Recombinant Pomoxis nigromaculatus cytochrome b (mt-cyb) is a truncated protein derived from the mitochondrial cytochrome b gene (MT-CYB) of the black crappie fish. Engineered for research purposes, this recombinant variant is expressed in Escherichia coli with an N-terminal His tag to facilitate purification and downstream applications . The protein spans residues 1–85 of the full-length cytochrome b, which is a critical subunit of mitochondrial complex III in the respiratory chain .
Cytochrome b is the sole mitochondrially encoded subunit of complex III, which mediates electron transfer from ubiquinol to cytochrome c during oxidative phosphorylation . Mutations in MT-CYB disrupt complex III activity, leading to impaired ATP production and mitochondrial disorders . The recombinant Pomoxis nigromaculatus variant serves as a model to study:
Structural dynamics of cytochrome b in complex III.
Mutational effects on electron transport efficiency.
A novel missense mutation (m.14757T>C) in MT-CYB was identified in a patient with DCM, highlighting cytochrome b’s role in maintaining cardiac mitochondrial function. The mutation replaced methionine with threonine (M4T), disrupting hydrophobic interactions critical for protein stability .
Polymorphisms in MT-CYB (e.g., rs527236194, rs28357373) were linked to reduced sperm motility in subfertile males. These variants may impair mitochondrial ATP production in sperm, affecting motility .
A heteroplasmic mutation (m.14864T>C) caused a cysteine-to-arginine substitution (C40R), leading to MELAS-like symptoms (migraines, epilepsy, neuropathy). This underscores cytochrome b’s broader role in neuroenergetic pathways .
The recombinant protein is purified via affinity chromatography due to its His tag, ensuring >85% purity as confirmed by SDS-PAGE . Its expression in E. coli allows scalable production for:
Pomoxis nigromaculatus Cytochrome b (mt-cyb) is a mitochondrial protein encoded by the mt-cyb gene (also known as cob, cytb, or mtcyb) in Black crappie fish . It functions as a critical component of Complex III (ubiquinol-cytochrome c reductase) in the mitochondrial electron transport chain, catalyzing electron transfer from ubiquinol to cytochrome c . This process is essential for cellular respiration and ATP production.
The protein consists of 380 amino acids in its full length, with the amino acid sequence of the expression region (1-85) being: "LTGLFLAMHYTSDIATAFSSVAHICRDVNYGWLIRNIHANGASFFFICIYLHIGRGLYYGSYLYKETWNVGVVLLLLVMMTAFVG" . As a subunit of Complex III, it is also referred to as Complex III subunit 3, Complex III subunit III, or Cytochrome b-c1 complex subunit 3 .
Recombinant Pomoxis nigromaculatus Cytochrome b is produced through molecular cloning and expression systems rather than being isolated directly from Black crappie tissue. The recombinant protein maintains the same amino acid sequence as the native protein, but may include additional tag sequences to facilitate purification and detection in experimental settings .
The recombinant protein is typically produced with specific tag information that is determined during the production process and is stored in a Tris-based buffer with 50% glycerol, optimized for protein stability . While the native protein exists within the mitochondrial membrane environment, the recombinant version is isolated and purified, making it suitable for various in vitro applications, including antibody production, protein-protein interaction studies, and structural analyses.
For optimal stability and activity retention, recombinant Pomoxis nigromaculatus Cytochrome b should be stored at -20°C, with more extended storage recommended at either -20°C or -80°C . The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which helps maintain protein stability during freeze-thaw cycles.
To minimize protein degradation, repeated freezing and thawing should be avoided. When working with the protein, it is advisable to prepare small working aliquots that can be stored at 4°C for up to one week . This approach preserves the integrity of the remaining stock while providing convenient access for ongoing experiments.
When using recombinant Cytochrome b for phylogenetic studies, researchers should follow this methodological approach:
Sequence Alignment: Use software such as MAFFT (version 7.475 or later) to align cytochrome b nucleotide sequences from target species . For comprehensive analysis, include both the species of interest and appropriate outgroups.
Model Selection: Employ ModelFinder in IQ-TREE to identify the best-fit nucleotide model for your cytochrome b dataset . This ensures appropriate evolutionary model selection for accurate phylogenetic reconstruction.
Tree Construction: Implement both Maximum Likelihood (ML) and Bayesian Inference (BI) approaches for robust phylogenetic tree reconstruction:
Visualization: Use FigTree or similar software to visualize and interpret the resulting phylogenetic trees .
This methodological approach provides a comprehensive framework for utilizing cytochrome b in evolutionary studies, as demonstrated in research on various species including marine bivalves .
To effectively implement recombinant Cytochrome b in ELISA-based detection systems, researchers should consider the following methodological approach:
Protein Preparation: Utilize the recombinant protein (supplied at 50 μg concentration) directly from storage after allowing it to reach room temperature . Avoid repeated freeze-thaw cycles by working with small aliquots.
Coating Optimization: Determine optimal coating concentration through titration experiments, typically starting with 1-10 μg/mL of the recombinant protein in carbonate buffer (pH 9.6), and incubating overnight at 4°C.
Blocking Protocol: Block non-specific binding sites using 3-5% BSA or non-fat dry milk in PBS or TBS with 0.05% Tween-20 for 1-2 hours at room temperature.
Antibody Incubation: If detecting antibodies against Cytochrome b, dilute test samples appropriately and incubate for 1-2 hours at room temperature or overnight at 4°C, followed by species-appropriate labeled secondary antibody incubation.
Detection and Quantification: Develop using appropriate substrate (TMB for HRP-conjugated antibodies) and measure absorbance at the recommended wavelength. Generate standard curves using known positive controls for quantitative analysis.
This protocol should be optimized for each specific application, with particular attention to antigen coating concentration, sample dilution, and incubation times to achieve maximum sensitivity and specificity.
Several complementary techniques can be employed to analyze structural changes in Cytochrome b resulting from mutations:
Computational Prediction Methods:
Homology modeling using tools like SWISS-MODEL or Phyre2 to predict protein structure
Molecular dynamics simulations to analyze potential conformational changes
In silico mutagenesis followed by energy minimization to predict structural stability changes
Experimental Structural Analysis:
Circular dichroism (CD) spectroscopy to assess secondary structure changes
X-ray crystallography for high-resolution structural determination
Nuclear magnetic resonance (NMR) spectroscopy for solution structure analysis
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to examine protein dynamics and solvent accessibility
Functional Assays:
Enzyme kinetics measurements to determine changes in catalytic efficiency
Thermal stability assays to assess protein folding and stability
Protein-protein interaction analyses to evaluate complex formation capacity
When investigating mutations like those described in clinical cases (e.g., m.14864 T>C mutation that changes a conserved cysteine to arginine at position 40) , researchers should combine structural predictions with experimental validation to comprehensively characterize the impact of amino acid substitutions on protein structure and function.
Cytochrome b serves as a reliable molecular marker for species identification and phylogenetic analysis due to several key characteristics:
Evolution Rate: Mitochondrial genes like cytochrome b evolve 7-12 times faster than their nuclear counterparts, making them suitable for resolving relationships between closely related species . This higher mutation rate provides sufficient variation for distinguishing between recently diverged lineages.
Conservation Pattern: Cytochrome b exhibits a pattern of both conserved and variable regions. Functionally critical regions remain conserved across distant taxa, while other regions accumulate mutations at predictable rates, allowing for comparison across evolutionary distances .
Phylogenetic Resolution: Research has demonstrated cytochrome b's utility in resolving relationships at various taxonomic levels. For example, phylogenetic trees constructed using cytochrome b sequences have successfully established relationships within families like Cardiidae with strong statistical support (bootstrap values ≥90, Bayesian posterior probability = 1) .
Limitations: Despite its utility, cytochrome b analysis alone may have limitations. Multiple studies suggest that combining cytochrome b with other markers provides more robust phylogenetic signals than single-gene approaches . Additionally, phenomena like horizontal gene transfer, introgression, and incomplete lineage sorting can complicate interpretations based solely on mitochondrial sequences.
For optimal results, researchers should consider using cytochrome b in conjunction with nuclear markers to overcome potential biases introduced by maternal inheritance and to provide complementary evolutionary information.
Evidence for positive selection on the Cytochrome b gene has been documented in several species, with specific methodological approaches identifying adaptive evolution:
This evidence suggests that cytochrome b is not merely a neutral marker but has played an adaptive role in species evolution, particularly in response to environmental challenges affecting mitochondrial function.
Cytochrome b pseudogenes can significantly impact phylogenetic analyses, creating challenges that require specific methodological approaches to mitigate:
Impact on Phylogenetic Analysis:
Erroneous Relationships: Nuclear pseudogenes (numts) of cytochrome b can be inadvertently amplified and included in analyses, leading to incorrect phylogenetic reconstructions.
Evolutionary Rate Discrepancies: Pseudogenes evolve differently than functional genes. Research has shown that mitochondrial genes evolve 7-12 times faster than their nuclear counterparts, creating divergent evolutionary signals .
Hybrid Signals: Pseudogenes may originate from highly divergent mitochondrial lineages, as observed in chamois species, potentially reflecting ancient hybridization events that complicate phylogenetic interpretations .
Mitigation Methods:
Sequence Verification: Carefully examine sequences for frameshift mutations and premature stop codons, which are characteristic of pseudogenes. In the case of chamois, researchers identified pseudogenes containing a frameshift and a stop codon .
RT-PCR Validation: Use reverse transcription PCR to specifically amplify expressed genes rather than pseudogenes.
Mitochondrial Enrichment: Perform mitochondrial isolation before DNA extraction to reduce nuclear DNA contamination.
Phylogenetic Analysis of Both Sequences: When pseudogenes are detected, analyze both functional and pseudogene sequences as independent evolutionary units. This approach can provide insights into gene evolution and potential hybridization events.
Multiple Markers: Use additional mitochondrial and nuclear markers to corroborate phylogenetic patterns and identify discordant signals.
By implementing these strategies, researchers can minimize the confounding effects of pseudogenes and potentially leverage their presence to gain additional evolutionary insights, as demonstrated in studies of Rupicapra species .
Several mutations in the mitochondrial DNA cytochrome b gene (MTCYB) have been associated with human diseases, with specific molecular mechanisms:
MELAS-Related Mutations:
The novel mutation m.14864 T>C changes a highly conserved cysteine to arginine at amino acid position 40 of cytochrome b and has been associated with a clinical picture resembling MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) .
This mutation was found to be heteroplasmic (present in varying proportions) in muscle, blood, fibroblasts, and urinary sediment from the patient but absent in tissues from the asymptomatic mother .
The molecular mechanism likely involves disruption of electron transport within Complex III, leading to impaired oxidative phosphorylation and increased reactive oxygen species production.
Isolated Mitochondrial Myopathy:
Multisystem Disorders:
Molecular Consequences:
Most pathogenic mutations disrupt the proper folding or function of cytochrome b within Complex III (ubiquinol-cytochrome c reductase).
This disruption impairs electron transfer from ubiquinol to cytochrome c, reducing ATP production and increasing oxidative stress.
The heteroplasmic nature of many mutations creates a threshold effect, where clinical symptoms manifest only when the mutation burden exceeds a tissue-specific threshold.
These findings demonstrate that MTCYB must be included in the analysis of mitochondrial DNA genes associated with MELAS and related mitochondrial disorders .
Differentiating between pathogenic mutations and neutral polymorphisms in the Cytochrome b gene requires a multi-faceted approach:
Conservation Analysis:
Assess evolutionary conservation of the affected amino acid position across species
Highly conserved residues (like the cysteine at position 40 changed in the m.14864 T>C mutation) are more likely to be functionally important and thus pathogenic when altered
Utilize conservation scoring tools like ConSurf or GERP++ to quantify evolutionary constraint
Functional Domain Assessment:
Determine if the mutation affects known functional domains within cytochrome b
Mutations in regions involved in ubiquinol binding, electron transfer, or protein-protein interactions within Complex III are more likely to be pathogenic
Use protein structure databases and visualization tools to map mutations onto 3D structures
Heteroplasmy Quantification:
Biochemical Studies:
Assess Complex III activity in patient samples or in vitro models
Measure electron transfer rates and reactive oxygen species production
Determine the impact on mitochondrial membrane potential and ATP synthesis
Clinical Correlation:
Establish clear association between the mutation and clinical phenotype
Perform family studies to demonstrate maternal inheritance patterns
Compare with previously reported cases with similar mutations or clinical presentations
Prediction Algorithms:
Utilize computational tools specifically designed for mitochondrial variants (MitoTIP, MitImpact)
Combine multiple prediction algorithms to improve accuracy
By integrating these approaches, researchers can build a comprehensive evidence base to classify Cytochrome b variants according to their likelihood of pathogenicity, which is essential for accurate diagnosis and genetic counseling.
Positive selection signals in Cytochrome b provide valuable insights into environmental adaptation that can be leveraged through several methodological approaches:
Correlative Environmental Analysis:
Identify specific amino acid sites under positive selection using programs like CodeML within the PAML package
Correlate these sites with environmental variables (temperature, oxygen levels, depth, salinity) across species' habitats
Apply statistical methods such as canonical correlation analysis or environmental association analysis to quantify relationships
Experimental Validation of Adaptive Hypotheses:
Express recombinant Cytochrome b variants with selected mutations in cellular systems
Measure functional parameters (electron transfer rates, oxygen consumption, ROS production) under varying environmental conditions
Compare performance metrics between variants to assess adaptive advantages
Comparative Analysis Across Environmental Gradients:
Sample populations across environmental gradients (e.g., depth, latitude, temperature)
Sequence Cytochrome b and analyze selection patterns specific to environmental transitions
Construct selection landscapes that map fitness effects of mutations across environmental parameters
Integration with -Omics Data:
Combine Cytochrome b selection analysis with transcriptomics and proteomics data
Identify co-evolving genes within the mitochondrial and nuclear genomes
Construct adaptive networks that reveal system-level responses to environmental challenges
Temporal Analysis of Selection:
Use ancient DNA techniques to analyze historical samples of the same species
Quantify changes in selection pressure over time in relation to documented environmental changes
Model future evolutionary trajectories under climate change scenarios
This integrative approach enables researchers to move beyond merely detecting positive selection to understanding its functional significance in environmental adaptation. The evidence of positive selection on the cytochrome b gene in marine bivalves like Keenocardium buelowi suggests this methodology could be particularly valuable for studying adaptation to changing marine environments.
The integration of cytochrome b data with other molecular markers for comprehensive phylogenomic analyses has advanced significantly with several cutting-edge methodological approaches:
Multi-gene Concatenation and Partitioning:
Combine cytochrome b with other mitochondrial genes (like cox1) and nuclear markers
Implement partitioned models that allow different evolutionary parameters for each gene region
Use software like PartitionFinder to determine optimal partitioning schemes
This approach provides stronger phylogenetic signals than single-gene analyses, as demonstrated in studies of Cardiidae
Species Tree Methods:
Apply coalescent-based methods (ASTRAL, StarBEAST2) that account for gene tree discordance
These methods recognize that cytochrome b (as a mitochondrial marker) represents only the maternal evolutionary history
Incorporate nuclear markers to capture biparental inheritance patterns
This approach addresses limitations stemming from horizontal gene transfer, introgression, and incomplete lineage sorting
Bayesian Total Evidence Approaches:
Integrate molecular data (including cytochrome b) with morphological characters and fossil calibrations
Implement tip-dating methods for time-calibrated phylogenies
Use programs like MrBayes or BEAST2 with appropriate evolutionary models
This approach provides robust estimates of divergence times, as seen in the dating of lineage splits (e.g., 44.5 MYA between K. buelowi and V. flavum)
Phylogenetic Network Analysis:
Integrative Taxonomic Approaches:
Combine cytochrome b data with genomic, morphological, and ecological information
Apply machine learning algorithms to identify diagnostic character combinations
This approach provides a more comprehensive understanding of species boundaries and relationships
These advanced techniques address the limitations of cytochrome b as a single marker while leveraging its rapid evolution rate to resolve relationships between closely related species. The integration of multiple data types produces more robust phylogenies that better reflect the complex evolutionary history of organisms.