Recombinant Eulemur macaco Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c Oxidase Subunit 2 and what is its function in Eulemur macaco?

Cytochrome c oxidase subunit II (MT-CO2) is one of the core components of the mitochondrial electron transport chain, specifically of complex IV (cytochrome c oxidase). In Eulemur macaco, as in other primates, MT-CO2 contains a dual core CuA active site that serves as the primary electron acceptor from cytochrome c . This site facilitates electron transfer to the catalytic center of the enzyme, where oxygen is reduced to water.

The MT-CO2 protein plays a crucial role in cellular respiration, contributing to the proton gradient across the inner mitochondrial membrane that drives ATP synthesis. In lemurs like Eulemur macaco, MT-CO2 may have evolved specific adaptations reflecting their unique evolutionary history and ecological niche on Madagascar. The protein is encoded by mitochondrial DNA and typically consists of approximately 227 amino acids with a molecular mass around 26 kDa .

Why is studying MT-CO2 in Eulemur macaco specifically valuable for evolutionary research?

Studying MT-CO2 in Eulemur macaco provides several distinct research advantages. As a lemuriform primate, Eulemur macaco represents an early-diverging lineage in primate evolution, having evolved in isolation on Madagascar. This makes their MT-CO2 particularly valuable for comparative evolutionary analyses across the primate order .

Research has demonstrated that higher primates (monkeys and apes) exhibit accelerated evolutionary rates in MT-CO2 compared to lemurs and other primates, with nearly a two-fold increase in amino acid replacement rates . By studying Eulemur macaco MT-CO2, researchers can investigate this differential evolution pattern and its functional implications. The sequence and structural differences in MT-CO2 between lemurs and higher primates, particularly at functionally significant sites like positions 114 and 115, provide insights into how mitochondrial proteins adapt over evolutionary time .

Additionally, as Eulemur macaco faces conservation challenges, studying its genetic diversity at functional loci like MT-CO2 can inform conservation genetics approaches and contribute to preservation efforts for this vulnerable species.

How does the structure of MT-CO2 differ between Eulemur macaco and higher primates?

• The amino terminal region shows greater variability, with higher primates exhibiting increased amino acid replacements compared to lemurs .

• Key substitutions at positions 114 and 115 represent one of the most significant differences. In Eulemur macaco and other lemurs, these positions typically contain carboxyl-bearing residues (glutamate and aspartate), whereas in higher primates, these have been replaced with different amino acids .

• These substitutions at positions 114 and 115 are particularly noteworthy as they may explain the poor enzyme kinetics observed in cross-reactions between cytochromes c and cytochrome c oxidases between higher primates and other mammals .

• The CuA binding domain, critical for electron transfer function, maintains conserved copper-binding motifs across primates, but may exhibit subtle structural differences affecting redox potential or electron transfer efficiency.

These structural differences likely reflect different selective pressures and adaptive pathways during primate evolution, with potential implications for mitochondrial function and energy metabolism.

What are the optimal protocols for cloning the Eulemur macaco MT-CO2 gene?

For successful cloning of Eulemur macaco MT-CO2, researchers should follow these methodological steps:

• Primer design: Design primers based on conserved regions in lemur MT-CO2 sequences. If Eulemur macaco MT-CO2 sequence is unavailable, align MT-CO2 sequences from related lemur species to identify conserved regions for primer design. Include appropriate restriction sites to facilitate subsequent cloning.

• Sample collection and nucleic acid isolation: Extract DNA from a suitable tissue sample (blood, muscle, or hair follicle). For RNA-based approaches, extract total RNA from fresh tissue and synthesize cDNA using reverse transcriptase with oligo(dT) or random hexamer primers.

• PCR amplification: Optimize PCR conditions for the full-length MT-CO2 gene (approximately 684 bp based on typical primate MT-CO2) . Typical conditions include:

  • Initial denaturation: 95°C for 5 minutes

  • 30-35 cycles of: 95°C for 30 seconds, 55-60°C for 30 seconds, 72°C for 1 minute

  • Final extension: 72°C for 10 minutes

• Cloning strategy: After gel purification of the PCR product, perform TA cloning into a vector like pGEM-T Easy for sequencing verification. For expression purposes, subclone the verified sequence into an expression vector like pET-32a, which has been successful for COXII expression .

• Verification: Perform colony PCR screening followed by plasmid isolation and restriction digestion to confirm insert presence. Sequence multiple clones to identify and select error-free constructs.

This approach has been successfully applied to COXII genes from other species and can be adapted specifically for Eulemur macaco MT-CO2 .

What expression systems yield optimal results for recombinant Eulemur macaco MT-CO2?

The expression of functional recombinant Eulemur macaco MT-CO2 requires careful consideration of expression systems and conditions:

• Bacterial expression systems:

  • E. coli is the most commonly used system, with specialized strains like Transetta(DE3), Rosetta(DE3), or BL21(DE3)pLysS recommended for membrane proteins .

  • Expression vectors with solubility-enhancing fusion tags are essential; pET-32a with thioredoxin fusion has shown success with COXII proteins .

  • Optimal expression conditions typically include induction with 0.1-0.5 mM IPTG at lower temperatures (15-25°C) for 16-24 hours to enhance proper folding.

• Eukaryotic expression systems:

  • For studies requiring post-translational modifications, insect cell systems (Sf9, Sf21) with baculovirus vectors may yield more appropriately modified protein.

  • Mammalian expression (CHO or HEK293 cells) can be considered for studies requiring mammalian-specific folding machinery.

• Cell-free expression systems:

  • For rapid screening or when toxicity is an issue, cell-free protein synthesis with added detergents or lipids can be effective for membrane proteins.

• Expression optimization:

  • Codon optimization of the MT-CO2 sequence for the chosen expression system can significantly improve yields.

  • Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ) can enhance proper folding.

  • Supplementation with copper in the growth medium may promote proper formation of the CuA center.

Expression verification should be performed using SDS-PAGE and western blotting with either anti-His antibodies or specific antibodies against MT-CO2. The expected molecular weight is approximately 26.2 kDa for the native protein or 44 kDa when expressed as a fusion with thioredoxin .

What purification strategy provides the highest purity and yield of functional recombinant MT-CO2?

A multi-step purification approach is recommended to obtain high-purity, functional Eulemur macaco MT-CO2:

• Initial clarification and solubilization:

  • Lyse cells in buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, protease inhibitors, and mild detergent (0.5-1% Triton X-100 or n-dodecyl β-D-maltoside).

  • Clarify by centrifugation at 16,000-20,000 × g for 30 minutes at 4°C.

• Affinity chromatography:

  • For His-tagged constructs, use Ni²⁺-NTA agarose affinity chromatography with the following buffer system :

    • Binding buffer: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10-20 mM imidazole, 10% glycerol, 0.1% detergent

    • Wash buffer: Same as binding buffer but with 40-50 mM imidazole

    • Elution buffer: Same as binding buffer but with 250-300 mM imidazole

• Tag removal (if required):

  • Cleave fusion tags using specific proteases (thrombin, TEV, etc.)

  • Remove the cleaved tag by a second pass through the affinity column

• Ion exchange chromatography:

  • Apply the protein to an ion exchange column based on its predicted pI value (approximately 6.37 for COXII proteins)

  • For MT-CO2, anion exchange (Q Sepharose) at pH 8.0 is typically effective

• Size exclusion chromatography:

  • As a final polishing step, apply the protein to a Superdex 75 or 200 column equilibrated in buffer containing detergent at concentrations just above CMC (critical micelle concentration)

• Quality assessment:

  • Analyze purity by SDS-PAGE (>95% is desirable)

  • Confirm identity by western blotting and/or mass spectrometry

  • Assess copper incorporation using UV-visible spectroscopy

  • Verify oligomeric state by native PAGE or analytical size exclusion

This purification scheme typically yields 5-10 mg of purified protein per liter of bacterial culture with approximately 50 μg/mL final concentration .

How can researchers verify the functionality of purified recombinant MT-CO2?

Verifying the functionality of recombinant Eulemur macaco MT-CO2 requires multiple complementary approaches:

• Spectrophotometric enzyme activity assay:

  • Monitor the oxidation of reduced cytochrome c at 550 nm as described previously for COXII proteins

  • Reaction mix typically contains:

    • 10-50 nM purified MT-CO2

    • 50 μM reduced cytochrome c

    • 50 mM potassium phosphate buffer (pH 7.4)

    • 0.1% detergent (n-dodecyl β-D-maltoside)

  • Calculate specific activity as μmol cytochrome c oxidized/min/mg protein

• UV-visible spectroscopy:

  • Properly folded MT-CO2 with incorporated copper shows characteristic absorption features in the 400-700 nm region

  • Compare the spectrum with published data for cytochrome c oxidase subunits

• Structural integrity assessment:

  • Circular dichroism spectroscopy to verify secondary structure content

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to examine folding quality

• Binding assays:

  • Surface plasmon resonance (SPR) to measure binding kinetics with cytochrome c

  • Microscale thermophoresis (MST) as an alternative approach for binding studies

• Inhibitor sensitivity tests:

  • Verify sensitivity to known cytochrome c oxidase inhibitors (e.g., cyanide, azide)

  • Establish dose-response curves and determine IC₅₀ values

• Comparative functional analysis:

  • Compare activity of Eulemur macaco MT-CO2 with recombinant MT-CO2 from other primates

  • Test cross-reaction with cytochrome c from different species to evaluate specificity

UV-spectrophotometer analysis should confirm that recombinant MT-CO2 can catalyze the oxidation of substrate cytochrome c, similar to what has been demonstrated for other COXII proteins .

How can structural studies of MT-CO2 from Eulemur macaco contribute to understanding primate evolution?

Structural studies of MT-CO2 from Eulemur macaco provide unique insights into primate evolution through several research approaches:

• Three-dimensional structure determination:

  • X-ray crystallography or cryo-electron microscopy of recombinant Eulemur macaco MT-CO2 can reveal the precise molecular architecture

  • Comparing this structure with those from higher primates can identify structural adaptations that evolved during primate radiation

• Structure-function correlation with evolutionary rates:

  • Mapping sites of accelerated evolution in higher primates onto the MT-CO2 structure can identify regions of functional significance

  • Particularly important are positions 114 and 115, where higher primates have replaced carboxyl-bearing residues that are conserved in lemurs

• Molecular dynamics simulations:

  • Simulations using the determined structure can predict how specific amino acid substitutions alter protein dynamics and function

  • Comparative simulations between lemur and other primate MT-CO2 can reveal functional consequences of evolutionary changes

• Electron transfer kinetics modeling:

  • The CuA center structure in Eulemur macaco MT-CO2 can be compared with that of other primates to understand how evolutionary changes affect electron transfer

  • Quantum mechanical calculations can predict how differences in the protein environment around the CuA center influence redox potential

• Protein-protein interaction interfaces:

  • Structural analysis of the interface between MT-CO2 and cytochrome c can reveal how co-evolution maintains efficient electron transfer despite sequence divergence

  • This is particularly relevant given the poor enzyme kinetics observed in cross-reactions between higher primate and other mammalian components

These structural studies provide a molecular lens through which to view primate evolution, connecting sequence differences to functional adaptations and potentially explaining the nearly two-fold increase in amino acid replacement rates observed in higher primate MT-CO2 .

What techniques are most effective for studying interactions between recombinant MT-CO2 and other components of the respiratory chain?

Investigating interactions between recombinant Eulemur macaco MT-CO2 and other respiratory chain components requires specialized techniques that account for the membrane-associated nature of these proteins:

• Reconstitution systems:

  • Nanodiscs: Incorporate purified MT-CO2 into membrane nanodiscs with defined lipid composition

  • Proteoliposomes: Co-reconstitute MT-CO2 with interaction partners in liposomes

  • These systems provide a membrane-like environment for physiologically relevant interaction studies

• Advanced biophysical methods:

  • Surface plasmon resonance (SPR) with captured MT-CO2 in detergent or nanodiscs

  • Microscale thermophoresis (MST) for measuring interactions in solution

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Förster resonance energy transfer (FRET) for measuring distances between labeled proteins

• Chemical cross-linking coupled to mass spectrometry:

  • Cross-link MT-CO2 with putative interaction partners

  • Digest cross-linked complexes and identify cross-linked peptides by MS/MS

  • Map interaction interfaces to the primary sequence and structural models

• Functional coupling assays:

  • Oxygen consumption measurements using respirometry

  • Membrane potential measurements using potentiometric dyes

  • These assays can detect functional consequences of specific interactions

• Comparative analysis across primates:

  • Compare interaction properties of MT-CO2 from Eulemur macaco with those from other primates

  • Correlate interaction differences with sequence variations, particularly at positions 114 and 115

  • Test cross-species interactions to evaluate the functional impacts of evolutionary changes

• Computational docking and molecular dynamics:

  • In silico docking of MT-CO2 with electron transport chain components

  • Molecular dynamics simulations of predicted complexes

  • Energy calculations to identify key residues at interaction interfaces

These approaches collectively provide insights into how evolutionary changes in MT-CO2 affect interactions with other respiratory chain components, potentially explaining the functional consequences of the accelerated evolution observed in higher primate MT-CO2 .

How can studies of recombinant MT-CO2 from Eulemur macaco inform conservation genetics approaches?

Recombinant MT-CO2 studies can significantly enhance conservation genetics for Eulemur macaco through multiple research applications:

• Functional assessment of genetic variants:

  • Express and characterize MT-CO2 variants identified in wild populations

  • Measure enzymatic activity, stability, and interaction capacity of each variant

  • Correlate functional parameters with genetic diversity patterns

• Evolutionary fitness implications:

  • Determine how specific MT-CO2 variants affect energy metabolism efficiency

  • Assess whether certain variants provide adaptive advantages in changing environments

  • Identify potentially deleterious mutations that might impact population viability

• Population-specific adaptations:

  • Compare MT-CO2 variants between geographically distinct Eulemur macaco populations

  • Identify population-specific adaptations that might be important for local adaptation

  • Evaluate whether these adaptations should influence conservation management units

• Hybridization assessment:

  • Express and characterize MT-CO2 from hybrids between Eulemur macaco and related species

  • Evaluate whether hybridization affects MT-CO2 function through cytonuclear incompatibilities

  • Use this information to develop strategies for managing hybrid populations

• Environmental stress response:

  • Test how different MT-CO2 variants perform under stress conditions (temperature, pH, oxidative stress)

  • Identify variants with enhanced resilience to climate change-related stressors

  • Incorporate this information into vulnerability assessments

• Non-invasive monitoring methods development:

  • Design primers for MT-CO2 that can be used with DNA from non-invasive samples (feces, hair)

  • Develop assays to detect MT-CO2 variants from field-collected samples

  • Create portable testing methods for on-site genetic analysis in remote field locations

These applications bridge molecular and conservation biology, providing functional context for genetic data and enhancing the scientific basis for conservation decisions for this vulnerable lemur species.

What approaches can identify post-translational modifications in recombinant versus native MT-CO2?

Identifying and comparing post-translational modifications (PTMs) between recombinant and native MT-CO2 from Eulemur macaco requires a comprehensive analytical approach:

• Mass spectrometry-based proteomics:

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

  • Top-down approach: Analysis of intact protein to preserve PTM combinations

  • PTM-specific enrichment methods:

    • Phosphorylation: Titanium dioxide or immobilized metal affinity chromatography

    • Glycosylation: Lectin affinity chromatography or hydrazide chemistry

    • Acetylation: Anti-acetyl lysine antibodies

• Site-specific analysis:

  • Targeted multiple reaction monitoring (MRM) for quantifying specific modifications

  • Parallel reaction monitoring (PRM) for improved selectivity

  • ETD/ECD fragmentation for labile PTM preservation during MS/MS

• Comparative workflow:

  • Side-by-side analysis of native MT-CO2 (isolated from Eulemur macaco tissue) and recombinant protein

  • Differential isotopic labeling to distinguish samples

  • Statistical analysis to identify consistent differences

• Functional correlation:

  • Enzymatic introduction of PTMs to recombinant protein using appropriate modifying enzymes

  • Site-directed mutagenesis to create PTM-mimicking mutations

  • Activity assays comparing native, recombinant, and PTM-modified recombinant proteins

• Structural impact assessment:

  • Hydrogen-deuterium exchange mass spectrometry to detect structural changes from PTMs

  • NMR spectroscopy for localized structural analysis

  • Molecular dynamics simulations to predict PTM effects on protein dynamics

• Visualization methods:

  • PTM-specific staining (Pro-Q Diamond for phosphorylation, periodic acid-Schiff for glycosylation)

  • Western blotting with modification-specific antibodies

  • 2D gel electrophoresis to separate modified protein species

These approaches can identify differences between native and recombinant MT-CO2, guiding strategies to introduce relevant PTMs to recombinant proteins or engineer expression systems that properly modify the protein.

How can researchers address solubility challenges when working with recombinant MT-CO2?

Recombinant MT-CO2 from Eulemur macaco often presents solubility challenges due to its hydrophobic regions and membrane association. Researchers can implement these evidence-based strategies:

• Fusion protein optimization:

  • Thioredoxin fusion has proven successful for other COXII proteins, as demonstrated in the pET-32a expression system

  • Alternative tags to consider include SUMO, MBP, and GST

  • Test both N-terminal and C-terminal tag placements to determine optimal configuration

• Expression condition modifications:

  • Lower induction temperature (15-20°C) significantly improves solubility for membrane proteins

  • Reduce IPTG concentration to 0.1-0.2 mM for gentler induction

  • Extend expression time to 16-24 hours at lower temperatures

  • Implement oxygen limitation during expression to reduce inclusion body formation

• Buffer optimization:

  • Screen multiple detergents (LDAO, DDM, Triton X-100, CHAPS) at concentrations just above CMC

  • Include stabilizing additives:

    • 10-20% glycerol as a stabilizing agent

    • 50-300 mM sodium chloride to maintain ionic strength

    • 1-5 mM reducing agents (DTT or TCEP) to prevent oxidation

    • Arginine (50-100 mM) to reduce aggregation

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Consider co-expression with other cytochrome c oxidase subunits

• Refolding approaches (if inclusion bodies persist):

  • Solubilize inclusion bodies in 8 M urea or 6 M guanidine hydrochloride

  • Perform step-wise dialysis with decreasing denaturant and addition of detergent

  • Add copper ions during refolding to facilitate CuA center formation

  • Use on-column refolding during affinity purification

• Construct engineering:

  • Remove non-essential hydrophobic regions if they don't affect the research question

  • Create truncated constructs focusing on the soluble domains

  • Introduce solubility-enhancing point mutations based on computational prediction

Implementation of these strategies has enabled successful expression and purification of soluble and functional COXII proteins, as demonstrated by previous studies where recombinant COXII was shown to catalyze the oxidation of substrate cytochrome c .

What approaches can resolve inconsistent activity measurements with recombinant MT-CO2?

When faced with inconsistent activity measurements of recombinant Eulemur macaco MT-CO2, researchers should implement a systematic troubleshooting approach:

• Protein quality assessment:

  • Verify protein purity by SDS-PAGE (>95% purity recommended)

  • Confirm protein identity by western blotting or mass spectrometry

  • Check for degradation products using anti-His tag antibodies and MT-CO2-specific antibodies

  • Assess protein aggregation state by size exclusion chromatography

  • Verify copper incorporation using atomic absorption spectroscopy or specific spectral features

• Assay standardization:

  • Standardize reaction conditions:

    • Buffer composition and pH (typically 50 mM phosphate buffer, pH 7.4)

    • Temperature control (±0.5°C)

    • Consistent substrate preparation (fresh reduction of cytochrome c before each assay)

  • Establish standard curves with commercial cytochrome c oxidase as positive control

  • Determine the linear range of the assay with respect to both enzyme concentration and time

• Statistical robustness improvements:

  • Increase technical replicates (minimum n=5) to improve precision

  • Perform biological replicates with independent protein preparations

  • Apply appropriate statistical tests (ANOVA with post-hoc tests) to analyze variation

  • Implement blinded experimental designs when possible

  • Use multivariate analysis to identify factors contributing to variability

• Reaction component optimization:

  • Test multiple sources of cytochrome c substrate

  • Evaluate the effect of different detergents on activity

  • Assess the impact of additives (glycerol, salt concentration)

  • Measure activity at varying enzyme:substrate ratios

• Instrument validation:

  • Calibrate spectrophotometers before each measurement session

  • Verify temperature control in reaction chambers

  • Use standard compounds to validate instrument performance

  • Consider implementing automated mixing to ensure consistency

• Alternative assay methods:

  • Complement spectrophotometric assays with oxygen consumption measurements

  • Use fluorescence-based assays for increased sensitivity

  • Implement continuous rather than endpoint measurements when possible

By systematically addressing these factors, researchers can identify sources of variability and establish robust protocols for consistent activity measurements of recombinant Eulemur macaco MT-CO2 .

How can evolutionary rate analyses of MT-CO2 be optimized for phylogenetic studies of lemurs?

Optimizing evolutionary rate analyses of MT-CO2 for lemur phylogenetic studies requires specialized approaches that account for the unique evolutionary history of these primates:

• Sequence sampling strategy:

  • Include comprehensive sampling of lemur species, particularly within Eulemur

  • Incorporate strategically selected outgroups (other strepsirrhines, higher primates)

  • Sample multiple individuals per species to capture intraspecific variation

  • Consider including ancient DNA if available from subfossil lemurs

• Alignment optimization:

  • Use translation-aware alignment algorithms for coding sequences

  • Manually inspect and refine alignments, particularly at indel boundaries

  • Test multiple alignment algorithms and parameters to evaluate alignment stability

  • Consider structure-based alignment using available crystal structures of MT-CO2

• Model selection approach:

  • Test multiple substitution models and select the best-fit model using AIC, BIC, or DT

  • Implement codon-based models for analyzing selection pressure

  • Test for site-specific rate heterogeneity using gamma-distributed rates

  • Consider mixture models that can detect distinct evolutionary categories

• Phylogenetic inference optimization:

  • Implement both maximum likelihood and Bayesian inference methods

  • Use partition models to allow different evolutionary parameters for different protein domains

  • Test for and account for compositional heterogeneity

  • Evaluate the impact of recombination using appropriate tests

• Selection pressure analysis:

  • Calculate dN/dS ratios to detect selective pressures

  • Implement site-specific models to identify functionally important residues

  • Apply branch-site models to detect lineage-specific selection patterns

  • Test specifically for selection differences between lemurs and other primates as suggested by previous research

• Rate variation analysis:

  • Test for significant evolutionary rate differences between lemur lineages

  • Compare lemur rates with those of higher primates to verify the previously reported nearly two-fold rate increase in higher primates

  • Investigate whether specific domains show different rate patterns

  • Correlate rate variations with ecological or life history traits

• Structural and functional context:

  • Map sites of interest (variable sites, selected sites) onto the MT-CO2 structure

  • Pay particular attention to positions 114 and 115, which show significant differences between higher primates and other mammals

  • Correlate evolutionary patterns with functional data from recombinant protein studies

These approaches enable rigorous evolutionary analyses of MT-CO2 that can contribute to understanding lemur phylogeny while providing insights into functional adaptation across primates .

What experimental design is optimal for comparing recombinant MT-CO2 properties across multiple primate species?

A robust experimental design for comparative analysis of recombinant MT-CO2 from multiple primate species, including Eulemur macaco, should incorporate these elements:

• Species selection strategy:

  • Include strategically selected primates representing major evolutionary lineages:

    • Lemurs (Eulemur macaco and at least one other lemur species)

    • Lorisiforms (e.g., slow loris)

    • New World monkeys (e.g., marmoset)

    • Old World monkeys (e.g., macaque)

    • Apes (e.g., human, chimpanzee)

  • This design enables testing of the hypothesis regarding accelerated evolution in higher primates

• Standardized molecular methods:

  • Use identical cloning strategies, expression vectors, and tags for all species

  • Express all proteins in the same host system under identical conditions

  • Purify all proteins using identical protocols to eliminate method-based variation

  • Process all species in parallel to minimize batch effects

• Comprehensive property assessment:

  • Measure multiple parameters for each recombinant protein:

    • Enzyme kinetics (kcat, KM) with cytochrome c substrate

    • Thermal stability (melting temperature)

    • pH optimum and stability

    • Redox potential of the CuA center

    • Inhibitor sensitivity profiles

    • Protein-protein interaction affinity with cytochrome c

• Cross-species interaction analyses:

  • Test each MT-CO2 protein with cytochrome c from multiple species

  • Create a matrix of cross-reactivity to identify species-specific adaptation

  • This approach can directly test the hypothesis about poor enzyme kinetics in cross-reactions between higher primates and other mammals

• Data analysis plan:

  • Implement multivariate statistical analyses to identify patterns across species

  • Use phylogenetic comparative methods to account for shared evolutionary history

  • Correlate functional differences with key sequence variations, particularly at positions 114 and 115

  • Perform principal component analysis to identify major sources of variation

• Structural investigation integration:

  • Determine or model the structure of each species' MT-CO2

  • Map functional differences to structural variations

  • Use molecular dynamics simulations to predict the impact of species-specific variations

• Validation strategy:

  • Create site-directed mutants to verify the impact of specific residues

  • Develop chimeric proteins to identify domains responsible for species differences

  • Validate in vitro findings with available physiological data from each species

This comprehensive experimental design enables rigorous testing of evolutionary hypotheses regarding MT-CO2 function across primates while controlling for methodological variables that could confound comparative analyses .