Recombinant Lepisosteus oculatus Cytochrome c oxidase subunit 2 (mt-co2)

What is the function of Cytochrome c Oxidase Subunit 2 in Lepisosteus oculatus?

Cytochrome c Oxidase Subunit 2 (mt-CO2) in Lepisosteus oculatus functions as a critical component of the mitochondrial respiratory chain, specifically in Complex IV. This subunit contains copper centers that facilitate electron transfer from cytochrome c to molecular oxygen in the final step of the electron transport chain . In gar fish, this protein plays an essential role in cellular respiration and energy production through oxidative phosphorylation. The specific properties of gar mt-CO2 make it valuable for comparative studies of respiratory chain evolution across fish species, particularly when examining the divergence of ancient fish lineages such as the Lepisosteiformes from teleosts .

How does the structure of gar mt-CO2 compare to other vertebrate species?

Lepisosteus oculatus mt-CO2 exhibits structural features that reflect its evolutionary position as a non-teleost ray-finned fish. The protein contains characteristic copper-binding domains similar to those found in other vertebrates, but with distinct sequence variations reflective of its evolutionary history. When comparing gar mt-CO2 to teleost fishes like zebrafish (Danio rerio) or more distantly related vertebrates, researchers can observe conserved functional domains alongside lineage-specific adaptations . These structural differences can be visualized through protein threading analysis similar to methods used for other membrane proteins, where surface-exposed residues often display greater variability than those involved in core catalytic functions or interactions with other subunits .

What expression systems are typically used for producing recombinant Lepisosteus oculatus mt-CO2?

For the expression of recombinant Lepisosteus oculatus mt-CO2, researchers typically employ prokaryotic systems like E. coli or eukaryotic systems depending on research requirements. E. coli systems (similar to those used for other recombinant proteins) utilize vectors containing appropriate promoters and purification tags . The expressed protein is typically formulated in a buffer system containing stabilizing agents such as glycerol and reducing agents like DTT to maintain protein integrity . Eukaryotic expression systems may be necessary when post-translational modifications are required for functionality studies. A typical expression protocol would include gene optimization for the chosen expression system, vector construction with appropriate tags (His-tag being common), transformation, culture optimization, and purification through affinity chromatography .

What are the storage and stability conditions for recombinant L. oculatus mt-CO2?

Recombinant L. oculatus mt-CO2 requires specific storage conditions to maintain stability and functionality. Based on protocols for similar recombinant proteins, the purified protein is typically stored at -20°C in a buffer solution containing stabilizing agents . A recommended formulation would include 20mM Tris-HCl buffer (pH 8.0), 0.1M NaCl, 10% glycerol, and 2mM DTT to maintain protein integrity and prevent oxidation of sensitive residues . For longer-term storage, aliquoting the protein to minimize freeze-thaw cycles is advisable. When handling the protein, it should be kept on ice and transported using blue ice to maintain temperature stability . Stability testing under various conditions is an important preliminary step for any research protocol involving this recombinant protein.

How can the functional interaction between recombinant L. oculatus mt-CO2 and cytochrome c be accurately measured in vitro?

Measuring the functional interaction between recombinant L. oculatus mt-CO2 and cytochrome c requires specialized biochemical assays that assess electron transfer kinetics. A robust methodological approach involves spectrophotometric assays monitoring the oxidation rate of reduced cytochrome c at 550 nm, which directly correlates with mt-CO2 activity. Researchers should establish a baseline using purified components in a controlled buffer system (typically 50 mM phosphate buffer, pH 7.4) with precise protein concentrations determined through Bradford or BCA assays .

For kinetic analysis, the following protocol is recommended:

• Prepare reduced cytochrome c by adding sodium dithionite and removing excess reductant via gel filtration

• Add recombinant mt-CO2 at varying concentrations (0.1-10 μg/ml)

• Monitor absorbance changes at 550 nm over time at physiologically relevant temperatures (20-25°C for gar-based studies)

• Calculate initial reaction rates and derive kinetic parameters (Km, Vmax) through Lineweaver-Burk or Eadie-Hofstee plots

This methodology allows researchers to compare the functional characteristics of gar mt-CO2 with those of other species, providing insights into evolutionary adaptations of the respiratory chain in different vertebrate lineages .

What strategies can overcome expression challenges when producing recombinant L. oculatus mt-CO2?

Expression of recombinant L. oculatus mt-CO2 presents challenges due to its hydrophobic regions and potential toxicity to host cells. To overcome these challenges, researchers should employ a multi-faceted approach:

For particularly difficult constructs, a cell-free expression system may be employed, which allows for the direct incorporation of the protein into nanodiscs or liposomes . When conventional approaches fail, researchers might consider expressing individual domains separately or creating chimeric constructs with better-expressing homologs from related species while maintaining the key functional regions of interest.

How can molecular dynamics simulations enhance our understanding of L. oculatus mt-CO2 evolutionary adaptations?

Molecular dynamics (MD) simulations offer powerful insights into the evolutionary adaptations of L. oculatus mt-CO2 by revealing atomic-level behavior under different conditions. To implement this approach effectively, researchers should:

• Begin by creating a high-quality structural model through homology modeling based on crystallographic structures of cytochrome c oxidase from other species, using threading methods to identify key structural features .

• Embed the protein model in a simulated lipid bilayer that mimics the mitochondrial inner membrane, with appropriate phospholipid composition.

• Perform equilibrium MD simulations (typically 100-500 ns) under physiologically relevant conditions, comparing simulations at different temperatures (10-30°C) to examine temperature adaptations specific to the gar's environment.

• Analyze specific parameters including:

  • Proton transfer pathway dynamics

  • Conformational flexibility of key residues

  • Water molecule organization within channels

  • Interaction energies with other subunits

• Compare simulation results with those from other species to identify unique features that may represent evolutionary adaptations .

This computational approach complements experimental data by providing mechanistic hypotheses that can be subsequently tested through site-directed mutagenesis or biochemical assays, creating a powerful iterative research approach.

What are the best approaches for studying the assembly of L. oculatus mt-CO2 into functional respiratory complexes?

Studying the assembly of L. oculatus mt-CO2 into functional respiratory complexes requires specialized techniques that can track protein-protein interactions and complex formation. A comprehensive methodological approach includes:

• Co-immunoprecipitation studies: Using antibodies against mt-CO2 or epitope tags to pull down interaction partners, followed by mass spectrometry identification of the components. This approach can identify both stable and transient interactions during complex assembly .

• Blue Native PAGE analysis: This technique separates intact respiratory complexes while preserving their native interactions. By incorporating a first-dimension BN-PAGE with a second-dimension SDS-PAGE, researchers can visualize both the intact complexes and their subunit composition .

• Pulse-chase experiments: These can track the incorporation of newly synthesized mt-CO2 into assembling complexes over time, providing insights into the assembly kinetics and sequencing.

• Fluorescence microscopy with split-GFP constructs: By tagging mt-CO2 and other subunits with complementary GFP fragments, researchers can visualize complex assembly in living cells through the reconstitution of fluorescent GFP when the proteins interact.

• Crosslinking mass spectrometry (XL-MS): This advanced technique uses chemical crosslinkers to capture protein-protein interactions, followed by mass spectrometry to identify interaction interfaces at amino acid resolution .

These approaches should be conducted in systems that closely mimic the native environment, such as isolated mitochondria or suitable cell culture models expressing the gar proteins.

How can recombinant L. oculatus mt-CO2 serve as a model for understanding respiratory chain evolution?

Lepisosteus oculatus (spotted gar) occupies a unique evolutionary position as a non-teleost ray-finned fish that diverged from the teleost lineage before the teleost-specific genome duplication. This makes recombinant L. oculatus mt-CO2 an invaluable model for understanding respiratory chain evolution through comparative analysis . Researchers can leverage this model by:

• Conducting phylogenetic analysis of mt-CO2 sequences across vertebrate lineages to identify conserved regions versus lineage-specific adaptations.

• Performing enzymatic activity comparisons under varying conditions (temperature, pH, salt concentration) to correlate sequence differences with functional adaptations.

• Using site-directed mutagenesis to introduce teleost-specific or tetrapod-specific residues into the gar protein and assess their impact on function.

• Examining the co-evolution of nuclear-encoded and mitochondrial-encoded subunits of the cytochrome c oxidase complex, which can reveal constraints on the evolution of the respiratory chain .

This evolutionary perspective is particularly valuable for understanding how environmental adaptations have shaped mitochondrial function across vertebrate lineages, with the gar representing an important reference point between teleosts and tetrapods.

What are the specific challenges in purifying active recombinant L. oculatus mt-CO2 and how can they be addressed?

Purifying active recombinant L. oculatus mt-CO2 presents several challenges due to its hydrophobic nature and requirement for proper cofactor incorporation. These challenges and their solutions include:

Additionally, researchers should consider implementing a multi-step purification strategy including initial IMAC (immobilized metal affinity chromatography) using the His-tag, followed by ion-exchange and size-exclusion chromatography to separate properly folded, active protein from aggregates or misfolded species . Activity assays should be performed at each purification step to track specific activity and recovery of functional protein.

How can differential scanning fluorimetry be applied to optimize stability conditions for recombinant L. oculatus mt-CO2?

Differential scanning fluorimetry (DSF) is a powerful technique for optimizing stability conditions for recombinant L. oculatus mt-CO2. This approach involves using environment-sensitive fluorescent dyes that bind to hydrophobic regions exposed during protein unfolding, allowing researchers to determine melting temperatures (Tm) under various conditions. A methodological approach includes:

• Prepare purified recombinant mt-CO2 at concentrations of 0.1-0.5 mg/mL in various test buffers.

• Add SYPRO Orange or similar fluorescent dye at appropriate dilutions (typically 5X final concentration).

• Perform thermal ramping (25-95°C) while monitoring fluorescence emission in a real-time PCR instrument or specialized thermal shift analyzer.

• Analyze melting curves to determine Tm values under each condition.

• Systematically test multiple parameters:

  • Buffer composition (HEPES, Tris, phosphate, MES) at pH range 6.0-8.5

  • Salt type and concentration (NaCl, KCl at 0-500 mM)

  • Additives (glycerol 5-20%, reducing agents 1-10 mM)

  • Detergents (type and concentration)

  • Metal ions (particularly copper at 1-100 μM)

This approach can generate a stability landscape for the protein, identifying optimal conditions that significantly extend shelf-life and maintain functional activity . The resulting optimized buffer system can then be validated through long-term activity assays to confirm that thermal stability correlates with functional longevity.

What techniques can effectively measure the interaction between L. oculatus mt-CO2 and potential inhibitors?

Measuring interactions between L. oculatus mt-CO2 and potential inhibitors requires sensitive biophysical techniques that can detect binding events and their functional consequences. An effective methodological approach includes:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified mt-CO2 on a sensor chip via His-tag or biotinylation

  • Flow potential inhibitors at varying concentrations (1 nM to 100 μM)

  • Measure association and dissociation kinetics

  • Calculate binding constants (KD, kon, koff)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine binding stoichiometry, enthalpy (ΔH), and entropy (ΔS)

  • Distinguish between enthalpy-driven and entropy-driven interactions

• Microscale Thermophoresis (MST):

  • Label protein with fluorescent dye or use intrinsic tryptophan fluorescence

  • Measure changes in thermophoretic mobility upon inhibitor binding

  • Requires minimal protein amounts (typically <100 nM)

• Functional inhibition assays:

  • Measure cytochrome c oxidation rates in presence of varying inhibitor concentrations

  • Determine IC50 values and inhibition mechanisms (competitive, non-competitive)

  • Correlate binding data with functional impact

• Thermal shift assays:

  • Measure changes in protein thermal stability upon inhibitor binding

  • Quick screening method for multiple compounds

  • Can indicate binding mode through stabilization or destabilization effects

These techniques provide complementary information about inhibitor interactions, from pure binding parameters to functional consequences, allowing researchers to develop comprehensive models of inhibitor action .

What are the most effective quality control measures for evaluating recombinant L. oculatus mt-CO2 preparations?

Ensuring the quality of recombinant L. oculatus mt-CO2 preparations requires multiple complementary assessment techniques. An effective quality control protocol should include:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (target >95% purity)

  • Western blotting with specific antibodies or against purification tags

  • Size-exclusion chromatography to detect aggregates or degradation products

• Identity confirmation:

  • Mass spectrometry (LC-MS/MS) peptide mapping against the theoretical sequence

  • N-terminal sequencing to confirm proper processing

  • Immunological detection with subunit-specific antibodies

• Structural integrity:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Fluorescence spectroscopy to assess tertiary folding

  • Limited proteolysis to verify proper folding (correctly folded proteins show distinct, reproducible digestion patterns)

• Functional activity:

  • Cytochrome c oxidation assays with standardized conditions

  • Oxygen consumption measurements

  • Spectroscopic analysis of metal centers (copper absorption features)

• Homogeneity assessment:

  • Dynamic light scattering to evaluate size distribution

  • Analytical ultracentrifugation to detect multiple oligomeric states

  • Blue native PAGE to assess complex assembly

Each batch of purified protein should be evaluated against established specifications, with documented acceptance criteria for each parameter. This comprehensive approach ensures that experimental results can be reliably attributed to the protein's intrinsic properties rather than preparation artifacts .

How can researchers effectively address the challenges of low expression yields for L. oculatus mt-CO2?

Low expression yields are a common challenge when producing recombinant L. oculatus mt-CO2. Researchers can implement several strategies to overcome this limitation:

• Vector optimization:

  • Test multiple promoter systems (T7, tac, araBAD) to find optimal expression levels

  • Incorporate enhancer elements or optimize the ribosome binding site

  • Consider using vectors with tight regulation to minimize toxicity during culture growth

• Fusion partner screening:

  • Evaluate different fusion tags beyond standard His-tags (MBP, GST, SUMO, Trx)

  • Position tags at either N- or C-terminus to determine optimal configuration

  • Include precision protease sites for tag removal that don't leave residual amino acids

• Culture optimization:

  • Test expression in specialized media formulations (e.g., Terrific Broth, Auto-induction media)

  • Optimize induction parameters (inducer concentration, temperature, duration)

  • Implement fed-batch cultivation to achieve higher cell densities before induction

• Co-expression strategies:

  • Co-express with chaperones (GroEL/ES, DnaK/J) to assist proper folding

  • Include key interacting partners that might stabilize the protein

  • Co-express with enzymes required for cofactor synthesis or incorporation

• Alternative expression systems:

  • Evaluate eukaryotic systems (yeast, insect cells) if prokaryotic expression fails

  • Consider cell-free expression systems for toxic proteins

  • Test expression in mitochondria-like bacterial systems (Paracoccus denitrificans)

By systematically exploring these options and documenting the impact of each modification, researchers can develop an optimized production protocol that significantly improves yields of functional protein .

What strategies can researchers employ to study post-translational modifications of L. oculatus mt-CO2?

Studying post-translational modifications (PTMs) of L. oculatus mt-CO2 requires specialized techniques to identify, locate, and functionally characterize these modifications. An effective methodological approach includes:

• PTM identification and mapping:

  • High-resolution mass spectrometry (MS) with multiple fragmentation techniques (CID, ETD, HCD)

  • Enrichment strategies for specific modifications (phosphopeptide enrichment, glycopeptide capture)

  • Site-specific antibodies for common PTMs (phosphorylation, acetylation)

  • Targeted multiple reaction monitoring (MRM) MS for quantitative analysis of specific modified sites

• Temporal dynamics of modifications:

  • Pulse-chase experiments with metabolic labeling

  • Time-course analysis following cellular stimulation

  • In vitro modification using purified modifying enzymes

• Functional significance assessment:

  • Site-directed mutagenesis of modified residues (mimicking modifications or preventing them)

  • Activity assays comparing modified and unmodified forms

  • Structural analysis to determine how modifications affect protein conformation

• Modification enzymes identification:

  • Co-immunoprecipitation coupled with proteomic analysis

  • Activity-based protein profiling for enzyme identification

  • Inhibitor studies to link specific enzymes to observed modifications

• Comparative analysis across species:

  • Examine conservation of modification sites in homologous proteins

  • Compare modification patterns between gar and other vertebrates under similar conditions

  • Correlate differences in modifications with functional or environmental adaptations

This comprehensive approach provides insights into the regulatory mechanisms controlling mt-CO2 function through post-translational modifications, potentially revealing unique aspects of respiratory chain regulation in non-teleost fishes like Lepisosteus oculatus.

What are the best approaches for developing specific antibodies against L. oculatus mt-CO2 for research applications?

Developing specific antibodies against L. oculatus mt-CO2 requires careful antigen design and validation strategies. A comprehensive approach includes:

• Antigen design strategy:

  • Identify unique, surface-exposed peptide regions through sequence analysis and structural prediction

  • Focus on regions with low sequence conservation across species for species-specific antibodies

  • Target conserved regions for pan-specific antibodies that work across multiple species

  • Consider both full-length protein and synthetic peptide approaches

• Production methods:

  • For polyclonal antibodies: Immunize rabbits or other host animals with purified recombinant protein or KLH-conjugated peptides

  • For monoclonal antibodies: Screen hybridoma libraries against the target antigen

  • For recombinant antibodies: Perform phage display selection against the target protein

• Purification approach:

  • Implement affinity purification using antigen-conjugated columns

  • Perform negative selection against homologous proteins from other species to enhance specificity

  • Characterize antibody isotypes and subclasses for optimal application performance

• Validation protocol:

  • Western blotting against recombinant protein and native tissue extracts

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Immunohistochemistry with appropriate controls (pre-immune serum, blocking peptides)

  • Cross-reactivity testing against homologous proteins from related species

• Application optimization:

  • Determine optimal working dilutions for each application (Western blot, immunoprecipitation, immunohistochemistry)

  • Establish appropriate buffer conditions and blocking agents

  • Document lot-to-lot variability and establish validation criteria for each batch

This methodical approach ensures the development of reliable antibody tools that can be used for multiple research applications, from protein localization to interaction studies and functional analyses of L. oculatus mt-CO2 .

How might CRISPR/Cas9 genome editing be utilized to study L. oculatus mt-CO2 function in vivo?

CRISPR/Cas9 genome editing offers powerful approaches for studying L. oculatus mt-CO2 function in vivo, despite the challenges of editing mitochondrial genes. Researchers can implement several strategies:

• Nuclear-encoded reporter systems:

  • Create reporter constructs that respond to mt-CO2 activity or assembly

  • Edit nuclear genes encoding mt-CO2 interacting partners to study their effects on function

  • Develop inducible systems to modulate expression of assembly factors

• Allotopic expression models:

  • Engineer nuclear-encoded versions of mt-CO2 with mitochondrial targeting sequences

  • Introduce mutations or tags for functional analysis

  • Use CRISPR to knockout or modify nuclear factors that influence mt-CO2 expression or activity

• Study design approaches:

  • Implement tissue-specific or temporally controlled gene editing using conditional Cas9 systems

  • Create knock-in models with fluorescent or affinity tags to track protein localization and interactions

  • Generate heterozygous models to study gene dosage effects on respiratory function

• Phenotypic analysis methods:

  • Measure oxygen consumption in isolated mitochondria from edited cells

  • Assess metabolic shifts using stable isotope labeling

  • Evaluate reactive oxygen species production and mitochondrial membrane potential

  • Examine cellular and organismal phenotypes under normal and stressed conditions

• Comparative editing strategies:

  • Perform parallel edits in multiple species to compare the impact of conserved residues

  • Create chimeric constructs between gar and other species to map functional domains

These approaches circumvent the technical challenges of directly editing mitochondrial DNA while still providing valuable insights into mt-CO2 function, assembly, and regulation in the context of living cells and organisms.

What potential applications exist for using L. oculatus mt-CO2 as a model for studying mitochondrial disorders?

Lepisosteus oculatus mt-CO2 offers unique advantages as a model for studying mitochondrial disorders due to its evolutionary position. Potential research applications include:

• Comparative pathogenicity assessment:

  • Introduce known human pathogenic mutations into the corresponding gar residues

  • Assess biochemical consequences in reconstituted systems

  • Compare outcomes with human and other model systems to identify conserved disease mechanisms

  • Evaluate compensatory mechanisms that may exist in different species

• Drug screening platforms:

  • Develop assay systems using recombinant gar mt-CO2 to screen compounds that rescue mutant phenotypes

  • Compare drug efficacy across species-specific versions of mt-CO2

  • Identify compounds that specifically modulate cytochrome c oxidase activity or assembly

• Evolution-guided therapy development:

  • Identify naturally occurring variations in gar mt-CO2 that confer resistance to stressors

  • Study the molecular basis of these protective mechanisms

  • Apply these insights to develop therapeutic approaches for human disorders

• Mitochondrial assembly model:

  • Use the gar system to study conserved and divergent aspects of Complex IV assembly

  • Identify species-specific assembly factors and their potential therapeutic relevance

  • Develop interventions that promote proper assembly of defective oxidase complexes

• Environmental susceptibility research:

  • Examine how environmental factors (temperature, pH, oxygen tension) affect wild-type versus mutant mt-CO2

  • Identify conditions that exacerbate or ameliorate defects

  • Develop mitochondrial stress tests with translational relevance to human disorders

These applications leverage the unique evolutionary features of gar mt-CO2 to provide insights into fundamental mechanisms of mitochondrial disease and potential therapeutic approaches that might not be apparent from studying mammalian systems alone.

How can structural biology techniques be optimized for determining the high-resolution structure of L. oculatus mt-CO2?

Determining the high-resolution structure of L. oculatus mt-CO2 requires optimized structural biology approaches tailored to this challenging membrane protein. A comprehensive methodological strategy includes:

• Crystallography optimization:

  • Screen detergent/lipid combinations systematically (including detergent:protein:lipid ratios)

  • Implement lipidic cubic phase (LCP) crystallization for membrane proteins

  • Use antibody fragments or nanobodies to stabilize flexible regions and promote crystal contacts

  • Explore fusion protein approaches (T4 lysozyme or BRIL insertions) to enhance crystallizability

• Cryo-EM approach refinement:

  • Optimize sample vitrification conditions (blotting times, grid types, humidity)

  • Implement GraFix or amphipol reconstitution to improve particle orientation distribution

  • Consider focused refinement approaches for flexible regions

  • Use multi-body refinement to capture conformational heterogeneity

• Hybrid method integration:

  • Combine lower-resolution cryo-EM maps with high-resolution X-ray structures of domains

  • Implement hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Use solid-state NMR to determine key distance constraints in the membrane-embedded regions

  • Integrate computational models with experimental restraints

• Sample preparation strategies:

  • Express the protein with stabilizing mutations identified through directed evolution

  • Co-purify with natural binding partners to stabilize native conformations

  • Implement nanodiscs or amphipols to maintain a native-like lipid environment

  • Screen metal ions and small molecule binders that might stabilize specific conformations

• Data collection and processing optimization:

  • Implement advanced direct electron detector technologies for cryo-EM

  • Use microfocus beamlines for small crystals

  • Apply machine learning approaches for particle picking and classification

  • Implement Bayesian approaches for dealing with conformational heterogeneity