Recombinant Drosophila tolteca Cytochrome c oxidase subunit 2 (mt:CoII)
Description
Definition and Biological Context
mt:CoII is one of three mitochondrial DNA-encoded subunits (MT-CO1, MT-CO2, MT-CO3) of COX, which catalyzes the electron transfer from cytochrome c to oxygen, producing water . In Drosophila tolteca, the recombinant mt:CoII retains structural and functional homology to its endogenous counterpart, enabling its use in biochemical and structural studies.
Functional Role
mt:CoII facilitates electron relay through its CuA center, acting as the primary acceptor for electrons from cytochrome c. In Drosophila, genetic knockdown of mt:CoII (via RNAi) results in:
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COX Activity Reduction: Enzymatic activity drops to ~55% of controls .
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Structural Defects: Co-purification assays show reduced binding to COX4, a core subunit of COX .
Production and Purification
Recombinant mt:CoII is synthesized via heterologous expression systems:
| Parameter | E. coli System | Baculovirus System |
|---|---|---|
| Protein Form | Full-length (1–229 aa) | Partial (truncated) |
| Tag | N-terminal 10xHis | Variable (determined during production) |
| Purity | >90% (SDS-PAGE) | >85% (SDS-PAGE) |
| Yield | High | Moderate |
| Buffer | Tris/PBS-based, 6% trehalose | Custom (e.g., Tris, glycerol) |
Purification Steps:
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Ni-NTA Affinity Chromatography (His-tag binding).
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Size-Exclusion Chromatography (SEC) for oligomer resolution.
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Lyophilization or storage in glycerol (50%) at -20°C/-80°C .
Enzymatic Assays
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COX Activity Measurements: mt:CoII is reconstituted with other COX subunits to study electron transfer kinetics .
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Copper Center Analysis: Spectroscopic studies (e.g., EPR) probe CuA center geometry .
Diagnostic Tools
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ELISA Kits: Recombinant mt:CoII serves as an antigen for detecting anti-COX antibodies in mitochondrial disorders .
Key Research Findings
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Recombinant Drosophila tolteca Cytochrome c oxidase subunit 2 (mt:CoII) is a component of cytochrome c oxidase (Complex IV, CIV), the terminal enzyme in the mitochondrial electron transport chain responsible for oxidative phosphorylation. This chain comprises three multi-subunit complexes: succinate dehydrogenase (Complex II, CII), ubiquinol-cytochrome c oxidoreductase (Complex III, CIII), and cytochrome c oxidase (CIV). These complexes work cooperatively to transfer electrons from NADH and succinate to molecular oxygen, generating an electrochemical gradient across the inner mitochondrial membrane that drives ATP synthesis and transmembrane transport. Cytochrome c oxidase catalyzes the reduction of oxygen to water. Electrons from reduced cytochrome c in the intermembrane space are transferred through the CuA center of subunit 2 and heme A of subunit 1 to the active site (a binuclear center, BNC) in subunit 1, composed of heme a3 and CuB. The BNC utilizes four electrons from cytochrome c and four protons from the mitochondrial matrix to reduce molecular oxygen to two water molecules.
Q&A
What is Cytochrome c oxidase subunit 2 (mt:CoII) and what is its role in Drosophila tolteca?
Cytochrome c oxidase subunit 2 (mt:CoII) is a mitochondrial DNA-encoded protein that forms a critical component of the cytochrome c oxidase (CcO) complex, which functions as the terminal enzyme in the electron transport chain of cellular respiration. In Drosophila tolteca, mt:CoII (UniProt ID: P67796) is a 229-amino acid protein that contributes to the catalytic core of the holoenzyme . As a component of complex IV of the respiratory chain, mt:CoII plays an essential role in aerobic energy production by catalyzing the reduction of molecular oxygen to water while simultaneously contributing to the proton gradient used for ATP synthesis .
How does the structure of recombinant D. tolteca mt:CoII compare to homologous proteins in other Drosophila species?
Comparative analysis of mt:CoII across Drosophila species reveals considerable conservation of structure and function. While D. tolteca mt:CoII shares significant sequence homology with counterparts in other Drosophila species, such as D. melanogaster and D. yakuba, there are species-specific variations that may reflect evolutionary adaptations .
In D. yakuba, for example, the mitochondrial genome has been extensively sequenced, revealing that COII is transcribed from the same strand as several other mitochondrial genes, including tRNAleuUUR, tRNAlys, tRNAasp, URFA6L, ATPase6, COIII, and tRNAgly, in a specific arrangement . Unique codon usage patterns have been observed across Drosophila species, with the triplet AGA specifying different amino acids in various positions but never arginine . These variations may have functional implications for the protein's activity and interactions within the respiratory complex.
What expression systems are most effective for producing recombinant D. tolteca mt:CoII?
Based on available research, recombinant D. tolteca mt:CoII has been successfully expressed in both prokaryotic (E. coli) and eukaryotic (yeast) expression systems . The choice of expression system depends on specific research requirements:
E. coli expression system:
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Typically used for producing His-tagged recombinant mt:CoII
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Advantages include high yield, rapid growth, and cost-effectiveness
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May require optimization of codon usage and growth conditions to maximize protein folding and solubility
Yeast expression system:
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Often preferred for mitochondrial proteins due to the presence of eukaryotic post-translational modification machinery
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Generally produces protein with more native-like folding and modifications
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Typically results in lower yield compared to bacterial systems but may provide higher quality protein
What are the optimal storage and handling conditions for recombinant D. tolteca mt:CoII?
To maintain the structural integrity and biological activity of recombinant D. tolteca mt:CoII, the following storage and handling conditions are recommended:
| Storage Condition | Recommendation | Purpose |
|---|---|---|
| Short-term storage | 4°C for up to one week | Maintains protein integrity for immediate use |
| Long-term storage | -20°C or -80°C | Prevents degradation over extended periods |
| Storage buffer | Tris-based buffer with 50% glycerol, pH 8.0 | Stabilizes protein structure |
| Handling | Avoid repeated freeze-thaw cycles | Prevents denaturation and activity loss |
| Aliquoting | Small volumes for single use | Minimizes exposure to freeze-thaw cycles |
| Reconstitution | Deionized sterile water to 0.1-1.0 mg/mL | Ensures proper solubility for experimental use |
For optimal results, the lyophilized protein should be briefly centrifuged before opening to ensure all material is at the bottom of the vial . After reconstitution, adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended before aliquoting and freezing for long-term storage .
How can researchers verify the activity and integrity of recombinant D. tolteca mt:CoII?
Verification of recombinant D. tolteca mt:CoII activity and integrity can be performed using several complementary approaches:
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SDS-PAGE analysis: Confirms protein purity (>90% is typically expected) and molecular weight
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Western blot analysis: Verifies protein identity using antibodies specific to mt:CoII or the His-tag
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Spectrophotometric assays: Measures cytochrome c oxidation rates to assess enzymatic activity
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Oxygen consumption measurements: Evaluates the functional capacity of mt:CoII in reconstituted systems
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Circular dichroism (CD) spectroscopy: Analyzes secondary structure elements to confirm proper folding
For standardized activity measurements, researchers should establish appropriate positive controls, such as commercially available cytochrome c oxidase, and negative controls, such as heat-inactivated enzyme preparations .
What experimental approaches can be used to study interactions between mt:CoII and other components of the respiratory chain?
Several methodologies are valuable for investigating the interactions between mt:CoII and other respiratory chain components:
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Co-immunoprecipitation: Identifies direct protein-protein interactions between mt:CoII and other subunits
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Blue Native PAGE: Preserves native protein complexes and allows visualization of intact cytochrome c oxidase complexes
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Crosslinking studies: Captures transient interactions within the respiratory chain complexes
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Surface plasmon resonance (SPR): Quantifies binding kinetics and affinity between mt:CoII and potential interacting partners
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Cryo-electron microscopy: Visualizes structural relationships within assembled respiratory complexes
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Proteoliposome reconstitution: Evaluates functional interactions in a membrane environment that mimics physiological conditions
These approaches provide complementary information about both structural and functional relationships between mt:CoII and other components of the respiratory chain .
How can recombinant D. tolteca mt:CoII be used to study age-related mitochondrial dysfunction?
Recombinant D. tolteca mt:CoII serves as a valuable tool for investigating age-related mitochondrial dysfunction, building on established research in related Drosophila species. Studies in D. melanogaster have demonstrated that CcO activity declines progressively with age by approximately 33%, with specific losses in various subunits ranging from 11% to 40% . These findings suggest that CcO, and specifically mt:CoII, represents a key intra-mitochondrial site of age-related deterioration.
Research approaches using recombinant D. tolteca mt:CoII for aging studies may include:
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Comparative activity assays: Measuring enzymatic activity of native mt:CoII extracted from Drosophila at different ages against recombinant protein standards
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Reconstitution experiments: Adding recombinant mt:CoII to aged mitochondrial preparations to assess restoration of function
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Post-translational modification analysis: Examining how age-related modifications affect protein function using recombinant proteins as controls
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Structure-function analysis: Investigating how specific domains contribute to stability and function during aging using site-directed mutagenesis of recombinant proteins
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Protein-protein interaction studies: Evaluating how aging affects interactions between mt:CoII and other respiratory chain components
These approaches can provide insights into the molecular mechanisms underlying age-related mitochondrial dysfunction and potentially identify targets for intervention .
What is known about the evolutionary conservation of mt:CoII across Drosophila species and how can this inform research?
The evolutionary conservation of mt:CoII across Drosophila species provides valuable insights for comparative studies. Analyses of mitochondrial genomes have revealed both conserved features and species-specific variations:
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Conserved functional domains: Core catalytic regions are highly conserved, reflecting their essential roles in electron transport
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Variable regions: Some segments show greater sequence divergence, potentially indicating areas under different selective pressures
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Codon usage patterns: Unique patterns have been observed, such as the use of the AGA codon to specify different amino acids across species
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Gene arrangement: The position of COII relative to other mitochondrial genes is conserved in many Drosophila species, with COII typically following tRNAleuUUR and preceding tRNAlys
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Start codon variations: Unlike some mitochondrial genes that show variation in start codons (such as CCG, ATC, and GTG in other genes), COII typically uses a standard start codon but has several nucleotide residues before the ORF begins
Researchers can leverage this evolutionary information to:
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Design experiments targeting conserved versus variable regions
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Develop cross-species compatible reagents and assays
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Interpret functional studies in an evolutionary context
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Identify natural variants for structure-function analyses
How do mutations in mt:CoII affect cytochrome c oxidase activity and what are the implications for mitochondrial function?
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Catalytic efficiency: Mutations in conserved regions can alter the protein's ability to transfer electrons efficiently, reducing the rate of oxygen reduction
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Assembly defects: Some mutations prevent proper integration of mt:CoII into the cytochrome c oxidase complex, leading to incomplete or unstable enzyme assemblies
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Proton pumping: Certain mutations affect proton translocation pathways, uncoupling electron transport from proton pumping and reducing ATP synthesis efficiency
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Reactive oxygen species (ROS) generation: Dysfunction in mt:CoII can increase electron leakage and ROS production, potentially exacerbating cellular damage
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Lifespan effects: Studies in D. melanogaster have shown that decreased CcO activity correlates with shortened lifespan, suggesting that mt:CoII integrity is critical for normal aging
Methodologically, researchers can use recombinant D. tolteca mt:CoII with site-directed mutations to:
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Assess the functional consequences of specific amino acid changes
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Study the effects of naturally occurring polymorphisms
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Investigate how mutations affect interactions with other subunits
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Examine the impact of mutations on protein stability and turnover
What are the common technical challenges in working with recombinant D. tolteca mt:CoII and how can they be addressed?
Researchers working with recombinant D. tolteca mt:CoII frequently encounter several technical challenges:
| Challenge | Solution Approach | Rationale |
|---|---|---|
| Poor solubility | Use detergents or lipid nanodiscs for reconstitution | mt:CoII is a membrane protein requiring hydrophobic environment |
| Improper folding | Express in eukaryotic systems like yeast | Provides appropriate chaperones and post-translational machinery |
| Low activity | Optimize buffer conditions (pH, salt, metal ions) | Ensures optimal environment for functional conformation |
| Aggregation | Add stabilizing agents (glycerol, trehalose) | Prevents protein-protein interactions that lead to aggregation |
| Storage instability | Aliquot and store with 50% glycerol at -80°C | Minimizes freeze-thaw damage and oxidation |
| Batch variability | Implement rigorous quality control testing | Ensures consistent protein characteristics across preparations |
Additionally, the hydrophobic nature of mt:CoII makes it challenging to maintain in solution without appropriate detergents or lipid environments. Researchers should consider using specialized techniques for membrane protein purification and handling to maximize protein quality and experimental reproducibility .
What are promising future research directions involving D. tolteca mt:CoII?
Several promising research directions involving D. tolteca mt:CoII could advance our understanding of mitochondrial function and dysfunction:
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Comparative aging studies: Investigating whether the age-related decline in CcO activity observed in D. melanogaster (33% reduction) is conserved in D. tolteca and other Drosophila species could provide evolutionary insights into mitochondrial aging mechanisms
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Structural biology approaches: Determining high-resolution structures of D. tolteca mt:CoII alone and in complex with other subunits would enhance our understanding of species-specific variations in cytochrome c oxidase architecture
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Genetic manipulation: Developing systems for expressing mutated versions of mt:CoII in Drosophila could allow in vivo assessment of structure-function relationships
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Mitochondrial disease models: Creating D. tolteca models with mutations that mimic human mitochondrial diseases affecting COII might provide valuable experimental systems for therapeutic development
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Environmental adaptation studies: Examining how mt:CoII sequence and function vary across Drosophila species adapted to different environments could reveal mechanisms of mitochondrial adaptation to environmental stressors
These research directions would benefit from continued refinement of recombinant protein production techniques and the development of species-specific analytical tools.
