Recombinant Tamias dorsalis Cytochrome c oxidase subunit 2 (MT-CO2)

What is the biological function of MT-CO2 in Tamias dorsalis?

MT-CO2 (mitochondrially encoded cytochrome c oxidase subunit 2) in Tamias dorsalis is a critical component of the respiratory chain complex IV involved in cellular respiration. This protein contributes to cytochrome-c oxidase activity and participates in mitochondrial electron transport from cytochrome c to oxygen . Specifically, MT-CO2 contains the dinuclear copper A center (CuA) that facilitates the initial transfer of electrons from cytochrome c in the intermembrane space to the active site in subunit 1 . This process is crucial for the production of ATP through oxidative phosphorylation. The protein is encoded by the mitochondrial genome and is highly conserved across species, though with notable variations that make it useful for evolutionary studies .

How is recombinant Tamias dorsalis MT-CO2 expressed and purified for research applications?

Recombinant Tamias dorsalis MT-CO2 protein is typically expressed in bacterial systems such as E. coli using recombinant DNA technology . The expression process involves:

• Generating a construct containing the MT-CO2 coding sequence (positions 1-227) with an N-terminal His-tag for purification purposes

• Transforming the construct into E. coli expression hosts

• Inducing protein expression under controlled conditions

• Lysing the cells and purifying the protein using affinity chromatography (typically Ni-NTA for His-tagged proteins)

• Further purification steps may include size exclusion or ion exchange chromatography to achieve >90% purity as determined by SDS-PAGE

The resulting recombinant protein is typically provided as a lyophilized powder with purity greater than 90% . While E. coli is the most common expression system for high yields and shorter turnaround times, alternative expression systems include yeast, insect cells with baculovirus, or mammalian cells when post-translational modifications are required for proper folding or activity .

What are the recommended storage and handling conditions for recombinant MT-CO2?

For optimal stability and activity of recombinant Tamias dorsalis MT-CO2, the following storage and handling recommendations should be followed:

• Store lyophilized protein at -20°C/-80°C upon receipt

• For extended storage, store at -20°C or -80°C

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) to working aliquots for long-term storage

• Working aliquots can be stored at 4°C for up to one week

• The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

Repeated freezing and thawing should be avoided as it may lead to protein denaturation and loss of activity. Centrifuge the vial briefly before opening to bring contents to the bottom .

How is MT-CO2 used in evolutionary studies of Tamias species?

MT-CO2 has proven valuable in evolutionary and phylogenetic studies of Tamias species due to several characteristics:

• Mitochondrial DNA introgression tracking: The MT-CO2 gene shows extensive mtDNA introgression among para- and sympatric species in the T. quadrivittatus group, making it useful for tracking evolutionary histories and hybridization events .

• Phylogenetic analyses: Combined with other mitochondrial genes like cytochrome b, MT-CO2 sequences contribute to resolving phylogenetic relationships within the Tamias genus. Research has documented:

  • Approximately 16% (298 out of 1871) of sequenced chipmunks exhibit introgressed mtDNA

  • In T. dorsalis specifically, 65% (35 out of 54) show evidence of mtDNA introgression from other species

• Divergence with gene flow studies: Sequence analysis of MT-CO2 helps establish evolutionary timescales and patterns of gene flow between species. For instance, studies have shown that T. dorsalis has experienced introgression from multiple source species, including T. cinereicollis, T. umbrinus, and T. quadrivittatus, with a maximum mtDNA divergence of 4.7% .

The data from these studies supports the concept of divergence with gene flow (DGF) in the generation of biological diversity, where lineage divergence occurs on a shorter timescale than reproductive isolation .

What experimental approaches are used to study MT-CO2's role in the electron transport chain?

Several methodological approaches are employed to investigate MT-CO2's function in the electron transport chain:

• Enzyme activity assays: Cytochrome c oxidase activity can be measured using spectrophotometric methods that monitor the oxidation of reduced cytochrome c. These assays provide quantitative data on the electron transfer efficacy of MT-CO2 in different experimental conditions .

• Oxygen consumption measurements: Techniques such as high-resolution respirometry can assess the role of MT-CO2 in oxygen reduction by measuring oxygen consumption rates in isolated mitochondria or cells expressing recombinant MT-CO2 .

• Site-directed mutagenesis: Strategic amino acid substitutions within the recombinant MT-CO2 protein can identify critical residues for electron transfer, metal binding, or protein-protein interactions, particularly at the dinuclear copper A center .

• Protein-protein interaction studies: Co-immunoprecipitation, crosslinking, or surface plasmon resonance can characterize the interactions between MT-CO2 and other components of the respiratory chain, especially cytochrome c .

• Structural biology approaches: Techniques such as X-ray crystallography or cryo-electron microscopy provide insights into the three-dimensional structure of MT-CO2 within the cytochrome c oxidase complex, revealing the spatial arrangement of functional domains.

How can recombinant MT-CO2 be used to study mitochondrial disease mechanisms?

Recombinant Tamias dorsalis MT-CO2 serves as a valuable model for investigating mitochondrial disease mechanisms through several research applications:

• Comparative functional studies: The recombinant protein can be used to compare functional differences between normal and disease-associated MT-CO2 variants. For example, MT-CO2 is associated with MELAS syndrome (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes) .

• In vitro reconstitution systems: Purified recombinant MT-CO2 can be incorporated into liposomes or nanodiscs with other cytochrome c oxidase subunits to study the functional consequences of specific mutations on enzyme activity and complex assembly.

• Structure-function relationship investigations: The effects of specific amino acid substitutions found in patient samples can be recreated in the recombinant protein to understand how structural changes affect function.

• Therapeutic screening platforms: The recombinant protein can serve as a target for high-throughput screening of compounds that might restore function in cases where MT-CO2 activity is compromised.

• Biomarker development: Antibodies generated against recombinant MT-CO2 can be used to develop detection methods for altered MT-CO2 expression or localization in patient samples, as MT-CO2 has been identified as a biomarker for conditions such as Huntington's disease and stomach cancer .

What methodological challenges exist in studying mitochondrially encoded proteins like MT-CO2?

Researchers face several significant challenges when working with mitochondrially encoded proteins such as MT-CO2:

• Genetic manipulation limitations: Unlike nuclear genes, direct genetic manipulation of mitochondrial DNA is extremely difficult. As noted in research: "the technique for transfection into mitochondrial DNA has not yet been established, it is impossible to overexpress or downregulate a mitochondrial gene" . This significantly limits gene editing approaches.

• Expression and folding challenges: Correct folding of recombinant mitochondrial proteins often requires specific chaperones and post-translational modifications that may be absent in bacterial expression systems. This necessitates careful selection of expression systems based on experimental needs .

• Functional reconstitution complexities: MT-CO2 functions as part of a multi-subunit complex (cytochrome c oxidase), making studies of isolated MT-CO2 potentially less physiologically relevant. Reconstituting the entire complex is technically challenging.

• Dual genetic control: The assembly of functional cytochrome c oxidase requires coordination between mitochondrially encoded subunits (like MT-CO2) and nuclear-encoded subunits. This dual genetic control complicates the interpretation of experimental results.

• Species-specific variations: While MT-CO2 is relatively conserved across species, there are important structural and functional differences that must be considered when using model systems or recombinant proteins from different species as research tools.

How does MT-CO2 contribute to apoptotic pathways and how can this be studied experimentally?

MT-CO2 plays a significant role in apoptotic pathways through its function in the cytochrome c oxidase complex. The relationship between MT-CO2 and apoptosis can be studied through several experimental approaches:

• Redox state analysis: Cytochrome c oxidase (including MT-CO2) affects the redox state of cytochrome c, which is critical for apoptosis. Research has shown that "only oxidized cytochrome c can activate the apoptosome, whereas reduced cytochrome c cannot" . Experimental methods to measure the redox state of cytochrome c in the presence of functional or altered MT-CO2 can provide insights into this mechanism.

• Caspase activation assays: Studies have demonstrated that downregulation of MT-CO1 (another subunit that works with MT-CO2) leads to reduced caspase-3 activity after irradiation, suggesting that cytochrome c oxidase activity is necessary for full activation of the caspase cascade . Similar approaches can be applied to studying MT-CO2's role.

• Apoptosis resistance models: Research has shown that altered expression of cytochrome c oxidase subunits can confer resistance to apoptotic stimuli. For example, one study found that "downregulation of MT-CO1 induced radioresistance by blocking activation of the caspase cascade in esophageal cancer cells" . Similar mechanisms involving MT-CO2 can be investigated.

• Mitochondrial membrane potential measurements: As MT-CO2 contributes to the proton gradient across the inner mitochondrial membrane, changes in its function can affect membrane potential, which is a key factor in cytochrome c release during apoptosis.

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation or proximity ligation assays can identify interactions between MT-CO2 and components of the apoptotic machinery, providing insights into potential regulatory mechanisms.

How does MT-CO2 sequence variation correlate with habitat adaptation in Tamias dorsalis populations?

Tamias dorsalis inhabits diverse environments across the southwestern United States and northern Mexico, ranging from elevations of 1500 to 3700 meters in various habitats including juniper patches, oak and maple forests, pine woodlands, lava fields, and even desert environments at lower elevations . Research examining MT-CO2 sequence variations across these populations reveals patterns that may reflect adaptive responses to these diverse habitats.

Sequence analysis can be conducted by:

• Collecting tissue samples from T. dorsalis populations across different habitat types

• Amplifying and sequencing the MT-CO2 gene using PCR and Sanger sequencing

• Aligning sequences to identify single nucleotide polymorphisms (SNPs) and amino acid substitutions

• Correlating sequence variations with habitat parameters (elevation, temperature, precipitation, vegetation)

• Conducting selection analyses to determine if specific codons are under positive, neutral, or purifying selection

Studies of other chipmunk species have shown that while most codons in MT-CO2 are under strong purifying selection (ω << 1), approximately 4% of sites appear to evolve under relaxed selective constraint (ω = 1) . Similar analyses in T. dorsalis populations could identify habitat-specific selective pressures.

How can contradictory data regarding MT-CO2 function in Tamias species be reconciled?

Researchers may encounter seemingly contradictory data regarding MT-CO2 function across different studies. A systematic approach to reconciling such contradictions includes:

• Methodological comparisons: Carefully analyze methodological differences between studies, including:

  • Expression systems used (E. coli vs. yeast vs. mammalian cells)

  • Purification methods

  • Assay conditions (temperature, pH, buffer compositions)

  • Detection methods

• Sequence verification: Confirm that the MT-CO2 sequence used in different studies is identical. Even minor sequence variations can lead to functional differences.

• Post-translational modification analysis: Investigate whether post-translational modifications differ between recombinant protein preparations. Mass spectrometry can be used to identify and characterize modifications.

• Context-dependent function analysis: MT-CO2 function may depend on its interaction with other proteins or cellular components. The presence or absence of these factors could explain functional differences between studies.

• Meta-analysis approach: Compile and statistically analyze data from multiple studies to identify patterns and potential sources of variation.

What approaches can be used to compare MT-CO2 function between Tamias species with different patterns of mitochondrial introgression?

To study functional differences in MT-CO2 between Tamias species with varying patterns of mitochondrial introgression, researchers can employ these methodological approaches:

• Comparative enzymatic assays: Express and purify recombinant MT-CO2 from multiple Tamias species (including T. dorsalis, T. ruficaudus, T. amoenus, and others with documented introgression) and compare their enzymatic activities using standardized cytochrome c oxidase activity assays.

• Hybrid protein studies: Create chimeric proteins containing domains from different species' MT-CO2 to identify which regions are responsible for functional differences.

• Co-evolution analysis: Examine whether nuclear-encoded interaction partners of MT-CO2 show evidence of co-evolution in species with introgressed MT-CO2. This can help identify potential compensatory changes that maintain function despite sequence divergence.

• Thermal stability comparisons: Compare the thermal stability of MT-CO2 from different species, as this can reflect functional adaptation to different environmental conditions.

• Interspecies compatibility testing: Test whether MT-CO2 from one species can functionally replace the protein in another species, either in vitro using reconstituted systems or in cell culture models.

Research has shown that in T. dorsalis, 65% of individuals exhibit introgressed mtDNA from other species, highlighting the importance of understanding the functional implications of these genetic exchanges . The table below summarizes introgression patterns in Tamias species based on extensive mtDNA sequencing:

This data indicates extensive mitochondrial introgression across Tamias species , providing an excellent system for studying the functional consequences of MT-CO2 variation.

How can carbon dioxide (CO2) research methodologies inform studies of cytochrome c oxidase subunit 2 (CO2)?

While there is potential for confusion due to the abbreviation "CO2" referring to both carbon dioxide and cytochrome c oxidase subunit 2, methodological approaches from carbon dioxide research can inform studies of the protein:

• Sequestration principles: Laboratory techniques for CO2 gas sequestration, such as those described for capturing CO2 through deposition at temperatures ≤135 K , conceptually parallel approaches for isolating cytochrome c oxidase complexes from cellular environments.

• Measurement precision: Advanced CO2 gas analyzers like the LI-7000 CO2/H2O gas analyzer demonstrate the importance of precise measurement technologies for studying both molecular entities. Similar precision is required when quantifying cytochrome c oxidase activity in enzymatic assays.

• Environmental influence assessment: Studies of environmental factors affecting carbon dioxide levels can inform approaches to understanding how environmental stressors affect MT-CO2 expression and function in different Tamias populations.

• Quantification frameworks: The conceptualization of carbon dioxide quantities (e.g., "a ton of CO2 would fill a cube 27 feet tall, wide, and long" ) provides a framework for effectively communicating abstract molecular concepts in MT-CO2 research.

• Experimental design principles: The rigorous control of variables in CO2 sequestration experiments, including temperature monitoring at multiple levels , exemplifies best practices for designing controlled experiments when studying MT-CO2 function.

What are the implications of MT-CO2 research for understanding broader mitochondrial diseases and evolutionary processes?

Research on Tamias dorsalis MT-CO2 has significant implications for understanding both mitochondrial diseases and evolutionary processes:

• Disease mechanism insights: MT-CO2 is associated with several mitochondrial disorders, including MELAS syndrome . Understanding the structure-function relationships in T. dorsalis MT-CO2 can provide comparative insights into human disease-causing mutations, particularly in relation to electron transport and energy production.

• Evolutionary adaptation models: The extensive mitochondrial introgression documented in Tamias species provides an excellent model for studying:

  • Selection pressures on mitochondrial genes

  • Co-evolution of nuclear and mitochondrial genomes

  • Adaptive responses to environmental changes

• Mitonuclear compatibility: Studies have shown that hybridization between chipmunk species with divergent MT-CO2 sequences can lead to fitness consequences, highlighting the importance of mitonuclear compatibility . This has implications for understanding reproductive isolation mechanisms.

• Biomarker development: As MT-CO2 serves as a biomarker for conditions such as Huntington's disease and stomach cancer , comparative studies across species can identify conserved features that might be targeted for diagnostic or therapeutic purposes.

• Climate adaptation insights: Given that cytochrome c oxidase function is affected by environmental factors such as temperature and oxygen availability, studying MT-CO2 across T. dorsalis populations from different elevations (1500-3700m) and habitats can provide insights into adaptation mechanisms relevant to climate change responses .