Recombinant Tarsius syrichta Cytochrome c oxidase subunit 2 (MT-CO2)

Basic Research Questions

• What is Cytochrome c oxidase subunit 2 (MT-CO2) and what is its role in Tarsius syrichta?

MT-CO2 (also abbreviated as COXII, COX2, or COII) is one of the core subunits of mitochondrial Cytochrome c oxidase (Complex IV) in the electron transport chain. It contains a dual core CuA active site that plays a significant role in physiological processes . In Tarsius syrichta (Philippine tarsier), MT-CO2 consists of 227 amino acids with a molecular mass of approximately 25.6 kDa, similar to other primate species .

The protein functions as part of the terminal enzyme in the respiratory chain, facilitating electron transfer from cytochrome c to oxygen, thereby contributing to ATP production through oxidative phosphorylation. MT-CO2's structure includes two transmembrane alpha-helices in its N-terminal domain and contains one redox center with a binuclear copper A center (CuA) located in a conserved cysteine loop .

• How is recombinant Tarsius syrichta MT-CO2 produced in laboratory settings?

Production of recombinant Tarsius syrichta MT-CO2 typically follows these methodological steps:

• Gene cloning: The full-length cDNA of the MT-CO2 gene (684 bp encoding 227 amino acids) is isolated and amplified from Tarsius syrichta tissue samples .

• Vector construction: The gene is subcloned into an expression vector such as pET-32a, which typically includes a His-tag for purification purposes .

• Expression: The recombinant plasmid is transformed into a bacterial expression system like E. coli Transetta (DE3) and protein expression is induced using isopropyl β-d-thiogalactopyranoside (IPTG) .

• Purification: The recombinant protein with 6-His tag is purified using affinity chromatography with Ni(2+)-NTA agarose . Western blotting can confirm the molecular weight and identity of the purified protein.

• Functional verification: UV-spectrophotometry and infrared spectrometer analysis can verify that the recombinant protein retains its ability to catalyze the oxidation of the substrate cytochrome c .

The final purified protein is typically stored in a Tris-based buffer with glycerol at -20°C/-80°C, with recommendations to avoid repeated freeze-thaw cycles .

• Why is MT-CO2 valuable as a molecular marker in primate evolutionary studies?

MT-CO2 has proven to be an excellent molecular marker for phylogenetic studies of primates for several reasons:

• Mitochondrial inheritance: Being encoded by mitochondrial DNA, MT-CO2 is maternally inherited without recombination, providing a clear evolutionary lineage .

• Optimal mutation rate: MT-CO2 exhibits an appropriate rate of sequence evolution that allows differentiation between closely related species while maintaining enough conservation for alignment across more distant taxa .

• Sequence characteristics: The gene contains both conserved functional domains and variable regions, making it informative for phylogenetic analysis at different taxonomic levels .

Studies have demonstrated MT-CO2's effectiveness in resolving phylogenetic relationships among tarsier species. For instance, research using MT-CO2 gene sequences revealed that Tarsius species from the same geographic area have closer genetic relationships, with genetic distance values of ≤20 between species from adjacent areas compared to ≥60 between species from distant regions .

The phylogenetic trees constructed using MT-CO2 sequences were consistent with analyses using other genes like cytochrome b, confirming its reliability as a molecular marker . This makes MT-CO2 particularly valuable for investigating the evolutionary history and diversification of primate lineages.

• What are the physical and biochemical properties of Tarsius syrichta MT-CO2?

Tarsius syrichta MT-CO2 has the following physical and biochemical properties:

The protein contains conserved domains including the CuA center, which is located in a conserved cysteine loop at positions equivalent to 196 and 200 in human MT-CO2, with a conserved histidine at position 204 . This CuA center functions as a critical electron acceptor from cytochrome c in the electron transport chain.

• How does the MT-CO2 sequence vary among different tarsier species?

Sequence analysis of MT-CO2 across different tarsier species has revealed important patterns of conservation and variation:

• Intraspecific variation: Within a single species such as T. tarsier, MT-CO2 sequences show minimal variation, typically 0.1 to 1.0% in intron sites and 0 to 1.5% in synonymous exon sites .

• Interspecific variation: Between different tarsier species, the sequence similarity reflects their evolutionary relationships and geographic distribution. Species from the same geographic region show higher sequence similarity than those from distant regions .

• Phylogenetic patterns: Analysis of MT-CO2 sequences has shown that:

  • T. tarsier is positioned as a basal lineage in intron and synonymous site trees

  • T. bancanus (Bornean tarsier) and T. syrichta (Philippine tarsier) show close relationship in these trees

  • In non-synonymous site phylogeny, the sequences group differently, with T. tarsier and T. syrichta clustering together, excluding T. bancanus

This pattern of sequence variation has provided important insights into tarsier evolution, suggesting that the last common ancestor of crown tarsiers may have had polymorphic MT-CO2 alleles, with implications for understanding their evolutionary history, geographical distribution, and speciation events .

Advanced Research Questions

• How can recombinant Tarsius syrichta MT-CO2 be used to investigate cytochrome c oxidase enzyme activity?

Recombinant Tarsius syrichta MT-CO2 can be employed in several sophisticated experimental approaches to study cytochrome c oxidase activity:

• Enzyme kinetics analysis:

  • The purified recombinant protein can be used to assess the catalytic activity of cytochrome c oxidase

  • UV-spectrophotometry can measure the oxidation rate of reduced cytochrome c substrate

  • Enzyme kinetic parameters (Km, Vmax, kcat) can be determined under varying conditions of temperature, pH, and substrate concentration

• Inhibitor and activator studies:

  • The effects of potential inhibitors or activators on enzyme function can be evaluated

  • Research has shown that compounds such as allyl isothiocyanate (AITC) can influence the catalytic activity of recombinant COXII

  • Molecular docking methods have revealed that a sulfur atom of AITC can form a hydrogen bond with specific amino acid residues (such as Leu-31), potentially explaining its modulatory effect

• Structure-function relationship investigation:

  • Site-directed mutagenesis can be performed on the recombinant protein to identify critical residues for enzyme function

  • The effects of mutations on electron transfer efficiency and proton pumping can be assessed

  • These studies can provide insights into the molecular mechanisms of cytochrome c oxidase function

• Comparative biochemical studies:

  • The properties of Tarsius syrichta MT-CO2 can be compared with those of other primate species

  • Differences in activity, stability, or regulation may reflect adaptations to different ecological niches or metabolic demands

  • This approach can contribute to understanding the evolution of mitochondrial function in primates

The ability to produce and analyze recombinant MT-CO2 provides researchers with a powerful tool to investigate the fundamental mechanisms of cellular respiration and energy production in this phylogenetically important primate species.

• What challenges exist in expressing and purifying functional recombinant MT-CO2 from Tarsius syrichta?

Expressing and purifying functional recombinant MT-CO2 from Tarsius syrichta presents several methodological challenges:

• Expression challenges:

  • Membrane protein solubility: As MT-CO2 contains transmembrane domains, it tends to form insoluble aggregates when overexpressed in bacterial systems

  • Codon usage bias: Differences in codon preference between tarsier and expression hosts (typically E. coli) can reduce expression efficiency

  • Post-translational modifications: Bacterial systems may not provide all necessary modifications required for proper folding and function

  • Toxicity to host cells: Overexpression of membrane proteins can disrupt bacterial membrane integrity, limiting yield

• Purification challenges:

  • Maintaining native conformation: Solubilization and purification procedures may disrupt the protein's native structure

  • Metal cofactor incorporation: Ensuring proper incorporation of copper ions into the CuA center is critical for function

  • Detergent selection: Identifying detergents that effectively solubilize the protein while preserving its activity requires extensive optimization

  • Yield limitations: The final yield of pure, functional protein is often low due to losses during multiple purification steps

• Methodological solutions:

  • Use of fusion tags (such as 6-His) to improve solubility and facilitate purification

  • Expression at lower temperatures (15-25°C) to slow protein synthesis and improve folding

  • Co-expression with molecular chaperones to assist proper folding

  • Use of specialized E. coli strains designed for membrane protein expression

  • Two-step purification combining affinity chromatography and size exclusion chromatography

  • Development of refolding protocols for proteins recovered from inclusion bodies

• Functional verification:

  • Western blotting to confirm protein identity and integrity

  • UV-spectrophotometry to assess enzyme activity by monitoring cytochrome c oxidation

  • Circular dichroism to evaluate secondary structure

  • Thermal stability assays to assess protein folding quality

Addressing these challenges requires careful optimization of each step in the expression and purification process, often necessitating multiple iterations to achieve sufficient yields of functional protein.

• How does MT-CO2 contribute to understanding the phylogeny and evolutionary history of tarsiers?

MT-CO2 has provided critical insights into tarsier phylogeny and evolution through several methodological approaches:

• Phylogenetic reconstruction:

  • Multiple sequence alignment and phylogenetic analysis of MT-CO2 sequences from different tarsier species has revealed their evolutionary relationships

  • Studies have employed Maximum likelihood methods to construct robust phylogenetic trees

  • These analyses have shown that MT-CO2 sequences group according to geographical distribution, with species from the same area having closer genetic relationships

• Genetic distance analysis:

  • Calculation of genetic distances between MT-CO2 sequences has provided quantitative measures of evolutionary divergence

  • Tarsier species from adjacent areas show genetic distance values ≤20, while those from distant regions have values ≥60

  • These patterns support theories of allopatric speciation in tarsiers, where geographical isolation has led to genetic divergence

• Sequence evolution patterns:

  • Analysis of synonymous versus non-synonymous substitutions in MT-CO2 genes has revealed selective pressures

  • Intron and synonymous site trees reflect the established phyletic relationships of tarsiers

  • Non-synonymous site trees show different patterns, suggesting functional constraints on amino acid changes

• Ancestral state reconstruction:

  • Comparative analysis of MT-CO2 sequences has helped reconstruct ancestral states and evolutionary trajectories

  • For example, the L opsin gene of T. tarsier clusters with the L opsin gene of T. syrichta in non-synonymous-site phylogeny, suggesting that alleles of the L/M opsin gene were present in the last common ancestor of crown tarsiers

  • This finding has implications for understanding the evolution of vision systems in tarsiers and other primates

These approaches have collectively enhanced our understanding of tarsier diversification, biogeographical distribution, and adaptation to different ecological niches, placing them in the broader context of primate evolution.

• What role does MT-CO2 play in mitochondrial energy metabolism and how can recombinant proteins help study this function?

MT-CO2 plays a critical role in mitochondrial energy metabolism as part of cytochrome c oxidase (Complex IV), the terminal enzyme in the electron transport chain. Recombinant proteins provide valuable tools for understanding this function:

Recombinant MT-CO2 thus serves as a powerful tool for investigating the molecular mechanisms of cellular respiration, energy production, and their regulation in different physiological contexts.

• How does the structure of Tarsius syrichta MT-CO2 compare with that of other primate species, and what evolutionary insights does this provide?

Comparative structural analysis of MT-CO2 across primate species yields important evolutionary insights:

• Phylogenetic positioning:

  • Tarsiers occupy a key phylogenetic position between strepsirrhines and anthropoid primates

  • MT-CO2 sequence analysis supports this intermediate position

  • The pattern of sequence conservation in MT-CO2 reflects the broader evolutionary relationships among primates

• Evidence of selection:

  • Studies have shown accelerated evolution of COX subunits in primate lineages

  • Nine of the thirteen COX subunits have demonstrated an accelerated amino acid replacement rate in anthropoid primates

  • This suggests adaptive evolution of the respiratory chain in certain primate lineages

• Functional implications:

  • Structural variations in MT-CO2 may reflect adaptations to different metabolic demands

  • Analysis of binding interactions between COX and cytochrome c has revealed evolutionary changes in anthropoid primates

  • In anthropoids, there appears to have been a reduction in electrostatic interaction between COX and cytochrome c, potentially affecting electron transfer efficiency

• Evolutionary model:

  • The pattern of MT-CO2 evolution supports a "domestication scenario" where nuclear-encoded subunits evolved to control the ancestral activity of mitochondrial-encoded subunits like MT-CO2

  • This represents an evolutionary adaptation allowing the host cell to regulate mitochondrial function more precisely

These structural comparisons provide a window into the evolutionary history of primates and the adaptive changes in their energy metabolism systems, positioning tarsiers as an important transitional group in primate evolution.

• What recent advances in bioinformatic approaches have enhanced MT-CO2 analysis in phylogenetic and evolutionary studies?

Recent bioinformatic advances have significantly improved MT-CO2 analysis in evolutionary studies:

• Advanced phylogenetic algorithms:

  • Maximum likelihood methods have become more sophisticated, allowing for more accurate phylogenetic tree construction

  • Bayesian inference approaches provide probability distributions for phylogenetic hypotheses

  • Coalescent-based methods better account for incomplete lineage sorting in closely related species

  • These advances have enhanced our ability to resolve the evolutionary relationships among tarsier species using MT-CO2 sequences

• Comparative genomic analysis:

  • Whole genome sequencing of Tarsius syrichta has provided a broader context for understanding MT-CO2 evolution

  • Integration of nuclear and mitochondrial gene data allows for more comprehensive evolutionary analyses

  • Analysis of transposable elements and mitochondrial genome insertions in the nuclear genome has revealed unusual evolutionary patterns in tarsiers

• Molecular evolution analysis tools:

  • Advanced methods for detecting positive selection, such as branch-site models, can identify specific amino acid positions under selection

  • Relaxed molecular clock models allow for variation in evolutionary rates across lineages

  • These tools have revealed patterns of accelerated evolution in COX subunits in certain primate lineages

• Structural bioinformatics:

  • Improved protein structure prediction algorithms allow for more accurate modeling of MT-CO2 structure

  • Molecular dynamics simulations can predict the functional effects of amino acid substitutions

  • Protein-protein interaction modeling helps understand the interaction between MT-CO2 and other proteins

• Ancestral sequence reconstruction:

  • Advanced algorithms can reconstruct the probable sequences of ancestral MT-CO2 proteins

  • This allows researchers to test hypotheses about the functional properties of ancestral proteins

  • Such analyses have suggested that the last common ancestor of crown tarsiers likely had polymorphic MT-CO2 alleles

• Population genetics integration:

  • Integration of phylogenetic and population genetic approaches provides insights into both deep evolutionary history and recent diversification

  • Analysis of genetic distances between MT-CO2 sequences from different tarsier populations has supported theories of allopatric speciation

These bioinformatic advances have collectively enhanced our understanding of MT-CO2 evolution and its role in tarsier phylogeny, providing methodological frameworks for future research in this field.