Recombinant Eschrichtius gibbosus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its role in mitochondrial function?

MT-ND4L (NADH-ubiquinone oxidoreductase chain 4L) is an essential mitochondrial-encoded protein that forms part of Complex I in the electron transport chain. This 98-amino acid protein plays a critical role in oxidative phosphorylation, specifically participating in the first step of electron transport by facilitating electron transfer from NADH to ubiquinone . As a component of Complex I, MT-ND4L contributes to establishing the electrochemical gradient across the inner mitochondrial membrane that drives ATP synthesis. The protein is embedded within the inner mitochondrial membrane along with other components of the respiratory chain complexes. In Eschrichtius gibbosus, MT-ND4L maintains a structure that enables efficient electron transport while accommodating the specific physiological demands of these marine mammals, particularly during deep-diving behaviors that require efficient oxygen utilization.

What is the amino acid sequence of Eschrichtius gibbosus MT-ND4L and its structural characteristics?

The complete amino acid sequence of Eschrichtius gibbosus MT-ND4L consists of 98 amino acids as follows:

MTLIHMNILMAFSMSLLGLLMYRSHLMSALLCLEGMMLSLFVLAALTILNSHFTLANMMPIILLVFAACEAAIGLALLVMVSNTYGTDYVQNLNLLQC

This highly hydrophobic protein contains multiple transmembrane domains that anchor it within the inner mitochondrial membrane. The protein's structural characteristics include:

• Multiple transmembrane alpha-helices that span the inner mitochondrial membrane

• Conserved domains involved in electron transport activity

• Hydrophobic regions essential for membrane integration

• Functional regions that interact with other subunits of Complex I

Structural analyses suggest that these features are conserved across cetacean species, though specific amino acid substitutions may reflect adaptations to different marine environments and diving behaviors.

How does MT-ND4L differ between Eschrichtius gibbosus and other marine mammals?

While the search results do not provide direct comparison of MT-ND4L across different marine mammals, we can draw parallels from myoglobin studies in these species. Comparative studies of myoglobin from Eschrichtius gibbosus show differences at multiple positions compared to other marine mammals: 12 positions different from sperm whale (Physeter catodon), 14 positions different from common porpoise (Phocoena phocoena) and Black Sea dolphin (Delphinus delphis), and 7 positions different from Amazon River dolphin (Inia geoffrensis) .

Similar patterns of variation likely exist in mitochondrial proteins like MT-ND4L, reflecting evolutionary divergence and adaptation to different ecological niches. Mitochondrial DNA analysis used in population structure studies of North Pacific gray whales indicates that protein-coding regions, including those potentially encoding MT-ND4L, show significant variation that can be used to distinguish population groups . These differences may affect protein function, stability, or interaction with other components of Complex I, potentially contributing to species-specific metabolic adaptations.

What expression systems are optimal for producing functional recombinant MT-ND4L?

Producing functional recombinant MT-ND4L presents significant challenges due to its hydrophobic nature and membrane-bound characteristics. Based on general protocols for recombinant mitochondrial proteins, the following expression systems offer distinct advantages:

• Bacterial Expression Systems (E. coli):

  • Most commonly used initial approach due to simplicity and cost-effectiveness

  • Requires optimization of codon usage for the whale sequence

  • Often requires fusion tags (His, GST, MBP) to improve solubility

  • May result in inclusion bodies requiring refolding protocols

• Yeast Expression Systems (Pichia pastoris):

  • Better suited for membrane proteins than bacterial systems

  • Provides eukaryotic post-translational processing

  • Can be scaled for larger yield preparations

• Insect Cell Systems (Baculovirus):

  • Superior folding of complex membrane proteins

  • More efficient for maintaining functional integrity of mitochondrial proteins

  • Higher production costs but potentially higher quality protein

Regardless of the chosen system, the tag type will need to be determined during the production process to optimize protein yield and activity . The recombinant protein should be stored in a Tris-based buffer with 50% glycerol at -20°C for routine use, or -80°C for extended storage, with recommendations against repeated freezing and thawing .

What purification strategies are most effective for recombinant MT-ND4L?

Purification of recombinant MT-ND4L requires specialized approaches due to its hydrophobic nature. The most effective strategies include:

• Affinity Chromatography:

  • Utilizing fusion tags (His, GST) for selective capture

  • Requires optimization of detergent conditions to maintain solubility

  • Often involves staged elution protocols to minimize co-elution of contaminants

• Size Exclusion Chromatography:

  • Critical for separating monomeric from aggregated forms

  • Useful for buffer exchange into final storage conditions

  • Provides quality control for protein homogeneity

• Ion Exchange Chromatography:

  • Can be employed as a polishing step

  • Separation based on the charge characteristics of the protein

  • Requires careful pH optimization

A typical purification workflow would begin with affinity chromatography, followed by size exclusion and possibly ion exchange as a final polishing step. Throughout purification, it's essential to maintain the protein in appropriate detergent-containing buffers to prevent aggregation. Final storage in a Tris-based buffer with 50% glycerol is recommended to stabilize the protein .

How can researchers verify the functional integrity of purified recombinant MT-ND4L?

Verifying the functional integrity of purified recombinant MT-ND4L requires multiple analytical approaches:

• Enzymatic Activity Assays:

  • NADH:ubiquinone oxidoreductase activity measurements

  • Electron transfer rate determination using artificial electron acceptors

  • Comparison with native Complex I activity from isolated mitochondria

• Structural Validation:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Limited proteolysis to assess proper folding

  • Thermal shift assays to evaluate protein stability

• Interaction Studies:

  • Co-immunoprecipitation with other Complex I subunits

  • Blue native PAGE to assess incorporation into Complex I

  • Surface plasmon resonance to quantify interaction with partner proteins

A comprehensive validation approach would incorporate multiple methods to ensure both structural integrity and functional activity of the recombinant protein before its application in experimental studies.

How can recombinant MT-ND4L be used to study mitochondrial disorders?

Recombinant MT-ND4L provides a valuable tool for investigating mitochondrial disorders, particularly those affecting Complex I function. Specific applications include:

• Mutation Analysis Studies:

  • In vitro reconstitution of pathogenic mutations identified in disorders like Leber hereditary optic neuropathy (LHON)

  • Assessment of mutation impact on protein stability and function

  • Structure-function relationship studies to identify critical functional domains

A particular mutation in MT-ND4L (T10663C or Val65Ala) has been identified in families with LHON . Recombinant protein containing this mutation could be used to study how this amino acid change affects protein function and interaction with other Complex I components.

• Drug Screening Platforms:

  • Development of high-throughput assays to identify compounds that rescue mutant protein function

  • Testing potential therapeutic compounds that enhance Complex I activity

  • Validation of compounds that stabilize mutant proteins

• Biomarker Development:

  • Generation of specific antibodies against recombinant MT-ND4L for detection in clinical samples

  • Development of assays to measure MT-ND4L levels or modifications in patient samples

  • Correlation studies between MT-ND4L alterations and disease progression

These applications can significantly advance our understanding of mitochondrial disorders and potentially lead to novel therapeutic approaches for conditions associated with Complex I dysfunction.

What insights can MT-ND4L provide about evolutionary adaptations in marine mammals?

MT-ND4L analysis can yield valuable insights into evolutionary adaptations in marine mammals, particularly regarding energy metabolism adaptations for deep diving and oxygen utilization:

• Comparative Sequence Analysis:

  • Alignment of MT-ND4L sequences across diverse marine mammal species

  • Identification of conserved and variable regions reflecting evolutionary pressures

  • Detection of positive selection signatures associated with diving adaptations

Mitochondrial DNA analysis has already been employed to study population structures in North Pacific gray whales, revealing significant genetic differentiation between eastern and western populations . Similar approaches focused specifically on MT-ND4L could reveal adaptations unique to different whale populations.

• Structure-Function Correlations:

  • Modeling of amino acid substitutions on protein structure and function

  • Correlation of sequence variations with diving capabilities across species

  • Investigation of changes affecting proton pumping efficiency and ROS production

• Physiological Context Studies:

  • Integration of MT-ND4L data with physiological parameters of different species

  • Correlation of sequence variations with metabolic rates and oxygen utilization

  • Assessment of adaptations that enhance mitochondrial efficiency under hypoxic conditions

These approaches can provide a molecular-level understanding of how marine mammals have adapted their energy production systems to their unique environmental challenges.

What techniques are most effective for analyzing MT-ND4L mutations and their functional impact?

Analysis of MT-ND4L mutations requires a multi-faceted approach combining genetic, biochemical, and structural methods:

• Genetic and Genomic Approaches:

  • Next-generation sequencing to identify novel mutations

  • Population genetics to determine mutation frequencies

  • Phylogenetic analysis to assess conservation and evolutionary significance

Techniques for mtDNA analysis, as described in the population studies of gray whales, include PCR amplification of specific regions using primers such as H00034 and L15812, followed by sequencing on platforms like Applied Biosystems models .

• Biochemical and Biophysical Analyses:

  • Enzyme kinetics to measure effects on electron transfer rates

  • Membrane potential assays to assess proton pumping efficiency

  • ROS production measurements to evaluate electron leakage

• Structural Biology Techniques:

  • Cryo-EM of reconstituted Complex I with mutant MT-ND4L

  • Molecular dynamics simulations to predict mutation effects

  • Hydrogen-deuterium exchange mass spectrometry to assess structural perturbations

• Cellular Models:

  • Cybrid cell lines incorporating mutant mitochondria

  • CRISPR-mediated introduction of mutations in model organisms

  • Patient-derived iPSCs differentiated into affected cell types

What critical quality control parameters should be monitored when working with recombinant MT-ND4L?

Ensuring high-quality recombinant MT-ND4L requires monitoring several critical parameters:

• Purity Assessment:

  • SDS-PAGE followed by Coomassie or silver staining (>95% purity recommended)

  • Western blotting with anti-MT-ND4L or anti-tag antibodies

  • Mass spectrometry to confirm protein identity and detect modifications

• Structural Integrity:

  • Circular dichroism spectroscopy to verify secondary structure elements

  • Fluorescence spectroscopy to assess tertiary structure

  • Dynamic light scattering to evaluate homogeneity and detect aggregation

• Functional Activity:

  • NADH:ubiquinone oxidoreductase activity assays

  • Electron transfer rate measurements

  • Reconstitution with other Complex I components to assess assembly

• Storage Stability:

  • Regular activity testing of stored protein aliquots

  • Avoidance of repeated freeze-thaw cycles as recommended in product guidelines

  • Use of working aliquots stored at 4°C for up to one week

Implementing these quality control measures ensures experimental reproducibility and valid research outcomes when working with this challenging membrane protein.

What are the most common technical challenges when working with recombinant MT-ND4L and how can they be addressed?

Researchers working with recombinant MT-ND4L encounter several technical challenges:

• Protein Aggregation and Precipitation:

  • Challenge: High hydrophobicity leads to aggregation during expression and purification

  • Solution: Optimize detergent type and concentration; consider mild solubilizing agents; use fusion partners known to enhance solubility

• Low Expression Yields:

  • Challenge: Membrane proteins often express poorly in heterologous systems

  • Solution: Test multiple expression systems; optimize codon usage; evaluate various fusion tags; consider specialized expression strains

• Loss of Activity During Purification:

  • Challenge: Functional integrity may be compromised during extraction and purification

  • Solution: Minimize exposure to harsh conditions; maintain appropriate detergent concentrations; include stabilizing agents; conduct rapid purification at 4°C

• Reconstitution into Functional Complexes:

  • Challenge: Difficulty incorporating recombinant protein into native-like complexes

  • Solution: Develop optimized reconstitution protocols; use nanodiscs or liposomes; co-express with partner proteins when possible

• Assay Interference:

  • Challenge: Detergents or buffer components may interfere with activity assays

  • Solution: Develop assay controls to account for buffer components; normalize results to appropriate standards; consider detergent removal prior to activity testing

Addressing these challenges requires iterative optimization of protocols specific to MT-ND4L, potentially drawing on approaches successful with other mitochondrial membrane proteins.

How should researchers interpret comparative data between wild-type and mutant MT-ND4L proteins?

Interpreting comparative data between wild-type and mutant MT-ND4L requires careful consideration of multiple parameters:

• Statistical Analysis Framework:

  • Employ appropriate statistical tests based on data distribution

  • Include sufficient biological and technical replicates (minimum n=3)

  • Report effect sizes alongside statistical significance

• Functional Parameter Interpretation:

  • Enzyme kinetics: Compare Km and Vmax values to quantify changes in substrate affinity and catalytic efficiency

  • Electron transfer rates: Assess percentage changes relative to wild-type

  • ROS production: Evaluate whether changes correlate with electron transport efficiency

• Structural Context Evaluation:

  • Map mutations onto known structural models of Complex I

  • Assess whether changes occur in functional domains, interaction interfaces, or membrane-spanning regions

  • Consider how structural perturbations might propagate through the protein

• Physiological Relevance Assessment:

  • Compare in vitro findings with cellular or organismal phenotypes when available

  • Consider whether observed effects would be significant under physiological conditions

  • Evaluate potential compensatory mechanisms that might mitigate mutation effects

When interpreting data related to the Val65Ala mutation associated with Leber hereditary optic neuropathy , researchers should consider how this amino acid substitution affects protein stability, interactions, and electron transport efficiency in the context of optic nerve vulnerability to mitochondrial dysfunction.

What bioinformatic approaches are most valuable for analyzing MT-ND4L sequence data?

Several bioinformatic approaches provide valuable insights when analyzing MT-ND4L sequence data:

• Comparative Sequence Analysis:

  • Multiple sequence alignment across species to identify conserved regions

  • Calculation of conservation scores to highlight functionally important residues

  • Identification of co-evolving residues that may function together

• Structural Prediction and Analysis:

  • Homology modeling based on related structures

  • Transmembrane topology prediction

  • Molecular dynamics simulations to assess conformational flexibility

• Evolutionary Analysis:

  • Phylogenetic tree construction to understand evolutionary relationships

  • Calculation of dN/dS ratios to detect selection pressures

  • Ancestral sequence reconstruction to track evolutionary changes

• Mutation Impact Prediction:

  • Use of algorithms like SIFT, PolyPhen, and PROVEAN to predict functional impacts

  • Energy calculation changes upon mutation

  • Protein stability predictions following amino acid substitutions

• Population Genetics Metrics:

  • Analysis of haplotype diversity and distribution

  • Calculation of fixation indices (FST) to assess population differentiation

  • Identification of migration patterns based on haplotype sharing

Similar approaches have been successfully applied in population studies of North Pacific gray whales, where analysis of mtDNA revealed significant genetic differentiation between eastern and western populations .