Recombinant Bubalus depressicornis Cytochrome c oxidase subunit 2 (MT-CO2)

What is Bubalus depressicornis and why is its MT-CO2 of research interest?

Bubalus depressicornis (Lowland Anoa) is an endangered endemic bovid species native to Sulawesi Island in the Wallacea biogeographical region. The population has declined significantly to fewer than 2,500 adults, with a population loss rate of up to 20% in the last 14-18 years due to poaching and deforestation . The International Union for Conservation of Nature (IUCN) categorizes this species as Endangered (EN), indicating a high level of threat and potential extinction risk . MT-CO2, the mitochondrially-encoded cytochrome c oxidase subunit 2, is of particular research interest because it represents a highly conserved protein essential for cellular respiration and can provide insights into evolutionary relationships, metabolic adaptations, and potential conservation strategies for this threatened species. The protein serves as a valuable molecular marker for population genetics studies and understanding the phylogenetic position of the Lowland Anoa within Bovidae.

How does MT-CO2 function within the cellular respiration process?

MT-CO2 functions as a critical component of Complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain. This complex is the terminal enzyme in the respiratory chain that catalyzes the reduction of oxygen to water . In the process, electrons from reduced cytochrome c in the intermembrane space (IMS) are transferred via the dinuclear copper A center (CU(A)) located on MT-CO2 and then through heme A of subunit 1 to reach the active site . The binuclear center (BNC) formed by heme A3 and copper B (CU(B)) reduces molecular oxygen to two water molecules using four electrons from cytochrome c and four protons from the mitochondrial matrix . This electron transfer process contributes to the electrochemical gradient across the inner membrane that drives ATP synthesis, making MT-CO2 essential for cellular energy production through oxidative phosphorylation.

What are the distinguishing structural features of Bubalus depressicornis MT-CO2 compared to other bovids?

While the search results don't provide specific structural information about Bubalus depressicornis MT-CO2, comparative analyses with related species such as Bubalus bubalis (water buffalo) suggest the protein likely maintains the highly conserved functional domains characteristic of mitochondrial cytochrome c oxidase subunits. The protein contains copper-binding domains essential for electron transfer and interacts directly with cytochrome c. As an endangered species with specific ecological adaptations to the Sulawesi environment, B. depressicornis may exhibit subtle but functionally significant amino acid substitutions in non-catalytic regions that reflect its evolutionary history and potentially its metabolic adaptations to its habitat. These variations could provide valuable insights into the molecular basis of physiological adaptations in this species compared to other bovids, including its close relative B. quarlesi (Mountain Anoa).

How is recombinant B. depressicornis MT-CO2 typically expressed in laboratory settings?

Recombinant B. depressicornis MT-CO2 can be expressed using several heterologous expression systems, with selection depending on research objectives. For functional studies requiring post-translational modifications, mammalian expression systems (HEK293 or CHO cells) are preferred, while high-yield production often employs bacterial systems like E. coli. Based on approaches used for related proteins, wheat germ cell-free expression systems represent an excellent compromise, providing eukaryotic translation machinery while avoiding membrane protein aggregation issues . The protocol typically involves isolating the MT-CO2 gene from B. depressicornis tissue samples, optimizing the coding sequence for the chosen expression system, and cloning it into an appropriate vector containing necessary regulatory elements and affinity tags for purification. Expression conditions must be optimized for temperature, induction timing, and duration to maximize properly folded protein yield while minimizing inclusion body formation that commonly occurs with membrane proteins.

What purification strategies are most effective for recombinant B. depressicornis MT-CO2?

Effective purification of recombinant B. depressicornis MT-CO2 requires a multi-step approach designed to address the hydrophobic nature of this membrane protein. Initial extraction typically employs mild detergents like n-dodecyl β-D-maltoside (DDM) or digitonin that maintain protein structure while solubilizing membrane components. For recombinant proteins expressed with affinity tags (His6, FLAG, or GST), immobilized metal affinity chromatography (IMAC) serves as an effective first purification step. This is typically followed by size exclusion chromatography to separate protein aggregates and remove detergent micelles. Ion exchange chromatography may further enhance purity by exploiting the protein's charge properties. When expressing the protein in systems similar to those used for human cytochrome c oxidase subunits, researchers should consider incorporating specialized detergent screening to identify optimal solubilization conditions, as the choice of detergent significantly impacts both yield and activity of the purified protein .

What are the key considerations for designing primers for B. depressicornis MT-CO2 amplification?

When designing primers for B. depressicornis MT-CO2 amplification, researchers should consider several critical factors. First, consensus regions should be identified by aligning MT-CO2 sequences from related bovid species, particularly B. bubalis, for which more genetic data is available . Primers should target conserved regions flanking the MT-CO2 gene while avoiding regions with high variability. The optimal primer length ranges from 18-30 nucleotides with a GC content of 40-60% and melting temperatures between 55-65°C. Terminal G/C bases ("GC clamp") improve annealing stability. For cloning purposes, primers should incorporate appropriate restriction sites with 3-6 additional nucleotides at the 5' end to ensure efficient enzyme cutting. When designing primers for an endangered species with limited available genetic information, researchers should test multiple primer pairs and optimize PCR conditions (particularly annealing temperature and magnesium concentration) to ensure specific amplification while minimizing non-specific products.

What advanced analytical techniques are most informative for studying B. depressicornis MT-CO2 structure?

The structural characterization of B. depressicornis MT-CO2 requires a multi-technique approach due to its complexity as a membrane protein. X-ray crystallography remains the gold standard but presents challenges due to the protein's hydrophobicity. Researchers should consider lipidic cubic phase crystallization methods that have proven successful with other membrane proteins. Cryo-electron microscopy (cryo-EM) offers a powerful alternative, especially when studying MT-CO2 in the context of the complete cytochrome c oxidase complex. For more dynamic structural information, hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides insights into protein flexibility and solvent accessibility without requiring crystallization. Nuclear magnetic resonance (NMR) spectroscopy, particularly solid-state NMR, can reveal local structural details and dynamics when applied to isotopically labeled protein samples. Computational approaches like molecular dynamics simulations complement experimental methods by predicting structural changes under varying conditions or in response to mutations. The integration of these techniques provides a comprehensive structural understanding that connects to functional studies and evolutionary analyses.

How can isothermal titration calorimetry (ITC) be applied to study binding interactions of B. depressicornis MT-CO2?

Isothermal titration calorimetry (ITC) offers a powerful approach for quantitatively characterizing the thermodynamic parameters of B. depressicornis MT-CO2 interactions with binding partners such as cytochrome c or copper ions. The experimental setup requires purified recombinant MT-CO2 maintained in appropriate detergent micelles or nanodiscs to preserve native conformation. For copper binding studies, researchers should prepare copper solutions with defined oxidation states (Cu+ or Cu2+) under anaerobic conditions to prevent oxidation. The ITC experiment involves titrating the binding partner into a cell containing MT-CO2 while measuring the heat released or absorbed during each injection. Data analysis yields binding affinity (Kd), stoichiometry (n), enthalpy change (ΔH), and entropy change (ΔS). Comparative analyses between B. depressicornis MT-CO2 and homologs from other species can reveal adaptations in binding energetics. Control experiments must account for heat of dilution, buffer mismatch, and protein stability throughout the titration. This methodology provides crucial insights into how evolutionary changes in MT-CO2 sequence might affect interaction energetics with conserved binding partners within the respiratory complex.

What is the optimal protocol for assessing MT-CO2 enzyme activity in vitro?

The optimal protocol for assessing B. depressicornis MT-CO2 enzyme activity in vitro requires measuring electron transfer capacity within the full cytochrome c oxidase complex. The procedure begins with either isolating native mitochondria from tissue samples (challenging for endangered species) or reconstituting the complex using recombinant components. The enzyme activity assay employs a Clark-type oxygen electrode or polarographic system to measure oxygen consumption rates at controlled temperature (typically 25°C or 37°C). The reaction mixture contains phosphate buffer (pH 7.4), reduced cytochrome c as electron donor, and the MT-CO2-containing enzyme complex. Oxygen consumption is recorded over time, with initial rates calculated from the linear portion of the curve. Specific activity is expressed as nmol O2 consumed per minute per mg protein. For mechanism studies, inhibitors like potassium cyanide can confirm specificity. Control experiments with denatured enzyme establish baseline activity. Comparative analyses between B. depressicornis and other bovid species can reveal adaptations in catalytic efficiency. This methodology provides quantitative insights into the functional consequences of sequence variations in this endangered species.

How should researchers design experiments to compare MT-CO2 expression levels across different tissues of B. depressicornis?

Designing experiments to compare MT-CO2 expression across B. depressicornis tissues requires careful consideration of sample acquisition, preservation, and analysis methods. Given the endangered status of the species, researchers should prioritize non-invasive sampling methods or coordinate with conservation programs for ethical access to samples. When tissue samples are available, immediate preservation in RNAlater or flash-freezing in liquid nitrogen is essential to prevent RNA degradation. For RNA extraction, specialized protocols for fibrous tissues may be necessary. Quantitative real-time PCR (qRT-PCR) represents the primary method for expression analysis, requiring B. depressicornis-specific MT-CO2 primers and appropriate reference genes validated for expression stability across the tissues studied. Western blotting provides protein-level validation using antibodies against conserved MT-CO2 epitopes. Experimental design should include biological replicates (n≥3) and technical replicates (n≥3) to ensure statistical validity. The following table outlines a systematic approach:

This comprehensive approach provides insights into tissue-specific energy requirements and potential metabolic adaptations in this endangered species.

What is the recommended approach for studying the impact of environmental stressors on B. depressicornis MT-CO2 function?

Studying environmental stressor impacts on B. depressicornis MT-CO2 function requires innovative approaches given the species' endangered status. Cell culture models offer an ethical alternative to direct animal studies. Researchers can develop primary cell lines or immortalized fibroblasts from minimally invasive tissue samples, then expose these cultures to relevant stressors including hypoxia, oxidative stress (H2O2), temperature variations, and pH changes that mimic potential habitat degradation scenarios. Following exposure, MT-CO2 function can be assessed through multiple parameters: oxygen consumption rates using Seahorse XF analyzers, mitochondrial membrane potential via JC-1 staining, ROS production using specific fluorescent probes, and ATP synthesis rates with luminescence-based assays. Complementary molecular analyses should include MT-CO2 expression levels (qRT-PCR), protein abundance (Western blotting), and post-translational modifications (mass spectrometry). For mechanistic insights, researchers should employ inhibitor studies and potentially develop CRISPR-Cas9 modified cell lines with tagged endogenous MT-CO2. This comprehensive approach connects environmental challenges facing this endangered species with molecular adaptations, providing valuable insights for conservation strategies while minimizing impact on wild populations.

How should researchers address contradictory results when comparing B. depressicornis MT-CO2 with homologs from other bovids?

When encountering contradictory results in comparative studies of B. depressicornis MT-CO2 with homologs from other bovids, researchers should implement a systematic troubleshooting and validation approach. First, experimental variables must be critically evaluated, including protein preparation methods, buffer compositions, and assay conditions that might affect protein behavior differently across species. Researchers should verify protein integrity through multiple methods (SDS-PAGE, mass spectrometry, circular dichroism) to rule out degradation or misfolding as sources of discrepancy. For functional contradictions, enzyme kinetics should be reassessed across a broader range of substrate concentrations and environmental conditions (pH, temperature, ionic strength) to identify species-specific optima. Post-translational modification differences should be characterized using phosphoproteomics or glycoproteomics approaches. When sequence-function relationships show unexpected patterns, researchers should consider phylogenetic context and potential convergent evolution events. Additional species should be included in the comparison to establish whether B. depressicornis represents an outlier or part of a broader evolutionary trend. Statistical approaches like bootstrapping can help determine the robustness of observed differences. By systematically investigating contradictory results rather than dismissing them, researchers may uncover previously unknown adaptations specific to this endangered species.

What statistical approaches are most appropriate for analyzing MT-CO2 sequence variation within B. depressicornis populations?

The analysis of MT-CO2 sequence variation within B. depressicornis populations requires statistical approaches suited to both conservation genetics and molecular evolution studies. Given the endangered status and likely limited sample sizes, researchers should begin with basic diversity metrics including nucleotide diversity (π), haplotype diversity (Hd), and the number of segregating sites (S). Population structure can be assessed through FST calculations and Analysis of Molecular Variance (AMOVA). For selection analysis, researchers should employ multiple methods to increase reliability: the McDonald-Kreitman test compares polymorphism to divergence ratios, dN/dS ratio analysis reveals selection pressure intensity, and Tajima's D and Fu's Fs tests can identify demographic events versus selection. Site-specific selection can be detected using maximum likelihood approaches implemented in software like PAML or HyPhy. Bayesian skyline plots can reconstruct historical population dynamics when temporal samples are available. For small populations typical of endangered species, researchers must account for potential biases from genetic drift using coalescent simulations. When integrating with geographic data, landscape genetic approaches like Mantel tests or circuit theory can reveal how habitat fragmentation impacts genetic connectivity. These combined approaches provide a robust framework for interpreting MT-CO2 variation in the context of both evolutionary history and conservation planning.

How can MT-CO2 sequence data contribute to conservation efforts for B. depressicornis?

MT-CO2 sequence data provides valuable contributions to B. depressicornis conservation through multiple applications in population genetics and evolutionary biology. As a mitochondrial gene, MT-CO2 enables maternal lineage tracking, helping conservation biologists identify distinct evolutionary significant units (ESUs) that require separate management strategies. Since the Lowland Anoa population has declined to fewer than 2,500 adults with a 20% population loss rate over 14-18 years , MT-CO2 can serve as a genetic marker to assess remaining genetic diversity and develop targeted breeding programs. The sequence data can identify population bottlenecks through patterns of reduced genetic variation, informing historical context for current conservation challenges. For law enforcement purposes, MT-CO2 sequences can help develop forensic tools to combat illegal poaching by identifying the origin of confiscated animal products. When combined with ecological data and species distribution modeling similar to that conducted for the species , MT-CO2 genetic diversity maps can identify priority areas for conservation that maintain maximum genetic diversity. Additionally, comparing MT-CO2 functional domains across populations can reveal local adaptations to specific environmental conditions, potentially identifying populations with unique adaptive potential that merit special conservation attention.

What methodological approaches are recommended for comparative analysis of MT-CO2 between B. depressicornis and B. quarlesi?

Comparative analysis of MT-CO2 between the closely related B. depressicornis (Lowland Anoa) and B. quarlesi (Mountain Anoa) requires an integrated methodology that combines molecular, biochemical, and computational approaches. Initially, researchers should sequence the complete MT-CO2 gene from multiple individuals of both species, ensuring adequate population sampling across their respective geographical ranges in Sulawesi. Multiple sequence alignment using MUSCLE or MAFFT should identify conserved and variable regions between species. Selection analysis using PAML or HyPhy can detect signatures of positive selection that might indicate adaptive divergence between lowland and mountain habitats. For functional comparison, recombinant proteins from both species should be expressed and characterized using identical conditions, measuring oxygen consumption rates, electron transfer efficiency, and thermal stability. Structural implications of sequence differences can be assessed through homology modeling followed by molecular dynamics simulations under conditions mimicking the different elevational habitats of these species. Researchers should also consider developing an enzyme kinetic profile across temperature ranges (15-40°C) to identify potential adaptations to thermal environments. This comprehensive comparative approach may reveal molecular mechanisms underlying the ecological divergence between these endangered sister species, providing insights into both evolutionary biology and targeted conservation strategies.

How does MT-CO2 variation in B. depressicornis compare to other endangered bovid species, and what are the evolutionary implications?

MT-CO2 variation in B. depressicornis should be examined within the broader context of endangered bovid species to understand evolutionary patterns and conservation implications. Comparative analyses should include phylogenetically diverse endangered bovids such as Bubalus mindorensis (Tamaraw), Bos sauveli (Kouprey), and Bos javanicus (Banteng) to establish variation baselines. Initial sequence comparison should quantify conservation levels across functional domains versus variable regions, identifying potential synapomorphies unique to island bovids. Selection analysis can reveal whether similar selective pressures operate across endangered bovids or if B. depressicornis shows unique patterns. Particular attention should focus on amino acid substitutions in copper-binding regions and cytochrome c interaction sites that might affect respiratory efficiency. Researchers should reconstruct the evolutionary history of MT-CO2 using Bayesian and maximum likelihood approaches, calibrated with fossil data to establish divergence timing. The resulting phylogenetic framework allows testing whether endangerment status correlates with particular evolutionary patterns in MT-CO2. Comparative population genetics metrics (nucleotide diversity, Tajima's D) across species can indicate whether B. depressicornis shows typical or unusual genetic diversity patterns for endangered bovids. This evolutionary context helps conservation biologists understand whether observed MT-CO2 variations represent recent adaptations to changing environments or deep evolutionary history, informing conservation priorities and management approaches.

What emerging technologies could advance our understanding of B. depressicornis MT-CO2 structure and function?

Several emerging technologies promise to significantly advance our understanding of B. depressicornis MT-CO2. Cryo-electron tomography now enables visualization of membrane proteins in their native cellular environment, potentially revealing MT-CO2's structural arrangement within intact mitochondrial membranes. AlphaFold2 and similar AI-based structure prediction tools can generate highly accurate models even with limited experimental data, particularly valuable for endangered species where sample availability is restricted. Single-molecule techniques including Förster resonance energy transfer (FRET) and optical tweezers can capture dynamic conformational changes during electron transfer, providing insights into the protein's mechanistic operation. For functional studies, genome editing technologies like prime editing offer precise modification of endogenous MT-CO2 in cell models while minimizing off-target effects. Nanopore sequencing enables long-read mitochondrial genome analysis from minimal DNA quantities, potentially allowing non-invasive genetic sampling from environmental DNA. Metabolic flux analysis using stable isotope labeling combined with mass spectrometry can connect MT-CO2 variants to whole-organism energy metabolism. The integration of these technologies with traditional approaches will provide unprecedented insights into how B. depressicornis MT-CO2 structure-function relationships may have adapted to the species' unique evolutionary history and ecological niche.

How might climate change impact B. depressicornis MT-CO2 function, and what experimental designs could address this question?

Climate change represents a significant threat to endangered species like B. depressicornis, potentially affecting MT-CO2 function through multiple mechanisms. Rising temperatures may impact protein stability and enzyme kinetics, while habitat degradation could introduce oxidative stressors affecting respiratory efficiency. To investigate these potential impacts, researchers should design a multi-faceted experimental approach using cell culture models derived from minimal tissue samples. The experimental design should incorporate climate projections specific to Sulawesi habitats, focusing on three primary variables: temperature increases (using precise thermal gradient incubators), oxygen tension fluctuations (controlled atmosphere chambers), and oxidative stress (calibrated H2O2 or paraquat exposure). Cell cultures would be exposed to either acute stress or chronic adaptive regimes, followed by comprehensive respiratory function assessment using high-resolution respirometry, ATP production assays, and ROS measurement. Transcriptomic and proteomic analyses would identify compensatory mechanisms and potential points of metabolic vulnerability. For genetic adaptation potential, researchers should sequence MT-CO2 from individuals across the species' current temperature range, identifying existing variants that might confer thermal tolerance. This data could inform machine learning models predicting vulnerability to specific climate scenarios. The findings would not only advance our understanding of mitochondrial adaptation to environmental change but also provide critical data for climate-informed conservation planning.

What opportunities exist for developing non-invasive methods to study MT-CO2 in wild B. depressicornis populations?

Developing non-invasive methods to study MT-CO2 in wild B. depressicornis populations represents a critical research frontier that balances scientific advancement with conservation ethics. Environmental DNA (eDNA) sampling from water sources and soil in Anoa habitats offers promising opportunities to isolate mitochondrial fragments containing MT-CO2 sequences. Advanced PCR techniques like droplet digital PCR can amplify these low-concentration targets with high sensitivity and specificity. For functional studies, researchers should develop methods to isolate intact exosomes from urine and fecal samples, as these membrane-bound vesicles contain mitochondrial proteins including respiratory complex components. Metabolomics analysis of fecal samples can identify downstream metabolites affected by MT-CO2 function. Remote thermal imaging combined with activity monitoring may provide indirect measures of metabolic efficiency linked to respiratory function. Camera traps equipped with hair collection devices can gather genetic material for MT-CO2 sequencing while simultaneously recording behavioral data. For more comprehensive studies, partnerships with wildlife rehabilitation centers caring for injured animals can provide opportunities for minimally invasive sampling during veterinary care. Collaborative approaches with indigenous communities who have traditional knowledge of Anoa behavior may improve sampling efficiency. By integrating these non-invasive techniques, researchers can develop a more complete understanding of MT-CO2 variation and function in wild populations without adding further stress to this endangered species.