Recombinant Cratogeomys castanops Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c oxidase subunit 2 (MT-CO2) and what is its function in Cratogeomys castanops?

Cytochrome c oxidase subunit 2 (MT-CO2) is a critical component of Complex IV of the mitochondrial respiratory chain in Cratogeomys castanops (Yellow-faced pocket gopher). This subunit is encoded by the mitochondrial genome (hence the MT prefix) and plays a crucial role in the terminal step of the electron transport chain, where it helps catalyze the reduction of oxygen to water while contributing to the proton gradient necessary for ATP synthesis. In Cratogeomys castanops, MT-CO2 consists of 227 amino acids and contains binding sites for both copper atoms and cytochrome c, making it essential for electron transfer during oxidative phosphorylation .

The functional domains of MT-CO2 include copper-binding sites that facilitate electron transfer, cytochrome c docking regions that enable interaction with this mobile electron carrier, and transmembrane domains that anchor the protein in the inner mitochondrial membrane. As a core subunit of cytochrome c oxidase, MT-CO2 plays an essential role in the bioenergetic function of mitochondria and is thus critical for cellular respiration and energy production.

How does MT-CO2 sequence data contribute to phylogenetic studies of pocket gophers?

MT-CO2 sequence data has proven invaluable in resolving evolutionary relationships among pocket gopher species. Mitochondrial DNA sequences evolve at a relatively rapid rate compared to nuclear genes, making them useful markers for distinguishing recently diverged species and populations. Studies utilizing MT-CO2 sequence data have successfully:

• Resolved relationships among members of the castanops species group within the genus Cratogeomys

• Identified five geographically distinct clades within the gymnurus species group

• Challenged existing taxonomic classifications by revealing discrepancies between genetic data and morphology-based classifications

For example, phylogeographic analysis using 1133 base pairs from the cytochrome b gene (another mitochondrial marker often analyzed alongside MT-CO2) showed that several nominal species (C. gymnurus and C. tylorhinus) are scattered among distinct genetic clades, suggesting the need for taxonomic revision . Additionally, peripherally isolated species (C. fumosus, C. neglectus, and C. zinseri) do not appear to be genetically distinct from other gymnurus species group taxa, further highlighting the value of molecular data in understanding pocket gopher systematics.

What expression systems are optimal for producing recombinant Cratogeomys castanops MT-CO2?

The choice of expression system for recombinant MT-CO2 depends on research objectives, with several systems offering different advantages:

Table 1: Comparison of Expression Systems for Recombinant MT-CO2

For bacterial expression, optimizing solubility typically requires fusion tags (His, GST, or MBP) and specialized protocols for membrane protein extraction. Yeast expression benefits from inducible promoters (AOX1 for P. pastoris, GAL1 for S. cerevisiae) and signal sequences for proper targeting. Each system requires tailored methodologies for optimal expression of this challenging membrane protein.

What buffer conditions and storage protocols are recommended for maintaining MT-CO2 stability?

Proper buffer formulation is critical for maintaining the stability and functionality of recombinant MT-CO2. Based on the product information and experimental experience with similar mitochondrial membrane proteins:

Extraction and Solubilization Buffers:

• pH range: 7.2-7.5 (typically HEPES or Tris-based)

• Salt concentration: 150-300 mM NaCl

• Glycerol: 10-20% to enhance stability

• Detergents: Mild non-ionic detergents like n-Dodecyl β-D-maltoside (DDM) at 0.5-1% for extraction

Storage Conditions:

• Short-term (up to one week): Store working aliquots at 4°C

• Long-term: Store at -20°C; for extended storage, -80°C is recommended

• Buffer: Tris-based buffer with 50% glycerol as indicated in the product information

• Avoid repeated freezing and thawing as this significantly reduces activity

The optimal storage buffer includes 50% glycerol in a Tris-based buffer that has been optimized for the specific protein . Creating small single-use aliquots minimizes freeze-thaw cycles that can lead to protein denaturation and activity loss.

What techniques are most effective for verifying the purity and identity of recombinant MT-CO2?

Multiple complementary techniques should be employed to verify the purity and identity of recombinant MT-CO2:

SDS-PAGE and Western Blotting:

• Confirms expected molecular weight (~26 kDa for mature protein)

• Assess purity through Coomassie or silver staining

• Western blotting with anti-MT-CO2 antibodies confirms identity

Mass Spectrometry:

• Peptide mass fingerprinting to confirm protein identity

• Intact protein mass analysis to verify full-length expression

• Can detect post-translational modifications or truncations

Spectroscopic Analysis:

• UV-visible spectroscopy to assess heme incorporation if applicable

• Circular dichroism to evaluate secondary structure content

• Fluorescence spectroscopy to monitor tertiary structure

Functional Verification:

• Copper binding assays using atomic absorption spectroscopy

• Cytochrome c binding assays using surface plasmon resonance

• Activity assays measuring electron transfer capabilities

A rigorous quality control protocol would combine these approaches to ensure both purity and functional integrity before proceeding with experimental applications.

How does cytochrome c interact with MT-CO2 during electron transfer?

The interaction between cytochrome c and MT-CO2 is a critical step in the electron transport chain. MT-CO2 contains specific binding sites for cytochrome c that facilitate efficient electron transfer:

• Binding interface: The interaction primarily involves electrostatic contacts between positively charged residues on cytochrome c and negatively charged residues on MT-CO2

• Electron transfer mechanism: Electrons move from reduced cytochrome c to the CuA center in MT-CO2, which then transfers them to heme a in subunit 1

• Binding kinetics: The interaction is characterized by rapid association and dissociation rates, allowing for efficient electron shuttling

Recent research has revealed that cytochrome c plays both functional and structural roles in cytochrome c oxidase assembly and stability. Beyond its electron carrier function, cytochrome c appears necessary for proper assembly of the COX complex in yeast studies . Experiments with inactive cytochrome c mutants demonstrated that even catalytically inactive cytochrome c can rescue COX assembly defects, suggesting a structural role independent of electron transfer capability .

Additionally, the interaction between cytochrome c and MT-CO2 is not strictly stoichiometric - studies in yeast showed that cytochrome c concentrations as low as 12% of normal levels can support up to 70% of normal COX expression, indicating that the structural role requires substoichiometric amounts of cytochrome c .

What role does MT-CO2 play in the assembly of the complete cytochrome c oxidase complex?

MT-CO2 plays a critical role in cytochrome c oxidase biogenesis, serving as an essential component for complex assembly and stability:

• Assembly sequence: While COX1 acts as the primary seed for assembly, MT-CO2 incorporation is a crucial early step in the assembly pathway

• Copper incorporation: MT-CO2 requires proper metallation of its CuA center, facilitated by specialized copper chaperones

• Interface formation: MT-CO2 forms critical interfaces with other subunits, particularly COX1 and COX3

The assembly of cytochrome c oxidase is a highly regulated process involving numerous assembly factors. Recent research has revealed sophisticated regulatory mechanisms controlling the concerted accumulation of COX subunits. In yeast, the synthesis of Cox1p (the equivalent of mammalian MT-CO1) is regulated by the availability of its assembly partners through a feedback mechanism involving Mss51p and Cox14p .

The biogenesis pathway also requires the correct import and folding of copper chaperones that contain characteristic twin CX9C motifs. These proteins are imported into the mitochondrial intermembrane space via the Mia40p pathway, which functions as a disulfide relay system catalyzing import through an oxidative folding mechanism . This process is connected to the respiratory chain through electron transfer from Erv1p to molecular oxygen via interaction with cytochrome c .

How can researchers experimentally distinguish between MT-CO2 expression defects versus assembly problems?

Distinguishing between expression defects and assembly problems requires systematic approaches:

Methodological Approach 1: Protein Level Analysis

• Western blotting of total cell lysates versus purified mitochondria

• Pulse-chase labeling to track synthesis and turnover rates

• Comparison of mRNA levels (qPCR) with protein levels to identify translational issues

Methodological Approach 2: Assembly State Analysis

• Blue Native PAGE to visualize assembly intermediates

• Immunoprecipitation with antibodies against different COX subunits

• Sucrose gradient ultracentrifugation to separate assembled versus unassembled subunits

Methodological Approach 3: Functional Complementation

• Expression of wild-type MT-CO2 in deficient systems

• Site-directed mutagenesis to identify critical residues

• Heterologous expression studies

An example of such experimental distinction comes from studies in yeast, where researchers demonstrated that cytochrome c mutants have characteristics of true COX assembly mutants, including lower levels of heme aa3 and reduced steady-state levels of COX mitochondrial subunits Cox1p, Cox2p, and Cox3p . Like other COX assembly mutants, failure of assembly in cytochrome c mutants results in down-regulation of Cox1p synthesis through a specific mechanism involving Mss51p and Cox14p .

How has MT-CO2 sequence data informed our understanding of pocket gopher evolution?

MT-CO2 sequence analysis has significantly advanced our understanding of pocket gopher evolution through several critical insights:

• Phylogenetic resolution: MT-CO2 sequences have helped resolve relationships within the closely related pocket gopher genera Pappogeomys and Cratogeomys

• Taxonomic revision: Molecular data has revealed five geographically distinct clades within the gymnurus species group that do not align with current taxonomy

• Biogeographic hypotheses: MT-CO2 data has supported the development of historical biogeographic hypotheses that can be further tested with nuclear DNA data

Analysis of 1133 base pairs from the cytochrome b gene (often studied alongside MT-CO2) revealed that members of the nominal species C. gymnurus and C. tylorhinus are scattered among these genetically distinct clades, indicating significant discordance between genetic data and morphology-based classifications . Furthermore, three peripherally isolated species (C. fumosus, C. neglectus, and C. zinseri) do not appear to be genetically distinct from other gymnurus species group taxa based on mitochondrial DNA evidence .

These findings have important implications for conservation and management, as they suggest that current taxonomic designations may not accurately reflect evolutionary history and genetic diversity in these species.

What methodological approaches are most effective for using MT-CO2 in phylogenetic studies?

Effective methodological approaches for MT-CO2 phylogenetic analysis include:

Sample Collection and DNA Extraction:

• Tissue sampling (typically muscle, liver, or ear clip)

• DNA extraction methods optimized for mitochondrial DNA

• Sample preservation protocols to maintain DNA integrity

PCR and Sequencing:

• Design of primers targeting conserved flanking regions

• Optimization of PCR conditions for high fidelity

• Bi-directional Sanger sequencing for accuracy

• Next-generation sequencing for population-level studies

Phylogenetic Analysis:

• Multiple sequence alignment with MUSCLE, MAFFT, or similar tools

• Model selection using AIC or BIC criteria

• Tree-building methods:

  • Maximum Likelihood (RAxML, IQ-TREE)

  • Bayesian Inference (MrBayes, BEAST)

  • Maximum Parsimony and Neighbor-Joining as complementary approaches

• Assessment of node support (bootstrap values, posterior probabilities)

Comparative Analysis:

• Integration with morphological data

• Comparison with nuclear gene phylogenies

• Molecular dating using fossil calibrations

• Tests for selection pressure (dN/dS ratios)

The complete mitochondrial sequence data analysis should be performed using rigorous methods, with appropriate outgroups and careful consideration of potential biases such as base composition heterogeneity or site saturation.

What evidence exists for adaptive evolution of MT-CO2 in pocket gophers?

While the specific search results don't directly address adaptive evolution of MT-CO2 in pocket gophers, we can outline methodological approaches to investigate this question:

Detecting Selection Signatures:

• Calculation of dN/dS ratios across the gene and at specific codons

• Branch-site models to identify episodic selection on particular lineages

• McDonald-Kreitman tests comparing polymorphism to divergence

• Phylogenetic ANOVA to correlate sequence variation with ecological factors

Functional Domain Analysis:

• Mapping selected sites onto structural models

• Comparison of selection patterns between functional domains

• Analysis of conservation at interaction interfaces

Ecological Correlations:

• Testing for correlations between sequence variation and:

  • Elevation gradients (hypoxia adaptation)

  • Thermal environments (metabolic adaptation)

  • Soil types (affecting burrowing energetics)

  • Geographic barriers (isolation and local adaptation)

Experimental Validation:

• Expression of variant proteins to assess functional differences

• Oxygen consumption measurements under different conditions

• Thermal stability assays of protein variants

What are common challenges in expressing and purifying recombinant MT-CO2?

As a mitochondrial membrane protein, MT-CO2 presents several challenges during recombinant expression and purification:

Expression Challenges:

• Membrane protein folding issues in heterologous systems

• Toxicity to host cells when overexpressed

• Codon bias affecting translation efficiency

• Post-translational modification differences

Purification Challenges:

• Detergent selection for efficient solubilization without denaturation

• Maintaining protein stability during purification steps

• Separating full-length protein from truncated products

• Achieving high purity without compromising activity

Methodological Solutions:

• Expression optimization:

  • Reduce expression temperature (16-18°C)

  • Use specialized expression hosts (C41/C43 E. coli strains)

  • Co-express chaperones to assist folding

  • Employ fusion tags that enhance solubility (MBP, SUMO)

• Purification strategies:

  • Screen multiple detergents at various concentrations

  • Include glycerol (10-20%) in all buffers

  • Add specific lipids that stabilize the protein

  • Use affinity chromatography followed by size exclusion

• Quality control:

  • Assess homogeneity by dynamic light scattering

  • Verify functional activity after each purification step

  • Monitor protein stability using thermal shift assays

When working with recombinant MT-CO2, researchers should consider storage in 50% glycerol and avoid repeated freeze-thaw cycles, as noted in the product information .

How can researchers overcome issues with antibody specificity for MT-CO2?

Antibody specificity issues are common when working with mitochondrial proteins like MT-CO2. Here are methodological approaches to overcome these challenges:

Common Specificity Problems:

• Cross-reactivity with homologous proteins from other species

• Non-specific binding to other mitochondrial proteins

• Epitope masking in native protein conformations

Mitigation Strategies:

• Antibody selection and validation:

  • Choose antibodies raised against species-specific regions

  • Validate with both positive controls (recombinant protein) and negative controls

  • Perform peptide competition assays to confirm specificity

  • Use multiple antibodies targeting different epitopes

• Sample preparation optimization:

  • Test different extraction buffers and detergents

  • Optimize protein denaturation conditions for Western blotting

  • Consider native versus denaturing conditions based on epitope accessibility

• Immunodetection protocol refinement:

  • Optimize blocking conditions (agent, time, temperature)

  • Titrate antibody concentrations to minimize background

  • Increase washing stringency to reduce non-specific binding

  • Consider signal amplification methods for low abundance proteins

• Alternative detection methods:

  • Mass spectrometry-based approaches for protein identification

  • Activity-based assays when antibody detection is problematic

  • Expression of tagged versions for detection via the tag

When interpreting results, researchers should always include appropriate controls and consider orthogonal methods to confirm antibody-based findings.

What experimental controls are essential when studying MT-CO2 function?

Robust experimental design for MT-CO2 functional studies requires comprehensive controls:

Positive Controls:

• Commercial cytochrome c oxidase preparations

• Native mitochondrial preparations containing endogenous COX

• Previously characterized recombinant MT-CO2 with verified activity

Negative Controls:

• Heat-inactivated enzyme preparations

• Specific inhibitors (potassium cyanide, sodium azide)

• Preparations lacking critical components (e.g., without cytochrome c)

• Buffer-only samples

Specificity Controls:

• Substrate specificity tests (reduced versus oxidized cytochrome c)

• Antibody specificity tests (pre-immune serum, isotype controls)

• Species specificity (comparing with MT-CO2 from related species)

Technical Controls:

• Instrument calibration standards

• Inter-assay standards to normalize between experiments

• Time-course measurements to ensure reaction linearity

• Concentration gradients to establish dose-response relationships

Experimental Design Considerations:

• Include biological replicates (n≥3) from independent protein preparations

• Perform technical replicates to assess measurement variability

• Randomize sample order to minimize systematic errors

• Consider blinding procedures where appropriate to prevent bias

Functional studies of MT-CO2 should pay particular attention to the role of cytochrome c, as research has shown that cytochrome c is required not only as an electron donor but also plays a structural role in COX assembly and stability . The interaction between cytochrome c and Erv1p is also important to consider, as it connects the disulfide relay import system with the mitochondrial respiratory chain .