Recombinant Balaena mysticetus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the biological function of MT-ND4L in mitochondrial complex I?

MT-ND4L (NADH dehydrogenase 4L) functions as an essential component of complex I in the mitochondrial respiratory chain. Methodologically, its function is determined through several approaches:

• Electron transport activity: MT-ND4L participates in the first step of the electron transport process, facilitating the transfer of electrons from NADH to ubiquinone within the inner mitochondrial membrane.

• Proton pumping assays: The protein contributes to creating an electrochemical gradient across the inner mitochondrial membrane by helping translocate protons.

• Energy production measurement: Through its role in oxidative phosphorylation, MT-ND4L indirectly supports ATP production, which can be quantified using luminescence-based ATP assays .

Complex I creates an unequal electrical charge across the inner mitochondrial membrane through the step-by-step transfer of electrons, and this difference in electrical charge provides the essential energy for ATP production. When studying MT-ND4L function, researchers should consider its interaction with other subunits of complex I and its position within the membrane domain of the complex .

What is the structure and sequence of recombinant Balaena mysticetus MT-ND4L?

The recombinant Balaena mysticetus (bowhead whale) MT-ND4L protein shares structural similarities with other mammalian MT-ND4L proteins. To analyze its structure:

• Primary structure analysis: While the exact sequence of Balaena mysticetus MT-ND4L is not provided in the search results, it can be compared to characterized sequences such as the Canis lupus MT-ND4L (MSMVYINIFLAFILSLMGMLVYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHLTLASMMPIVLLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC), which is 98 amino acids in length .

• Computational structure prediction: Homology modeling approaches can be used to predict structure based on templates from closely related species, as demonstrated for other MT-ND4L proteins using MODELLER software .

• Transmembrane domain prediction: MT-ND4L typically contains multiple transmembrane segments that can be predicted using algorithms such as TMHMM or HMMTOP.

Researchers should note that MT-ND4L is a highly hydrophobic protein embedded in the inner mitochondrial membrane, which affects experimental approaches for structural studies.

How should recombinant MT-ND4L proteins be stored and handled in laboratory settings?

Proper storage and handling of recombinant MT-ND4L is critical for maintaining protein integrity and experimental reproducibility:

• Storage temperature: Store at -20°C/-80°C upon receipt, with -80°C preferred for long-term storage .

• Aliquoting strategy: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade protein quality .

• Buffer composition: Optimal storage in Tris/PBS-based buffer with 6% Trehalose, pH 8.0 .

• Reconstitution protocol:

  • Briefly centrifuge vial prior to opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% recommended for optimal stability)

  • Aliquot for long-term storage

For working stocks, store aliquots at 4°C for up to one week, but avoid repeated freezing and thawing. When planning experiments, consider that protein activity may decrease over time even under optimal storage conditions, so freshly prepared protein is recommended for critical assays.

How can molecular dynamics simulations be utilized to study the impact of mutations in MT-ND4L on proton translocation?

Molecular dynamics (MD) simulations provide valuable insights into the functional consequences of mutations in MT-ND4L. To implement this approach:

• Homology modeling: Generate structural models of native and mutant MT-ND4L proteins using appropriate templates (e.g., respiratory complex I from Thermus thermophilus with 98% identity) .

• Transmembrane system building: Use Membrane Builder in platforms like CHARMM-GUI to create a realistic membrane system including:

  • Lipid bilayer composition

  • Pore water

  • Bulk water

  • Physiological ion concentrations (150 mM K⁺ and Cl⁻)

• Simulation parameters:

  • Timestep: 2 fs

  • Long-range electrostatics: Particle Mesh Ewald technique (PME)

  • Force field: Compatible with protein, lipids, water, and ions

  • Simulation length: Minimum 100 ns to observe conformational changes

• Analysis metrics:

  • RMSD/RMSF calculations to assess structural stability

  • Hydrogen bond network analysis to identify changes in proton pathways

  • Water molecule passage monitoring through the transmembrane region

Research has shown that mutations can disrupt proton translocation pathways through mechanisms such as the formation of aberrant hydrogen bonds. For example, studies of similar mutations showed interruption of translocation pathways by hydrogen bond formation between specific residues and restriction of water molecule passage through the transmembrane region .

What comparative approaches can reveal evolutionary adaptations in MT-ND4L across marine mammals?

To investigate evolutionary adaptations in MT-ND4L across marine mammals:

• Multiple sequence alignment: Align MT-ND4L sequences from diverse marine mammals including:

  • Balaena mysticetus (bowhead whale)

  • Balaenoptera bonaerensis (Antarctic minke whale)

  • Balaenoptera borealis (sei whale)

  • Megaptera novaeangliae (humpback whale)

  • Arctocephalus australis (South American fur seal)

  • Leptonychotes weddelli (Weddell seal)

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive selection

  • Implement programs like PAML, HyPhy, or MEME to detect episodic selection

• Structural mapping of variable sites:

  • Map conserved and variable residues onto 3D structural models

  • Correlate with functional domains, particularly those involved in proton translocation

• Physiological correlation:

  • Compare adaptive changes with species-specific diving capabilities

  • Analyze mitochondrial efficiency across species with different metabolic demands

This comparative approach allows researchers to understand how MT-ND4L has evolved to support the high oxygen demands and specialized metabolic needs of marine mammals that undergo prolonged dives and experience varying oxygen pressures.

What experimental approaches can be used to study the association between MT-ND4L mutations and mitochondrial diseases?

To investigate the pathogenic mechanisms of MT-ND4L mutations in mitochondrial diseases such as Leber hereditary optic neuropathy:

• Patient sample analysis:

  • Sequence MT-ND4L from patient samples to identify mutations

  • Quantify heteroplasmy levels using next-generation sequencing approaches

  • Correlate mutation load with clinical manifestations

• Functional assays:

  • Complex I activity measurements using spectrophotometric assays

  • Oxygen consumption rate determination using Seahorse XF analyzers

  • ATP production quantification under various substrate conditions

• Cellular models:

  • Generate cybrid cell lines containing patient-derived mitochondria

  • Introduce specific mutations using CRISPR-based mitochondrial DNA editing

  • Assess mitochondrial membrane potential using fluorescent dyes (JC-1, TMRM)

• Animal models:

  • Generate transgenic mice expressing mutant MT-ND4L

  • Evaluate tissue-specific effects, particularly in high-energy demanding tissues

  • Assess progression of phenotypes that mimic human disease

• Therapeutic screening:

  • Test compounds that may bypass complex I defects

  • Evaluate antioxidants that mitigate downstream effects of complex I dysfunction

  • Assess gene therapy approaches for mtDNA mutations

The identified T10663C mutation in MT-ND4L has been associated with Leber hereditary optic neuropathy and results in a valine to alanine substitution at position 65 (Val65Ala), providing a specific target for investigation .

How can recombinant MT-ND4L be integrated into structural studies of the complete mitochondrial complex I?

Structural investigation of mitochondrial complex I incorporating recombinant MT-ND4L requires:

• Protein preparation strategies:

  • Expression of recombinant MT-ND4L with appropriate tags (e.g., His-tag) for purification

  • Reconstitution into nanodiscs or liposomes to maintain native membrane environment

  • Co-expression with interacting partners to improve stability

• Structural determination techniques:

  • Cryo-electron microscopy (cryo-EM)

    • Sample vitrification protocols

    • Data collection parameters

    • Single-particle reconstruction approaches

  • X-ray crystallography

    • Lipidic cubic phase crystallization

    • Detergent screening for optimal crystal formation

  • NMR for specific domains or interactions

    • Isotope labeling strategies

    • Selective deuteration approaches

• Computational integration:

  • Molecular dynamics simulations to assess dynamics within the complex

  • Quantum mechanics/molecular mechanics (QM/MM) to study electron transfer

  • Docking studies to evaluate inhibitor binding

• Validation approaches:

  • Cross-linking mass spectrometry to confirm protein-protein interactions

  • Mutagenesis of key residues to verify functional importance

  • Hydrogen/deuterium exchange mass spectrometry to identify flexible regions

These approaches can help determine how MT-ND4L contributes to the fourth proton translocation pathway at the interface of the ND4L-ND6 subunit, as suggested by molecular dynamics studies .

What are the optimal conditions for expressing recombinant Balaena mysticetus MT-ND4L in E. coli systems?

For successful expression of recombinant MT-ND4L in E. coli:

• Expression vector selection:

  • Use vectors with strong inducible promoters (T7, tac)

  • Include appropriate tags (His-tag is commonly used) for purification

  • Consider codon optimization for E. coli expression

• Host strain optimization:

  • BL21(DE3) for general expression

  • C41(DE3) or C43(DE3) for membrane proteins

  • Rosetta or CodonPlus strains if codon bias is a concern

• Expression conditions:

  • Induction at OD600 0.6-0.8

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction temperature: 16-20°C for 16-20 hours (reduced temperature often improves membrane protein folding)

• Membrane fraction isolation:

  Step	Procedure	Parameters
  Cell lysis	Sonication or high-pressure homogenization	Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl
  Membrane separation	Ultracentrifugation	100,000 × g, 1 hour, 4°C
  Membrane solubilization	Detergent treatment	1% DDM or LMNG, 1 hour, 4°C
  Purification	IMAC chromatography	20 mM imidazole wash, 250 mM imidazole elution

• Quality control:

  • SDS-PAGE with Coomassie staining (>90% purity target)

  • Western blot with anti-His antibodies

  • Mass spectrometry to confirm protein identity

These conditions may require optimization for the specific properties of Balaena mysticetus MT-ND4L, as hydrophobic membrane proteins often present expression challenges.

How can researchers design experiments to investigate the proton translocation function of MT-ND4L?

To experimentally assess MT-ND4L's role in proton translocation:

• Proteoliposome reconstitution assays:

  • Purify recombinant MT-ND4L protein

  • Reconstitute into liposomes with defined lipid composition

  • Create pH gradient using buffer exchange

  • Monitor proton movement using pH-sensitive fluorescent dyes (ACMA, pyranine)

• Site-directed mutagenesis studies:

  • Target conserved residues potentially involved in proton pathways

  • Focus on residues identified in computational studies (e.g., Glu34)

  • Measure effects on proton translocation using the methods above

• Electrophysiological approaches:

  • Planar lipid bilayer recordings

  • Patch-clamp of mitochondrial membranes or proteoliposomes

  • Measurement of current changes under varying conditions

• Hydrogen/deuterium exchange mass spectrometry:

  • Map solvent-accessible regions

  • Identify potential water-filled channels

  • Compare exchange rates between wild-type and mutant proteins

• Complex I activity coupling:

  Measurement	Technique	Expected Result
  NADH oxidation	Spectrophotometric assay (340 nm)	Decreased rate with proton pathway disruption
  Membrane potential	Fluorescent dyes (TMRM, Rhodamine 123)	Reduced potential with impaired proton pumping
  Proton translocation	pH electrode or fluorescent probes	Altered H⁺/e⁻ stoichiometry with mutations

This multi-faceted approach allows researchers to correlate structural features with functional outcomes and determine how specific residues in MT-ND4L contribute to the proton translocation process that has been shown to be disrupted by mutations in related proteins .

How can researchers differentiate between primary effects of MT-ND4L mutations and secondary consequences in complex I dysfunction?

Distinguishing primary from secondary effects requires:

• Temporal analysis of consequences:

  • Measure immediate biophysical changes in protein structure

  • Track progressive alterations in complex I assembly/stability

  • Monitor delayed cellular responses to respiratory dysfunction

• Isolated vs. integrated approaches:

  • Study purified recombinant MT-ND4L (primary effects)

  • Examine assembled complex I (intermediate effects)

  • Assess whole mitochondrial function (secondary effects)

• Rescue experiments:

  • Expression of wild-type MT-ND4L in mutant backgrounds

  • Suppressor mutation screening

  • Pharmacological bypass of specific steps in electron transport

• Comparative analysis with known mutations:

  • Compare with established pathogenic mutations like T10663C (Val65Ala)

  • Analyze similar mutations in homologous positions across species

  • Correlate with mutations in interacting subunits

• Multi-omics integration:

  Approach	Technique	Information Gained
  Proteomics	Mass spectrometry	Changes in complex I subunit stoichiometry
  Metabolomics	LC-MS/GC-MS	Alterations in NADH/NAD+ ratio and downstream metabolites
  Transcriptomics	RNA-Seq	Compensatory gene expression responses

This systematic approach helps determine whether observed phenotypes result directly from altered MT-ND4L function or are downstream consequences of disrupted oxidative phosphorylation.

What are the most effective approaches for comparing MT-ND4L function across different species for evolutionary studies?

For comparative evolutionary analysis of MT-ND4L:

• Phylogenetic reconstruction:

  • Maximum likelihood or Bayesian methods

  • Time-calibrated trees using fossil evidence

  • Reconstruction of ancestral sequences at key evolutionary nodes

• Selection analysis:

  • Site-specific selection detection

  • Branch-site models to identify lineage-specific selection

  • Relaxation or intensification of selection analysis (RELAX)

• Structural comparison:

  • Homology modeling of MT-ND4L from diverse species

  • Comparative molecular dynamics simulations

  • Analysis of evolutionary rate variation mapped to structure

• Functional assays across species:

  • Standardized complex I activity measurements

  • Cross-species complementation experiments

  • Temperature and pH sensitivity profiles

• Correlation with ecological adaptations:

  Species Group	Adaptation	Expected MT-ND4L Features
  Deep-diving marine mammals (e.g., Balaena mysticetus)	Hypoxia tolerance	Modifications affecting proton leakage, oxygen affinity
  Arctic species	Cold adaptation	Altered thermal stability, modified lipid interactions
  Fast-swimming predators	High metabolic rate	Enhanced catalytic efficiency, thermal stability

By comparing recombinant MT-ND4L from various species like Balaena mysticetus, Balaenoptera borealis, Megaptera novaeangliae, and others available commercially , researchers can identify convergent adaptations and clade-specific innovations in mitochondrial function.

How might MT-ND4L contribute to unique metabolic adaptations in marine mammals like Balaena mysticetus?

Investigating MT-ND4L's role in the specialized metabolism of marine mammals:

• Hypoxia adaptation mechanisms:

  • Altered proton pumping efficiency during dive-induced hypoxia

  • Modified electron leak properties to reduce reactive oxygen species

  • Structural adaptations enhancing stability under pressure

• Tissue-specific expression patterns:

  • Compare MT-ND4L features in diving-relevant tissues (brain, heart, skeletal muscle)

  • Analyze potential post-translational modifications specific to marine mammals

  • Investigate tissue-specific interacting partners

• Thermal adaptation properties:

  • Cold-water adaptation features in Arctic species like Balaena mysticetus

  • Comparative thermal stability assays with terrestrial mammal homologs

  • Lipid interaction profiles optimized for different temperature ranges

• Experimental approaches:

  • Heterologous expression of Balaena mysticetus MT-ND4L in model systems

  • Mitochondrial function assessment under simulated diving conditions

  • Comparative respiratory measurements across temperature gradients

• Translational implications:

  Marine Mammal Adaptation	Potential Application	Research Approach
  Hypoxia tolerance	Ischemia-reperfusion injury protection	Human cell lines expressing whale MT-ND4L
  ROS management	Neurodegenerative disease models	Oxidative stress response comparison
  Pressure adaptation	High-pressure biological processes	Structural stability under pressure

Studying MT-ND4L from Balaena mysticetus may reveal unique adaptations that allow efficient mitochondrial function under the physiological extremes experienced during deep, prolonged dives in cold Arctic waters.

What are the implications of MT-ND4L research for developing mitochondrial therapeutics?

Research on MT-ND4L has significant translational potential:

• Therapeutic target identification:

  • Map disease-associated mutations like T10663C (Val65Ala) in Leber hereditary optic neuropathy

  • Identify druggable sites that could modulate proton translocation

  • Develop screening assays for compounds that may stabilize mutant proteins

• Gene therapy approaches:

  • Mitochondrially-targeted nucleic acid delivery systems

  • Allotopic expression of recoded MT-ND4L from the nucleus

  • CRISPR-based mitochondrial DNA editing strategies

• Bypass therapeutics:

  • Alternative electron transport chain entry points

  • Artificial electron carriers to bypass complex I defects

  • Short-circuit proton pumping pathways

• Structural insights for drug design:

  • Modeling of MT-ND4L interaction with other complex I subunits

  • Identification of small molecule binding pockets

  • Structure-based design of stabilizing compounds

• Biomarker development:

  Application	Methodology	Clinical Relevance
  Early disease detection	MT-ND4L mutation screening	Presymptomatic diagnosis of mitochondrial disorders
  Treatment monitoring	Complex I activity assays	Assessment of therapeutic efficacy
  Heteroplasmy quantification	Digital PCR	Determination of mutation threshold effects

Understanding the molecular mechanisms of MT-ND4L function, particularly how mutations disrupt proton translocation pathways , provides crucial information for developing targeted therapies for mitochondrial diseases like Leber hereditary optic neuropathy.