Recombinant Megalops atlanticus Cytochrome c oxidase subunit 2 (mt-co2)

What is Megalops atlanticus Cytochrome c oxidase subunit 2?

Megalops atlanticus Cytochrome c oxidase subunit 2 (mt-co2) is a mitochondrial protein found in Atlantic tarpon (Megalops atlanticus, also known as Clupea gigantea). This protein is the second subunit of cytochrome c oxidase (Complex IV), a critical enzyme in the mitochondrial respiratory chain involved in electron transfer from cytochrome c to oxygen. The protein contains 74 amino acids with the sequence MAHPSQLGLQDAASPVMEELLHFHDHALMIVFLISTLVLYIIVAMVSTKLTDKYTIDSQEIEIVWTVLPAVILI and has a UniProt accession number of P29660 .

How does mt-co2 from Megalops atlanticus compare to mt-co2 in other species?

Cytochrome c oxidase subunit 2 is highly conserved across eukaryotes but shows species-specific variations. In most eukaryotes, including M. atlanticus, the mt-co2 gene is encoded by the mitochondrial genome. The protein typically contains a binuclear copper A center (CuA) located in a conserved cysteine loop that serves as a redox center essential for electron transfer. Unlike some plant species such as soybean, where the mitochondrial cox2 gene has been transferred to the nuclear genome and the mitochondrial version is silent, the M. atlanticus mt-co2 remains mitochondrially encoded and functional .

What is the structural composition of Megalops atlanticus mt-co2?

The Megalops atlanticus mt-co2 protein consists of 74 amino acids with a structure that includes transmembrane domains. Like other cytochrome c oxidase subunit 2 proteins, it likely contains N-terminal transmembrane alpha-helices and a copper A center (CuA), which serves as a redox center. The CuA center is typically located in a conserved cysteine loop at specific amino acid positions and requires a conserved histidine for proper function. This structure is critical for the protein's role in the electron transport chain and cellular respiration .

How is recombinant Megalops atlanticus mt-co2 typically produced for research purposes?

Recombinant Megalops atlanticus mt-co2 is typically produced using standard molecular cloning techniques. The process involves:

• Isolating mitochondrial DNA from M. atlanticus tissue

• Amplifying the mt-co2 gene using PCR with specific primers

• Cloning the gene into an appropriate expression vector

• Transforming the construct into a suitable expression system (commonly E. coli, yeast, or insect cells)

• Inducing protein expression

• Purifying the recombinant protein using affinity chromatography

The recombinant protein is typically stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage to maintain stability .

What are the common applications of Megalops atlanticus mt-co2 in research?

Megalops atlanticus mt-co2 is utilized in various research contexts including:

• Evolutionary studies to understand phylogenetic relationships among fish species

• Biomarker development for population genetics and conservation biology

• Structural and functional analysis of mitochondrial respiratory complexes

• Comparative biochemistry of electron transport chains across species

• Development of immunological assays such as ELISA for detecting specific antibodies or antigens

• Molecular ecological studies of Atlantic tarpon populations

How can researchers differentiate between authentic Megalops atlanticus mt-co2 and potential contamination in experimental settings?

Differentiating authentic Megalops atlanticus mt-co2 from contamination requires a multi-faceted approach:

• Sequence verification: Complete sequencing of the gene or protein to confirm its identity against the known sequence (UniProt P29660)

• Mass spectrometry: Utilizing techniques such as MALDI-TOF to verify the exact molecular weight and peptide fingerprint

• Immunological verification: Using specific antibodies raised against M. atlanticus mt-co2 in Western blots

• Functional assays: Measuring the catalytic activity specific to cytochrome c oxidase

• Genetic markers: Using the 15 polymorphic microsatellite DNA loci identified for M. atlanticus to authenticate the species origin

Additionally, researchers should implement rigorous controls, including:

• Negative controls from closely related species

• Positive controls with verified M. atlanticus mt-co2

• Blank samples to detect reagent contamination

What are the implications of genetic variation in mt-co2 for Atlantic tarpon population studies?

Genetic variation in mt-co2 has significant implications for Atlantic tarpon population studies:

• Population structure assessment: Variations in mt-co2 sequences can help delineate distinct populations or subspecies

• Evolutionary history reconstruction: As a mitochondrial gene, mt-co2 can reveal maternal lineages and historical population bottlenecks

• Conservation management: Genetic diversity metrics derived from mt-co2 and nuclear markers provide insights for conservation strategies

• Adaptation signatures: Selection pressure analysis on mt-co2 can reveal adaptations to different environments

The microsatellite DNA analysis of M. atlanticus from Tampa Bay, Florida showed high polymorphism with an average of 7.7 alleles per locus (ranging from 2 to 24 alleles), demonstrating substantial genetic diversity. This diversity suggests that mt-co2 and associated genetic markers could be powerful tools for population studies .

How does the function of mt-co2 in Megalops atlanticus compare with nuclear-encoded versions in other species?

The function of mitochondrially encoded mt-co2 in Megalops atlanticus can be compared with nuclear-encoded versions (such as in soybean) through several aspects:

• Protein targeting and import: Nuclear-encoded versions typically contain an N-terminal extension that functions as a mitochondrial targeting sequence, which is cleaved upon import. This feature is absent in mitochondrially encoded versions.

• Post-transcriptional modifications: Mitochondrially encoded genes often undergo RNA editing, particularly C-to-U editing, which is not typically required for nuclear-encoded versions.

• Expression regulation: Nuclear-encoded versions are subject to nuclear transcriptional regulation and cytoplasmic translation, whereas mitochondrially encoded versions follow mitochondria-specific expression patterns.

• Evolutionary constraints: The transfer of genes from mitochondria to nucleus, as observed in soybean, represents an ongoing evolutionary process that affects protein structure and function.

• Functional adaptation: Despite these differences, both versions must maintain the essential electron transfer function within Complex IV, suggesting convergent constraints on functional domains.

Research indicates that in some species like soybean, the nuclear-encoded cox2 evolved from a mitochondrial gene transfer event via a C-to-U edited RNA intermediate, while in M. atlanticus, the gene remains mitochondrially encoded .

What are the optimal conditions for storing and handling recombinant Megalops atlanticus mt-co2?

The optimal conditions for storing and handling recombinant Megalops atlanticus mt-co2 involve several critical parameters:

• Storage temperature: Store at -20°C for regular use, and at -80°C for long-term preservation

• Buffer composition: Use Tris-based buffer with 50% glycerol, optimized for protein stability

• Aliquoting strategy: Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Working temperature: Maintain at 4°C for up to one week during active experimentation

• Thawing procedure: Thaw slowly on ice to prevent protein denaturation

• Handling precautions: Use non-metallic implements to avoid metal ion contamination that could affect the copper centers

Repeated freezing and thawing should be avoided as it significantly reduces protein activity. For experiments requiring extended use, it is recommended to keep working aliquots at 4°C for up to one week rather than repeatedly freezing and thawing the main stock .

What methodologies are recommended for studying the enzymatic activity of recombinant Megalops atlanticus mt-co2?

To study the enzymatic activity of recombinant Megalops atlanticus mt-co2 as part of cytochrome c oxidase, researchers should consider these methodological approaches:

• Spectrophotometric assays: Measure the oxidation of reduced cytochrome c at 550 nm, with activity calculated as the first-order rate constant.

• Oxygen consumption measurement: Use oxygen electrodes or optical sensors to quantify oxygen consumption rates in reconstituted systems.

• Electron transfer kinetics: Employ stopped-flow spectroscopy to determine electron transfer rates between cytochrome c and the copper A center.

• Proton pumping assays: Measure pH changes or use pH-sensitive fluorescent probes to detect proton translocation activity.

• Reconstitution in liposomes: Incorporate the protein into phospholipid vesicles to study its function in a membrane-like environment.

• Protein-protein interaction studies: Use techniques such as co-immunoprecipitation or crosslinking to identify interactions with other respiratory complex subunits.

Standard reaction conditions typically include:

• pH 7.0-7.4 buffer (commonly HEPES or phosphate)

• Temperature range of 25-37°C

• Presence of appropriate detergents for membrane protein solubilization

• Reduced cytochrome c as electron donor

• Oxygen as terminal electron acceptor

How can researchers address potential discrepancies in mt-co2 sequence data from different populations of Megalops atlanticus?

Addressing discrepancies in mt-co2 sequence data from different populations requires a systematic approach:

• Quality control protocols:

  • Implement bidirectional sequencing to verify accuracy

  • Use high-fidelity polymerases to minimize PCR errors

  • Establish minimum quality score thresholds for sequence acceptance

• Statistical analysis of variation:

  • Calculate nucleotide diversity (π) and haplotype diversity (Hd)

  • Perform tests for selective neutrality (Tajima's D, Fu's Fs)

  • Use analysis of molecular variance (AMOVA) to partition variation within and between populations

• Phylogenetic methods:

  • Construct haplotype networks to visualize relationships

  • Apply appropriate evolutionary models for phylogenetic reconstruction

  • Conduct bootstrap analyses to assess tree topology confidence

• Population genetics analysis:

  • Test for Hardy-Weinberg equilibrium across markers

  • Assess linkage disequilibrium between loci

  • Calculate fixation indices (FST) to quantify population differentiation

• Reconciliation approaches:

  • Consider heteroplasmy (multiple mitochondrial genomes within individuals)

  • Account for potential numts (nuclear mitochondrial DNA segments)

  • Evaluate RNA editing that might affect coding sequences

The microsatellite analysis of M. atlanticus demonstrated that one locus deviated significantly from Hardy-Weinberg equilibrium, illustrating the importance of thorough population genetic analysis when interpreting sequence variation data .

What bioinformatic tools are most appropriate for analyzing mt-co2 protein structure and function?

When analyzing mt-co2 protein structure and function, researchers should utilize these specialized bioinformatic tools:

• Sequence analysis tools:

  • BLAST/HMMER: For homology searches

  • Clustal Omega/MUSCLE: For multiple sequence alignment

  • MEGA/PAML: For evolutionary analysis and selection detection

• Structural prediction software:

  • AlphaFold/RoseTTAFold: For ab initio protein structure prediction

  • SWISS-MODEL/Phyre2: For homology-based structural modeling

  • ProCheck/QMEAN: For structural quality assessment

• Functional prediction tools:

  • InterProScan: For domain and motif identification

  • ConSurf: For conservation analysis and functional residue prediction

  • COACH/COFACTOR: For ligand-binding site prediction

• Molecular dynamics:

  • GROMACS/NAMD: For simulating protein dynamics in membrane environments

  • AMBER/CHARMM: For force field calculations

  • VMD/PyMOL: For visualization and analysis

• Systems biology approaches:

  • STRING/BioGRID: For protein-protein interaction prediction

  • Cytoscape: For network visualization and analysis

  • KEGG/Reactome: For pathway integration

For mt-co2 specifically, tools that account for transmembrane domains and metal-binding sites are particularly valuable due to the protein's role in the inner mitochondrial membrane and its copper-binding properties .

How can recombinant Megalops atlanticus mt-co2 be utilized in comparative studies of mitochondrial evolution?

Recombinant Megalops atlanticus mt-co2 serves as a valuable tool in comparative studies of mitochondrial evolution through several applications:

• Phylogenetic marker analysis:

  • The conserved nature of mt-co2 makes it useful for resolving deeper evolutionary relationships among fish taxa

  • Comparison of synonymous vs. non-synonymous substitution rates can reveal selection patterns

• Functional evolution studies:

  • Enzymatic assays comparing recombinant mt-co2 from different species can reveal functional adaptations

  • Site-directed mutagenesis to recreate ancestral sequences allows testing of evolutionary hypotheses

• Mitochondrial gene transfer research:

  • Unlike in some plants where cox2 has transferred to the nuclear genome, M. atlanticus retains mitochondrial encoding

  • This provides a comparative system to study the mechanisms and consequences of mitochondrial gene transfer

• Coevolution of nuclear and mitochondrial genomes:

  • Interactions between mt-co2 and nuclear-encoded subunits of Complex IV reveal coevolutionary constraints

  • These interactions can be studied using recombinant proteins in reconstitution experiments

• Adaptive evolution in aquatic environments:

  • Comparing mt-co2 from species in different aquatic environments can reveal adaptations to oxygen availability

  • Functional assays under varying oxygen tensions can test hypotheses about environmental adaptation

The study of mt-co2 evolution particularly benefits from comparing species like M. atlanticus (mitochondrial encoding) with species like soybean (nuclear encoding) to understand the evolutionary transition and its functional consequences .

What role does mt-co2 research play in conservation efforts for Atlantic tarpon populations?

Research on Megalops atlanticus mt-co2 contributes significantly to conservation efforts through multiple avenues:

• Population genetic structure assessment:

  • mt-co2 sequences help identify distinct management units for conservation

  • Microsatellite markers developed for M. atlanticus (with 2-24 alleles per locus) enable fine-scale population structure analysis

• Genetic diversity monitoring:

  • The high observed heterozygosity (mean 0.60) in Tampa Bay populations provides a baseline for monitoring genetic health

  • Regular sampling and genetic analysis can detect potential loss of diversity over time

• Phylogeographic patterns:

  • Mitochondrial markers like mt-co2 reveal historical population expansions and contractions

  • These patterns inform about historical population sizes and connectivity

• Adaptive potential evaluation:

  • Selection analysis on mt-co2 can identify locally adapted populations requiring special conservation consideration

  • Functional variations may indicate differential resilience to environmental stressors

• Non-invasive monitoring techniques:

  • Development of environmental DNA (eDNA) assays targeting mt-co2 allows population monitoring without capturing individuals

  • This is particularly valuable for vulnerable or endangered populations

The well-resolved and highly polymorphic nature of genetic markers in M. atlanticus makes them particularly suitable for conservation genetics applications, as demonstrated by the microsatellite analysis of Tampa Bay specimens .

What emerging technologies might enhance our understanding of Megalops atlanticus mt-co2 function?

Several cutting-edge technologies are poised to advance our understanding of M. atlanticus mt-co2 function:

• Cryo-electron microscopy (Cryo-EM):

  • High-resolution structural determination of the entire cytochrome c oxidase complex

  • Visualization of conformational changes during the catalytic cycle

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to track dynamic interactions between subunits

  • Optical tweezers to measure force generation during proton pumping

• CRISPR-based approaches:

  • Development of mitochondrial genome editing for in vivo studies

  • Creation of specific knockouts or point mutations to test functional hypotheses

• Integrative multi-omics:

  • Combination of proteomics, metabolomics, and transcriptomics to understand system-level responses

  • Machine learning approaches to identify patterns in complex datasets

• Advanced computational methods:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations of electron transfer

  • Enhanced sampling techniques to model rare events in the catalytic cycle

• Organoid and cell-free systems:

  • Development of fish-derived mitochondrial organoids for functional studies

  • Cell-free expression systems for studying membrane protein assembly

These emerging technologies will allow researchers to bridge the gap between structural information and functional understanding, potentially revealing novel aspects of mt-co2 contribution to cellular energetics .

How might research on mt-co2 contribute to our understanding of climate change effects on marine species?

Research on Megalops atlanticus mt-co2 can provide valuable insights into climate change impacts on marine species through several research avenues:

• Thermal adaptation studies:

  • Investigating how mt-co2 function varies across populations adapted to different temperature regimes

  • Thermal stability assays of recombinant mt-co2 to determine potential vulnerability to warming waters

• Oxygen utilization efficiency:

  • Examining how changing ocean oxygen levels affect cytochrome c oxidase efficiency

  • Comparing mt-co2 from species with different oxygen requirements to predict vulnerability

• Metabolic response to acidification:

  • Assessing how changing pH affects the proton gradient necessary for mt-co2 function

  • Measuring activity of recombinant mt-co2 under varying pH conditions mimicking ocean acidification

• Carbon dioxide processing:

  • While mt-co2 is not directly involved in carbon sequestration, understanding cellular respiration efficiency helps predict metabolic responses to changing CO2 levels

  • Experimental designs similar to those used in carbon sequestration studies (as seen in the laboratory prototype for CO2 deposition) could be adapted

• Evolutionary response prediction:

  • Analysis of historical mt-co2 sequence changes in response to past climate fluctuations

  • Laboratory evolution experiments to test adaptive potential

These research directions align with broader climate change research efforts, such as the Carbon Monitor project that tracks CO2 emissions, by providing biological mechanism insights that help predict species responses to changing environmental conditions .