Recombinant Damaliscus pygargus phillipsi Cytochrome c oxidase subunit 2 (MT-CO2)

What is the biological function of MT-CO2 in Damaliscus pygargus phillipsi?

MT-CO2 is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX), a crucial step in ATP production during cellular respiration. This highly conserved protein contains a dual core CuA active site and serves as one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in various physiological processes . In Damaliscus pygargus phillipsi (blesbok), as in other mammals, this protein maintains essential respiratory functions while potentially exhibiting subspecies-specific adaptations.

How is the MT-CO2 gene organized in the mitochondrial genome of blesbok?

While the specific organization in blesbok has not been extensively documented in the provided sources, MT-CO2 genes typically reside in the mitochondrial DNA between transfer RNA genes. In insect species like mosquitoes, for example, the COII gene is positioned between transfer RNA for Leu and Lys . The gene organization and direction of transcription are highly conserved across mammalian species, suggesting that blesbok MT-CO2 likely follows similar structural patterns with potential subspecies-specific variations.

What are the expected molecular characteristics of blesbok MT-CO2?

Based on comparative analysis with other species, blesbok MT-CO2 would likely possess the following characteristics:

• An open reading frame of approximately 680-700 base pairs

• A translated protein of approximately 220-230 amino acid residues

• Molecular mass of approximately 26 kDa

• Isoelectric point (pI) between 6.0-6.5

These predictions are supported by studies of COII in other species, such as Sitophilus zeamais, which has a COII protein with 227 amino acids, a molecular mass of 26.2 kDa, and a pI value of 6.37 .

How might genetic diversity in blesbok MT-CO2 compare with that of the closely related bontebok?

Research suggests that blesbok (Damaliscus pygargus phillipsi) likely possesses greater genetic diversity in MT-CO2 compared to bontebok (Damaliscus pygargus pygargus). This prediction is supported by studies showing significantly higher neutral genetic diversity in blesbok (Ho = 0.36 ± 0.068) compared to bontebok (Ho = 0.22 ± 0.073) . Bontebok populations experienced a severe bottleneck in the 1830s, which likely reduced their genetic diversity across both neutral and adaptive loci. While specific MT-CO2 diversity has not been directly measured in these subspecies, the pattern observed in other genetic markers suggests similar trends would apply to mitochondrial genes.

What evolutionary patterns might be detected in blesbok MT-CO2 sequences?

Evolutionary analysis of blesbok MT-CO2 would likely reveal patterns of purifying selection interrupted by occasional relaxed selective constraints or positive selection at specific sites. This pattern has been observed in other species, such as the marine copepod Tigriopus californicus, where approximately 4% of COII codons evolved under relaxed selective constraint while the majority were under strong purifying selection . Given the critical function of MT-CO2 in cellular respiration, most nonsynonymous substitutions would likely be deleterious, but some might be tolerated or even advantageous in specific ecological contexts, particularly at sites interacting with nuclear-encoded proteins.

How can recombinant MT-CO2 expression systems be optimized for functional studies?

Optimizing recombinant MT-CO2 expression requires careful consideration of expression vectors, host systems, and purification strategies. For blesbok MT-CO2, a bacterial expression system using pET-32a vector in E. coli Transetta (DE3) would be a reasonable starting point, following protocols similar to those used for Sitophilus zeamais COII . Induction using isopropyl β-d-thiogalactopyranoside (IPTG) and purification via affinity chromatography with Ni²⁺-NTA agarose for His-tagged constructs can yield functional protein. Key optimization parameters include:

• Codon optimization for the expression host

• Temperature and duration of induction (typically 16-25°C for 16-24 hours)

• IPTG concentration (typically 0.1-1.0 mM)

• Addition of copper ions during expression to facilitate CuA center formation

• Gentle lysis and purification conditions to maintain structural integrity

What functional assays can verify activity of recombinant blesbok MT-CO2?

Functional verification of recombinant blesbok MT-CO2 requires assays that measure its electron transfer capabilities. UV-spectrophotometric analysis can track the oxidation of reduced cytochrome c, providing quantitative measurements of electron transfer rates . Additionally, infrared spectrometer analysis can assess structural integrity and functional properties. Western blotting confirms protein expression and size, while circular dichroism spectroscopy evaluates secondary structure. For comprehensive functional characterization, reconstitution experiments with other cytochrome c oxidase subunits may be necessary to fully assess integrated electron transport activity.

What is the optimal cloning strategy for blesbok MT-CO2?

The optimal cloning strategy for blesbok MT-CO2 involves:

• DNA extraction from blesbok tissue samples

• PCR amplification of the complete MT-CO2 gene using primers designed from conserved flanking regions

• Gel purification of the PCR product

• Restriction enzyme digestion or Gibson assembly for insertion into an expression vector

• Transformation into a cloning strain (e.g., DH5α)

• Sequence verification

• Subcloning into expression vector (e.g., pET-32a) with appropriate tags

• Transformation into expression host (e.g., E. coli Transetta DE3)

For mitochondrial genes like MT-CO2, using DNA extracted from tissue rich in mitochondria (such as muscle) improves amplification success. PCR conditions typically include initial denaturation (95°C, 5 min), followed by 30-35 cycles of denaturation (95°C, 30 sec), annealing (55-58°C, 30 sec), and extension (72°C, 1 min), with a final extension (72°C, 10 min) .

How can the effects of nonsynonymous substitutions in blesbok MT-CO2 be assessed?

Assessing the effects of nonsynonymous substitutions in blesbok MT-CO2 requires a comprehensive approach combining computational prediction, in vitro analysis, and comparative studies:

• Computational analysis: Use algorithms like SIFT (Sorting Intolerant From Tolerant) to predict whether amino acid substitutions affect protein function . This approach identifies substitutions likely to be deleterious versus those likely to be tolerated.

• Structural modeling: Generate three-dimensional models of the MT-CO2 protein using programs like AlphaFold to visualize spatial positioning of mutations within the protein structure .

• Site-directed mutagenesis: Introduce specific mutations into recombinant MT-CO2 to directly test their functional impact.

• Enzymatic assays: Compare the electron transfer rates of wild-type and mutant proteins using spectrophotometric assays tracking cytochrome c oxidation.

• Protein stability assessment: Evaluate thermal stability and resistance to degradation to determine if mutations affect protein structural integrity.

What are the key considerations for purifying recombinant blesbok MT-CO2?

Purification of recombinant blesbok MT-CO2 presents several challenges requiring specific optimization:

• Solubility enhancement: MT-CO2 contains hydrophobic regions that may reduce solubility. Consider using fusion tags (e.g., SUMO, MBP) or detergents during purification.

• Affinity purification: His-tagged constructs can be purified using Ni²⁺-NTA affinity chromatography, typically yielding protein concentrations around 50 μg/mL .

• Tag removal: For functional studies, removing fusion tags with specific proteases (e.g., TEV protease for His-tags) may be necessary to avoid interference with enzyme activity.

• Protein verification: Western blotting confirms protein identity and size, with recombinant MT-CO2 fusion proteins typically appearing at approximately 44 kDa on SDS-PAGE .

• Metal incorporation: MT-CO2 requires copper for the CuA center. Consider incorporating copper refolding steps or supplementing growth media with copper ions.

• Storage conditions: Optimize buffer composition, pH, and storage temperature to maintain protein stability and activity during long-term storage.

How can recombinant blesbok MT-CO2 be used to study hybridization between blesbok and bontebok?

Recombinant MT-CO2 from blesbok and bontebok provides a valuable tool for investigating hybridization between these subspecies:

• Expression of variant proteins: Express MT-CO2 variants from both subspecies and potential hybrid sequences to compare biochemical properties.

• Functional differences: Assess whether subspecies-specific MT-CO2 variants show differences in electron transfer efficiency, potentially reflecting adaptation to different ecological conditions.

• Compatibility with nuclear-encoded partners: Test interaction between MT-CO2 variants and nuclear-encoded cytochrome c oxidase subunits to identify potential mitonuclear incompatibilities that might affect hybrid fitness.

• Heterologous expression systems: Create cell lines expressing different MT-CO2 variants to measure respiratory efficiency in a controlled cellular environment.

Studies show that bontebok and blesbok can produce fertile hybrid offspring, complicating conservation efforts for the near-threatened bontebok . Understanding functional differences in MT-CO2 might provide insights into the adaptive significance of subspecies differentiation.

What insights can comparative analysis of MT-CO2 sequences provide about evolutionary adaptation?

Comparative analysis of MT-CO2 sequences across Damaliscus subspecies and related bovids can reveal:

• Selection patterns: Regions under purifying selection (ω << 1) versus relaxed constraint (ω = 1) or positive selection (ω > 1) .

• Coevolution with nuclear partners: Compensatory mutations that maintain functional interaction with nuclear-encoded proteins like cytochrome c.

• Ecological adaptation signatures: Correlation between sequence variations and environmental variables such as altitude, temperature, or other factors affecting respiratory efficiency.

• Divergence timing: Molecular clock analyses to estimate when functional divergence between subspecies occurred.

The marine copepod Tigriopus californicus shows interpopulation divergence of nearly 20% at the COII locus, including 38 nonsynonymous substitutions, with evidence of positive selection at specific sites . Similar analyses in blesbok could reveal how MT-CO2 has evolved in response to specific selective pressures.

How can researchers overcome expression challenges for recombinant blesbok MT-CO2?

Expressing functional mitochondrial proteins presents several challenges with specific solutions:

• Codon bias: Synthesize codon-optimized gene sequences for the expression host to improve translation efficiency.

• Protein toxicity: Use tightly controlled inducible expression systems and lower growth temperatures (16-20°C) to reduce toxicity.

• Inclusion body formation: Test multiple solubility-enhancing fusion partners (SUMO, MBP, Trx) and optimize induction conditions (lower IPTG concentration, reduced temperature).

• Improper folding: Co-express molecular chaperones (GroEL/GroES, DnaK) to facilitate correct protein folding.

• Metal center formation: Supplement growth media with copper sulfate (typically 0.1-0.5 mM) to facilitate CuA center formation.

• Alternative expression systems: If bacterial expression fails, consider eukaryotic expression systems like yeast, insect cells, or mammalian cells that may better accommodate post-translational modifications.

What are the best approaches for studying interactions between recombinant MT-CO2 and other respiratory chain components?

Studying interactions between MT-CO2 and other respiratory components requires specialized techniques:

• Co-immunoprecipitation: Use tagged recombinant MT-CO2 to pull down interacting partners from mitochondrial extracts.

• Surface plasmon resonance (SPR): Quantitatively measure binding kinetics between MT-CO2 and purified cytochrome c or other subunits.

• Microscale thermophoresis (MST): Determine binding affinities in solution using minimal protein amounts.

• Reconstitution experiments: Combine purified subunits to rebuild functional complexes in vitro.

• Yeast two-hybrid or mammalian two-hybrid assays: Screen for protein-protein interactions in a cellular context.

• Molecular docking: Predict interaction interfaces using computational models, similar to how allyl isothiocyanate (AITC) binding sites were identified for Sitophilus zeamais COII through molecular docking .

These approaches provide complementary data about both physical interactions and functional consequences of those interactions.

How might recombinant blesbok MT-CO2 contribute to conservation genomics?

Recombinant MT-CO2 studies can enhance conservation genomics through:

• Functional conservation assessment: Determine if low genetic diversity in bontebok compared to blesbok (Ho = 0.22 ± 0.073 vs. Ho = 0.36 ± 0.068) translates to functional constraints in respiratory efficiency.

• Hybrid fitness evaluation: Test whether hybrid MT-CO2 variants maintain full functionality or show evidence of mitonuclear incompatibility.

• Selection marker development: Develop molecular markers based on MT-CO2 variation to identify pure subspecies versus hybrids in field samples.

• Adaptive potential assessment: Evaluate how existing MT-CO2 variation might affect adaptation to changing environmental conditions, particularly for the near-threatened bontebok.

• Genetic rescue planning: Guide genetic rescue efforts by identifying functionally important variations that should be preserved in conservation breeding programs.

Understanding the functional consequences of genetic diversity patterns is essential for effective conservation of Damaliscus subspecies, particularly given the hybridization threat to bontebok .

What experimental approaches could investigate the role of MT-CO2 in thermal adaptation?

Investigating MT-CO2's role in thermal adaptation requires multi-faceted approaches:

• Thermal stability assays: Compare the stability of recombinant MT-CO2 variants at different temperatures using circular dichroism or differential scanning fluorimetry.

• Temperature-dependent enzyme kinetics: Measure electron transfer rates across a temperature gradient to identify potential adaptive differences between subspecies.

• Respiratory efficiency in cellular models: Create cell lines expressing different MT-CO2 variants and assess respiratory function under various thermal conditions.

• Correlation with habitat data: Analyze whether MT-CO2 sequence variations correlate with temperature profiles of subspecies' native ranges.

• Directed evolution experiments: Subject recombinant MT-CO2 to in vitro evolution under thermal selection to identify potentially adaptive mutations.

Similar approaches have identified thermally adaptive mutations in cytochrome c oxidase subunits of other species adapted to different thermal environments, providing a framework for such investigations in blesbok.

Understanding the molecular basis of thermal adaptation is increasingly important for conservation planning in the context of climate change, particularly for species with restricted ranges like bontebok.