MT-ND3 research:
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Engineering specific mtDNA mutations: While base editing shows promise, creating mouse models with specific pathological mtDNA mutations remains technically challenging . Continued refinement of base editing techniques and delivery systems will be essential.
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Off-target effects: Higher off-target editing rates, particularly in neonatal animals, require improved editor specificity and better control methods .
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Tissue-specific editing: Current methods show variable efficiency across tissues; development of tissue-targeted delivery systems would enhance research capabilities.
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Functional analysis: Comprehensive assessment of how specific MT-ND3 mutations affect mitochondrial function requires standardized assays for parameters such as:
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Complex I assembly and activity
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ROS production
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ATP synthesis
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Mitochondrial membrane potential
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Cellular stress responses
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Translation to human disease: Bridging findings from model organisms to human pathology requires careful consideration of species-specific differences in mitochondrial biology and disease mechanisms.
