Recombinant Ommatophoca rossii NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L (Mitochondrially Encoded NADH:Ubiquinone Oxidoreductase Chain 4L) is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase, also known as Complex I. This protein plays a critical role in electron transfer from NADH through the respiratory chain, using ubiquinone as an electron acceptor . As part of the enzyme membrane arm, MT-ND4L is embedded in the lipid bilayer and directly involved in proton translocation across the inner mitochondrial membrane .

The protein functions within Complex I, which is responsible for the first step in the electron transport process during oxidative phosphorylation. Specifically, it facilitates the transfer of electrons from NADH to ubiquinone, contributing to the creation of an unequal electrical charge on either side of the inner mitochondrial membrane that provides energy for ATP production . MT-ND4L is among the most hydrophobic subunits of Complex I and helps form the core of the transmembrane region .

How is the MT-ND4L gene structured in mitochondrial DNA?

The MT-ND4L gene has several noteworthy structural characteristics:

• In humans, the gene is located in mitochondrial DNA spanning from base pair 10,469 to 10,765 .

• It encodes a relatively small protein of approximately 11 kDa, composed of 98 amino acids .

• MT-ND4L is one of seven mitochondrially encoded subunits of NADH dehydrogenase (ubiquinone), alongside MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, and MT-ND6 .

A particularly interesting feature of the human MT-ND4L gene is its unusual 7-nucleotide overlap with the MT-ND4 gene. The last three codons of MT-ND4L (5'-CAA TGC TAA-3' coding for Gln, Cys, and Stop) overlap with the first three codons of MT-ND4 (5'-ATG CTA AAA-3' coding for Met-Leu-Lys). With respect to the MT-ND4L reading frame (+1), the MT-ND4 gene starts in the +3 reading frame .

What are the available forms of recombinant MT-ND4L for experimental use?

Researchers have access to several recombinant forms of MT-ND4L, including:

• Recombinant Ommatophoca rossii (Ross seal) NADH-ubiquinone oxidoreductase chain 4L, which is commercially available .

• Human MT-ND4L recombinant protein antigen with an N-terminal His6-ABP tag, expressed in E. coli .

The human recombinant protein antigen has the amino acid sequence LLVSISNTYGLDYVHNLNLLQ and has been validated for antibody competition applications . This preparation is typically supplied in PBS and 1M Urea at pH 7.4, with purity >80% by SDS-PAGE and Coomassie blue staining .

How do mutations in MT-ND4L contribute to Leber Hereditary Optic Neuropathy (LHON) pathogenesis?

A specific mutation in the MT-ND4L gene, designated as T10663C or Val65Ala, has been identified in several families with Leber Hereditary Optic Neuropathy (LHON) . This mutation results in a single amino acid substitution where valine is replaced by alanine at position 65 of the NADH dehydrogenase 4L protein .

• Disruption of electron transport efficiency

• Increased production of reactive oxygen species (ROS)

• Compromised ATP synthesis in retinal ganglion cells

• Altered mitochondrial membrane potential

Research methodologies to investigate these mechanisms typically involve:

• Creating cellular models with the specific mutation using site-directed mutagenesis

• Measuring Complex I activity in patient-derived cells or model systems

• Assessing mitochondrial membrane potential using fluorescent probes

• Quantifying ATP production and ROS levels in affected tissues

• Comparative structural biology approaches to understand how the mutation affects protein folding and complex assembly

What techniques are most effective for analyzing the structure-function relationship of MT-ND4L in different species?

Investigating structure-function relationships of MT-ND4L across species requires a multi-faceted approach:

• Sequence Alignment and Phylogenetic Analysis:

  • Multiple sequence alignment of MT-ND4L from different species, including Ommatophoca rossii, humans, and other mammals

  • Identification of conserved domains and species-specific variations

  • Evolutionary rate analysis to identify selective pressure on specific regions

• Structural Biology Approaches:

  • Cryo-electron microscopy of intact Complex I to visualize MT-ND4L in its native conformation

  • Molecular dynamics simulations to predict the impact of sequence variations on protein folding and stability

  • Cross-linking mass spectrometry to map interaction interfaces with other subunits

• Functional Assays:

  • Respirometry to measure oxygen consumption rates in cells expressing wild-type or mutant MT-ND4L

  • Measurement of NADH:ubiquinone oxidoreductase activity using isolated mitochondria

  • Proton pumping assays to assess the impact of mutations on proton translocation

• Recombinant Protein Systems:

  • Expression of recombinant MT-ND4L variants in bacterial or eukaryotic systems

  • Reconstitution of minimal Complex I subassemblies to assess assembly dynamics

  • In vitro translation of mitochondrially-encoded subunits including MT-ND4L

What are the methodological considerations when using recombinant Ommatophoca rossii MT-ND4L in comparative mitochondrial studies?

When utilizing recombinant Ommatophoca rossii MT-ND4L for comparative mitochondrial research, several methodological considerations are essential:

• Expression System Selection:

  • E. coli expression systems are commonly used for MT-ND4L recombinant proteins , but may lack post-translational modifications

  • Consider mammalian expression systems for studies requiring authentic modifications

  • Evaluate codon optimization needs based on the expression host

• Protein Solubility and Stability:

  • MT-ND4L is highly hydrophobic and forms the core of the transmembrane region

  • Specialized detergents or lipid nanodiscs may be required to maintain native conformation

  • Storage conditions typically require -20°C with minimized freeze-thaw cycles

• Functional Assessment Challenges:

  • MT-ND4L functions within the context of Complex I, requiring complementary subunits

  • Consider using mitochondrial hybrid systems where host mitochondrial MT-ND4L is replaced with the Ross seal variant

  • Control experiments should include appropriate species-matched components

• Comparative Analysis Framework:

  • Establish a standardized framework for comparing MT-ND4L function across species

  • Account for different physiological adaptations (e.g., Ross seal adaptations to deep-diving)

  • Include appropriate evolutionary distance corrections in comparative analyses

How can researchers effectively study the interaction between MT-ND4L and other Complex I subunits?

Investigating the interactions between MT-ND4L and other Complex I subunits requires specialized approaches:

• Proximity-Based Protein Interaction Methods:

  • BioID or APEX2 proximity labeling with MT-ND4L as the bait protein

  • Chemical cross-linking followed by mass spectrometry (XL-MS) to identify interaction interfaces

  • FRET-based assays using fluorescently labeled subunits to measure real-time interactions

• Reconstitution Experiments:

  • Stepwise reconstitution of Complex I subunits to determine assembly dependencies

  • Identification of minimal subunit requirements for stable MT-ND4L incorporation

  • Analysis of subcomplex stability in the presence of wild-type versus mutant MT-ND4L

• Structural Visualization Techniques:

  • Cryo-EM of intact Complex I with gold-labeled MT-ND4L

  • Hydrogen-deuterium exchange mass spectrometry to identify protected regions

  • Computational modeling of subunit interfaces based on evolutionary coupling analysis

• Genetic Approaches:

  • CRISPR-mediated tagging of MT-ND4L and interacting partners

  • Suppressor mutation analysis to identify compensatory changes in interacting subunits

  • Heterologous expression of chimeric proteins to map interaction domains

What experimental approaches best characterize the role of MT-ND4L in proton translocation and mitochondrial membrane potential?

The involvement of MT-ND4L in proton translocation requires sophisticated biophysical and biochemical approaches:

• Membrane Potential Measurements:

  • Fluorescent probe-based assays (TMRM, JC-1) in cells expressing wild-type or mutant MT-ND4L

  • Patch-clamp electrophysiology of mitochondrial membranes

  • Potentiometric dyes to visualize real-time changes in membrane potential

• Proton Pumping Assays:

  • pH-sensitive fluorescent proteins targeted to mitochondrial compartments

  • Reconstituted proteoliposomes containing purified Complex I with wild-type or variant MT-ND4L

  • Stop-flow spectroscopy to measure proton translocation kinetics

• Site-Directed Mutagenesis Strategies:

  • Systematic mutation of conserved residues in MT-ND4L transmembrane domains

  • Introduction of proton-sensitive amino acids at key positions

  • Creation of chimeric proteins combining domains from different species

• Bioenergetic Profiling:

  • High-resolution respirometry to measure oxygen consumption linked to proton pumping

  • ATP synthesis rates in response to membrane potential changes

  • Measurement of proton leak and its relationship to MT-ND4L structure

What are the optimal conditions for expressing and purifying recombinant MT-ND4L for structural studies?

Optimizing expression and purification of recombinant MT-ND4L requires careful consideration of its hydrophobic nature:

• Expression System Selection:

  • E. coli BL21(DE3) with specialized vectors for membrane protein expression

  • Cell-free expression systems to avoid inclusion body formation

  • Consideration of fusion partners (MBP, SUMO) to enhance solubility

• Induction Conditions:

  • Lower temperatures (16-18°C) to slow expression and improve folding

  • Reduced IPTG concentrations (0.1-0.5 mM) for gentler induction

  • Extended expression times (18-24 hours) at lower temperatures

• Membrane Extraction:

  • Detergent screening panel including DDM, LMNG, and specialized fos-choline detergents

  • Evaluation of lipid-detergent mixed micelles to maintain native-like environment

  • Gentle solubilization procedures with extended incubation times

• Purification Strategy:

  • Tandem affinity purification using His6-tag and secondary affinity tag

  • Size-exclusion chromatography in detergent or amphipol-exchanged conditions

  • Quality control by analytical ultracentrifugation to confirm monodispersity

Storage of purified MT-ND4L typically requires specialized conditions, including storage at -20°C with minimized freeze-thaw cycles . For structural studies, maintaining >80% purity (as assessed by SDS-PAGE and Coomassie blue staining) is essential .

How can researchers effectively compare MT-ND4L function across different species?

Comparative functional analysis of MT-ND4L across species requires standardized approaches:

• Sequence-Based Comparative Analysis:

  • Multiple sequence alignment of MT-ND4L from target species

  • Identification of conserved vs. variable regions

  • Prediction of functional consequences of sequence variations

• Heterologous Expression Systems:

  • Expression of MT-ND4L variants from different species in a common host system

  • Creation of chimeric proteins to map species-specific functional domains

  • Complementation assays in MT-ND4L-deficient cell lines

• Functional Measurements:

  • Standardized assays for NADH:ubiquinone oxidoreductase activity

  • Oxygen consumption rates using high-resolution respirometry

  • ROS production measurements under controlled conditions

• Structural Comparisons:

  • Homology modeling based on available Complex I structures

  • Molecular dynamics simulations to predict species-specific conformational differences

  • Thermal stability analysis of purified proteins from different species

A comparison table for experimental design might include:

What controls and validation steps are essential when working with recombinant MT-ND4L in functional assays?

Robust experimental design for MT-ND4L functional studies requires:

• Expression Validation:

  • Western blotting with antibodies specific to MT-ND4L or epitope tags

  • Mass spectrometry confirmation of protein identity

  • RT-qPCR to confirm transcript levels when using gene expression systems

• Functional Controls:

  • Inclusion of known functional MT-ND4L variants as positive controls

  • Inactivated MT-ND4L (site-directed mutagenesis of key residues) as negative controls

  • Species-matched reference samples for comparative studies

• Assay-Specific Controls:

  • Complex I inhibitors (rotenone, piericidin A) to confirm specificity

  • Mitochondrial uncouplers to distinguish proton gradient effects

  • Antioxidants to control for ROS-mediated effects

• Validation Approaches:

  • Multiple complementary assays measuring the same functional parameter

  • Dose-response experiments to establish physiological relevance

  • Rescue experiments to confirm direct causality

When using recombinant MT-ND4L for antibody validation, it is recommended to employ the protein specifically as a blocking agent to confirm antibody specificity with the corresponding antibody .

How should researchers interpret variations in MT-ND4L sequences between species in the context of functional conservation?

Interpreting cross-species MT-ND4L variations requires:

• Evolutionary Conservation Analysis:

  • Calculation of conservation scores for each amino acid position

  • Identification of absolutely conserved residues likely essential for function

  • Mapping of variable regions that may reflect species-specific adaptations

• Structure-Function Correlation:

  • Mapping sequence variations onto known structural models

  • Assessment of whether variations occur in functional domains

  • Evaluation of physiochemical property changes (hydrophobicity, charge)

• Adaptive Evolution Assessment:

  • Analysis of positive selection signatures in specific lineages

  • Correlation with environmental adaptations or metabolic requirements

  • Consideration of co-evolution with interacting subunits

• Functional Impact Prediction:

  • In silico prediction of functional consequences using SIFT, PolyPhen

  • Molecular dynamics simulations to predict structural impacts

  • Integration with experimental functional data where available

For example, when comparing Ommatophoca rossii MT-ND4L with human MT-ND4L, researchers should consider the physiological adaptations of Ross seals to deep diving and cold environments, which may influence mitochondrial function requirements.

What statistical approaches are most appropriate for analyzing MT-ND4L-related experimental data?

Statistical analysis of MT-ND4L experimental data should consider:

• Appropriate Statistical Tests:

  • Paired t-tests for before/after interventions on the same samples

  • ANOVA with post-hoc tests for multiple experimental conditions

  • Non-parametric alternatives when normality assumptions are violated

• Dealing with Variability:

  • Mixed-effects models to account for batch and biological replicate variation

  • Normalization strategies appropriate to the experimental design

  • Robust statistical methods resistant to outliers

• Sample Size Considerations:

  • Power analysis to determine adequate sample sizes

  • Correction for multiple testing when examining multiple parameters

  • Consideration of biological vs. technical replicates

• Advanced Analytical Methods:

  • Principal component analysis for complex multivariate datasets

  • Hierarchical clustering to identify patterns in functional data

  • Machine learning approaches for predictive modeling of structure-function relationships

When reporting results, researchers should include complete statistical details including test selection rationale, p-values, confidence intervals, and effect sizes.

How is MT-ND4L research contributing to our understanding of mitochondrial diseases?

MT-ND4L research is advancing mitochondrial disease understanding through:

• Disease Mechanism Elucidation:

  • Characterization of how specific mutations like T10663C (Val65Ala) contribute to LHON

  • Investigation of the relationship between MT-ND4L mutations and increased BMI in adults

  • Understanding how Complex I dysfunction contributes to mitochondrial disease pathogenesis

• Genotype-Phenotype Correlations:

  • Identification of mutation-specific clinical manifestations

  • Investigation of why some mutations affect specific tissues despite ubiquitous expression

  • Analysis of nuclear-mitochondrial genetic interactions affecting disease penetrance

• Therapeutic Target Identification:

  • Screening for compounds that can rescue MT-ND4L mutant phenotypes

  • Investigation of bypass mechanisms to restore electron transport

  • Development of mitochondrially-targeted antioxidants to mitigate ROS-related damage

• Diagnostic Biomarker Development:

  • Creation of functional assays to assess MT-ND4L-related Complex I dysfunction

  • Development of mutation-specific antibodies for research and diagnostics

  • Identification of downstream metabolic signatures of MT-ND4L dysfunction

What emerging technologies are transforming MT-ND4L research?

Cutting-edge technologies advancing MT-ND4L research include:

• High-Resolution Structural Methods:

  • Cryo-electron microscopy achieving near-atomic resolution of Complex I

  • Integrative structural biology combining multiple experimental approaches

  • Time-resolved structural studies capturing dynamic conformational changes

• Advanced Genetic Engineering:

  • Mitochondrial-targeted CRISPR/Cas9 systems for precise MT-ND4L editing

  • Base editing technologies for introducing specific MT-ND4L mutations

  • Allotopic expression strategies for nuclear-encoded MT-ND4L

• Single-Cell Technologies:

  • Single-cell proteomics to detect MT-ND4L in individual cells

  • Live-cell imaging of labeled MT-ND4L to track dynamics

  • Single-mitochondrion functional assays to assess heterogeneity

• Computational Approaches:

  • AlphaFold2 and similar AI-driven structure prediction for protein modeling

  • Molecular dynamics simulations at extended timescales

  • Systems biology integration of multi-omics data related to MT-ND4L function