Recombinant Syncerus caffer Cytochrome c oxidase subunit 2 (MT-CO2)

What is Syncerus caffer Cytochrome c oxidase subunit 2 (MT-CO2)?

Syncerus caffer (Cape buffalo) Cytochrome c oxidase subunit 2 (MT-CO2), also known as COII or COXII, is one of the core subunits of mitochondrial Cytochrome c oxidase (CCO). The protein contains a dual core CuA active site and plays a significant role in cellular respiration and energy production. According to available data, the full-length protein consists of 227 amino acid residues with the UniProt accession number P50675 . Like other cytochrome c oxidase subunit II proteins, it functions in the electron transport chain, specifically in the terminal step of transferring electrons from cytochrome c to molecular oxygen. This process is essential for ATP production in aerobic organisms, making MT-CO2 a crucial component of cellular energy metabolism.

What is the molecular structure and biochemical properties of Syncerus caffer MT-CO2?

The recombinant Syncerus caffer MT-CO2 has a sequence of 227 amino acids with a molecular weight of approximately 26.2 kDa (based on comparable COXII proteins) . The amino acid sequence is: "MAYPMQLGFQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKLTHTSTMDAQEVETIWTILPAIILILIALPSLRILYMMDEINNPSLTVKTMGHQWYWSYEYTDYEDLSFDSYMVPTSELKPGELRLLEVDNRVVLPMEMTIRMLVSSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMSTRPGLYYGQCSEICGSNHSFMPIVILEMVPLKYFEKWASML" . The protein likely has a pI value around 6.37, similar to other COXII proteins . It contains hydrophobic transmembrane domains and the characteristic CuA binding site essential for electron transfer. The protein's structure incorporates metal cofactors, particularly copper ions at the CuA site, which are crucial for its electron transfer function. While Cape buffalo-specific structural data is limited, comparative analysis with related species suggests the presence of conserved functional domains that characterize cytochrome c oxidase subunit II proteins across species.

How does MT-CO2 function in the respiratory chain?

MT-CO2 functions as a critical component of the cytochrome c oxidase complex (Complex IV), which is the terminal enzyme in the electron transport chain of cellular respiration. The protein's dual core CuA active site receives electrons from cytochrome c and transfers them through the protein to the heme a-heme a₃-CuB center where oxygen reduction occurs . This electron transfer process is coupled to proton pumping across the inner mitochondrial membrane, contributing to the proton gradient that drives ATP synthesis. Specifically, MT-CO2 contains the binding site for cytochrome c and facilitates the initial electron transfer step in the complex. The protein's interaction with both cytochrome c and other subunits of the complex is essential for efficient electron flow and energy conversion. Mutations or structural alterations in MT-CO2 can significantly impact the efficiency of cellular respiration, potentially affecting the organism's energy metabolism and physiological functions.

What are the key considerations for expressing recombinant Syncerus caffer MT-CO2?

Expressing recombinant Syncerus caffer MT-CO2 requires careful optimization of multiple factors. Based on comparable studies with other COXII proteins, researchers should consider the following approach: First, select an appropriate expression vector - pET-32a has been successfully used for similar proteins . Second, choose a suitable E. coli strain; Transetta (DE3) has proven effective for COXII expression . Third, optimize induction conditions - typical protocols use isopropyl β-d-thiogalactopyranoside (IPTG) at concentrations between 0.5-1.0 mM, with induction temperatures of 16-30°C for 4-16 hours. Since MT-CO2 is naturally membrane-associated, solubility issues may arise during expression. To address this, fusion tags (such as thioredoxin or GST) or co-expression with chaperones may improve soluble protein yield. Expression levels should be monitored using SDS-PAGE and Western blotting with anti-His or specific anti-MT-CO2 antibodies. For scale-up production, fermentation parameters including aeration, media composition, and feed strategies must be systematically optimized.

What purification strategies are most effective for recombinant Syncerus caffer MT-CO2?

Effective purification of recombinant Syncerus caffer MT-CO2 typically employs a multi-step approach. Initial capture can be achieved using affinity chromatography with Ni²⁺-NTA agarose if the protein contains a 6-His tag . Optimization of binding and elution conditions is crucial - typically using imidazole gradients (20-250 mM) for elution. Following affinity purification, additional polishing steps may include:

If required, tag removal can be performed using specific proteases (TEV, thrombin, etc.), followed by a second affinity step to separate the cleaved protein. Quality control should include SDS-PAGE, Western blotting, mass spectrometry, and activity assays. For the recombinant protein, a final concentration of approximately 50 μg/mL has been achieved for similar COXII proteins , with higher concentrations possible through optimization.

How can the enzymatic activity of recombinant MT-CO2 be effectively measured?

Several approaches can be used to measure the enzymatic activity of recombinant MT-CO2:

• Spectrophotometric assays: UV-spectrophotometric analysis can monitor the oxidation of reduced cytochrome c by measuring the decrease in absorbance at 550 nm . The reaction buffer typically contains 10-50 mM phosphate buffer (pH 7.0-7.4), 0.1-0.2% detergent (if needed for protein solubility), and reduced cytochrome c (10-50 μM).

• Oxygen consumption: Polarographic methods using oxygen electrodes can directly measure oxygen consumption rates, providing real-time kinetic data.

• Infrared spectrometer analysis can provide insights into structural changes associated with substrate binding and catalysis .

For validation, controls should include:

• Heat-inactivated enzyme

• Reactions with known inhibitors (e.g., cyanide, azide)

• Comparison with native enzyme activity

Kinetic parameters (Km, Vmax) should be determined under standardized conditions and compared with published values for related proteins. Enzyme activity can be expressed as μmol cytochrome c oxidized per minute per mg protein. The influence of potential regulators or inhibitors, such as allyl isothiocyanate (AITC), can also be assessed using these assays .

What bioinformatic approaches are valuable for analyzing MT-CO2 conservation across bovid species?

For evolutionary analysis of MT-CO2 across bovids, several sophisticated bioinformatic approaches are recommended:

• Multiple sequence alignment (MSA): Tools like MUSCLE, MAFFT, or CLUSTAL Omega can align MT-CO2 sequences from various bovid species to identify conserved regions. Based on studies with other species, Syncerus caffer COXII would likely show high sequence identity with COXII from related bovids .

• Phylogenetic analysis: Maximum likelihood (RAxML, IQ-TREE) or Bayesian methods (MrBayes, BEAST) can establish evolutionary relationships and divergence times, with appropriate substitution models selected using tools like ModelTest.

• Selection analysis: Methods implemented in PAML, HyPhy, or MEGA can identify sites under positive, neutral, or purifying selection, revealing evolutionary constraints on protein function.

• Structural mapping: Conservation scores from tools like ConSurf can be mapped onto structural models of MT-CO2, visually highlighting functionally important regions.

• Coevolution analysis: Tools like CAPS or EVcouplings can identify coevolving residues, potentially revealing functional interactions between different regions of MT-CO2 or with other proteins.

These approaches together provide a comprehensive picture of evolutionary constraints on MT-CO2 structure and function, informing experimental studies and contributing to our understanding of bovid evolution.

How can researchers design effective antibodies against Syncerus caffer MT-CO2?

Designing effective antibodies against Syncerus caffer MT-CO2 requires a systematic approach:

• Epitope prediction: Analyze the protein sequence using algorithms that consider hydrophilicity, surface accessibility, and secondary structure. Tools like Bepipred, Ellipro, or ABCpred can identify potential B-cell epitopes.

• Antigen design strategies:

  • Full-length recombinant protein: Provides comprehensive epitope coverage but may present solubility challenges

  • Synthetic peptides: Target unique, accessible regions (15-25 amino acids)

  • Recombinant fragments: Focus on exposed, immunogenic domains

• Immunization approaches:

  • Polyclonal antibodies: Generate diverse antibodies recognizing multiple epitopes

  • Monoclonal antibodies: Produce highly specific antibodies using hybridoma technology or phage display

• Validation strategies:

  • Western blotting: Confirms specificity and molecular weight recognition

  • Immunoprecipitation: Tests antibody-antigen binding in solution

  • Immunohistochemistry/Immunofluorescence: Evaluates tissue localization

  • Cross-reactivity testing: Ensures species specificity

• Commercial considerations: Over 1000 cytochrome C oxidase antibodies are available across different suppliers , which might inform design strategies or provide alternatives to custom antibody production.

Optimal epitope selection should balance uniqueness to Syncerus caffer with conservation of functionally important regions, depending on the intended application of the antibodies.

What considerations are important when designing functional studies of MT-CO2 interactions?

Designing robust functional studies for MT-CO2 interactions requires careful attention to multiple factors:

• Protein preparation: Ensure the recombinant protein retains its native conformation using structural analyses (circular dichroism, thermal shift assays). Consider reconstitution in liposomes or nanodiscs to mimic the native membrane environment.

• Interaction analysis techniques:

  • Surface plasmon resonance (SPR): Provides real-time binding kinetics

  • Isothermal titration calorimetry (ITC): Measures thermodynamic parameters

  • Co-immunoprecipitation: Identifies protein-protein interactions

  • Fluorescence resonance energy transfer (FRET): Detects proximity between labeled interaction partners

• Cytochrome c interaction studies: Standardize conditions for enzymatic assays, including pH (typically 7.0-7.5), temperature (25-37°C), and ionic strength (50-150 mM). Varying these parameters can provide insights into the physiological relevance of interactions.

• Inhibitor studies: Molecular docking methods can predict interaction interfaces, as demonstrated with AITC binding to COXII . These predictions should be validated through site-directed mutagenesis of predicted interaction residues.

• Experimental controls:

  • Known inhibitors (cyanide, azide)

  • Mutated versions of MT-CO2 (particularly at the CuA site)

  • Denatured protein controls

  • Related proteins from other species

This multi-faceted approach ensures robust characterization of MT-CO2 interactions with substrates, regulators, and other components of the respiratory chain.

What are the optimal storage and handling conditions for maintaining recombinant MT-CO2 activity?

Maintaining the structural integrity and enzymatic activity of recombinant Syncerus caffer MT-CO2 requires careful attention to storage and handling conditions:

During experimental procedures:

• Thaw protein aliquots slowly on ice

• Maintain cold temperatures during handling when possible

• Include appropriate cofactors (copper ions) for activity assays

• Consider adding mild detergents or lipids to maintain native conformation

• Monitor protein stability over time using activity assays or biophysical methods

These conditions are essential to ensure reproducible results in experimental studies using the recombinant protein.

How can researchers troubleshoot expression and purification issues?

When troubleshooting expression and purification of recombinant Syncerus caffer MT-CO2, researchers should consider several factors:

Expression Issues:

• Poor expression levels:

  • Optimize codon usage for the expression host

  • Try different E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Adjust induction conditions (0.1-1.0 mM IPTG, 16-30°C, 4-24 hours)

  • Consider autoinduction media

• Inclusion body formation:

  • Lower induction temperature (16-20°C)

  • Co-express with chaperones (GroEL/ES, DnaK)

  • Use solubility-enhancing fusion tags (SUMO, MBP, Trx)

Purification Challenges:

• Low protein yield:

  • Optimize lysis conditions (sonication, pressure homogenization)

  • Try different buffer compositions and detergents

  • Increase imidazole in wash buffers to reduce non-specific binding

• Protein instability:

  • Include protease inhibitors during purification

  • Optimize buffer composition (pH, salt concentration)

  • Add stabilizing agents (glycerol, reducing agents)

Diagnostic Approaches:

• Track protein through purification using Western blotting

• Analyze fractions by SDS-PAGE with Coomassie and silver staining

• Verify protein identity by mass spectrometry

• Check activity at each purification step

Note that the expected size of the recombinant protein with a 6-His tag would be significantly larger than the native protein (which has a predicted molecular mass of approximately 26.2 kDa for similar COXII proteins) .

What considerations are important for comparative studies with other bovid species?

For comparative studies using recombinant Syncerus caffer MT-CO2 with other bovid species, researchers should consider:

• Standardization of methods:

  • Use identical expression systems and vectors for all species

  • Apply uniform purification protocols

  • Conduct functional assays under identical conditions

  • Analyze data using consistent statistical approaches

• Sequence and structural analyses:

  • Perform detailed sequence comparisons to identify species-specific variations

  • Pay particular attention to functional domains (CuA site, cytochrome c binding regions)

  • Create structural models to visualize the impact of sequence differences

• Experimental design considerations:

  • Include appropriate positive and negative controls

  • Analyze multiple independent protein preparations

  • Blind experimental conditions where possible

  • Consider using internal standards for normalizing activity data

• Complementary approaches:

  • Supplement recombinant protein studies with native tissue samples

  • Control for variables like age, sex, and physiological state

  • Consider cell-based assays to bridge biochemical and physiological insights

• Evolutionary context:

  • Correlate functional differences with phylogenetic relationships

  • Consider environmental adaptations of different bovid species

  • Relate molecular differences to physiological or ecological specializations

This comprehensive approach ensures valid cross-species comparisons while providing insights into how molecular evolution of MT-CO2 may contribute to species-specific physiological adaptations.

How can post-translational modifications of MT-CO2 be effectively studied?

Studying post-translational modifications (PTMs) of Syncerus caffer MT-CO2 requires a multi-faceted approach:

• Mass spectrometry-based proteomics:

  • Bottom-up approach: Enzymatic digestion followed by LC-MS/MS

  • Top-down approach: Analysis of intact protein

  • Targeted methods: Multiple reaction monitoring (MRM) for specific modifications

• Enrichment strategies for specific PTMs:

  • Phosphorylation: TiO₂ or IMAC enrichment

  • Glycosylation: Lectin affinity or hydrazide chemistry

  • Acetylation: Anti-acetyllysine antibodies

• Site-specific analysis:

  • Site-directed mutagenesis of modified residues

  • Expression of modified vs. unmodified forms

  • Activity comparison between modified and unmodified protein

• Physiological context:

  • Compare PTMs across different physiological states

  • Analyze tissue-specific modification patterns

  • Investigate regulatory enzymes (kinases, acetylases, etc.)

• Bioinformatic analysis:

  • Prediction tools for potential modification sites

  • Structural modeling of the impact of modifications

  • Comparison with known modifications in related species

Since MT-CO2 is involved in respiration, particular attention should be paid to modifications affecting the CuA center or interface with cytochrome c, as these could directly impact electron transfer efficiency and energy production.

How can Syncerus caffer MT-CO2 be utilized in evolutionary and conservation studies?

Syncerus caffer MT-CO2, being encoded by mitochondrial DNA, offers several advantages as a molecular marker for evolutionary and conservation studies:

• Population genetics applications:

  • Sequence MT-CO2 from different populations to assess genetic diversity

  • Analyze haplotype distribution for population structure

  • Use maternal inheritance pattern to track female lineages

  • Quantify genetic differentiation between populations (FST, AMOVA)

• Phylogenetic applications:

  • Reconstruct relationships among Syncerus populations

  • Estimate divergence times using molecular clock approaches

  • Compare with other bovid MT-CO2 sequences to clarify broader relationships

  • Combine with nuclear markers for comprehensive phylogenetic analysis

• Conservation applications:

  • Identify evolutionary significant units (ESUs) requiring separate conservation

  • Detect population bottlenecks or founder effects

  • Assess genetic erosion in threatened populations

  • Inform captive breeding programs to maintain genetic diversity

• Methodological considerations:

  • Sample across geographic range of Syncerus caffer

  • Use appropriate outgroups for phylogenetic analyses

  • Apply analytical methods suitable for mitochondrial markers

  • Integrate findings with ecological and morphological data

This multidisciplinary approach can provide comprehensive insights into the evolutionary history and conservation status of Syncerus caffer populations, contributing to evidence-based conservation strategies.

How can structural studies contribute to understanding species-specific adaptations?

Structural studies of Syncerus caffer MT-CO2 can provide valuable insights into adaptations in energy metabolism specific to this species:

• Structural determination approaches:

  • X-ray crystallography of the purified protein

  • Cryo-electron microscopy of the entire cytochrome c oxidase complex

  • NMR spectroscopy of specific domains

  • Homology modeling based on structures from related species

• Functional correlation analyses:

  • Link structural features to kinetic parameters

  • Identify species-specific variations in substrate binding sites

  • Analyze proton and electron transfer pathways

  • Investigate the structural basis for thermal stability

• Comparative structural biology:

  • Compare with MT-CO2 structures from species in different environments

  • Relate structural differences to ecological adaptations

  • Examine protein dynamics under varying conditions

  • Consider the impact of species-specific PTMs on structure

• Environmental adaptation insights:

  • Analyze how structural features might contribute to:

    • Heat tolerance in African environments

    • Metabolic efficiency during seasonal food scarcity

    • Adaptation to hypoxic conditions

    • Resistance to specific inhibitors or toxins

These structural insights have broader implications for understanding metabolic adaptation in large mammals across different ecological contexts, potentially informing conservation efforts and providing insights into evolutionary processes.