Recombinant Yarrowia lipolytica NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is the basic structure and function of Yarrowia lipolytica ND4L protein?

ND4L (NADH-ubiquinone oxidoreductase chain 4L) is a subunit of respiratory complex I in the mitochondrial electron transport chain. In Yarrowia lipolytica, ND4L is a small hydrophobic protein consisting of 89 amino acids with the sequence: MFIGTIILVLSFLGFVFNRRNIILAFICLETMLLGINLILLRNSVLFDDISGSLFAIVIIILAGVESAIGLSLLVSYYRLRGVINSYGI . The protein plays a crucial role in proton translocation across the inner mitochondrial membrane, contributing to the proton gradient necessary for ATP synthesis. Similar to ND4L proteins in other organisms, the Y. lipolytica ND4L forms part of the membrane domain of complex I and participates in energy conversion processes .

What is the evolutionary significance of ND4L across different species?

ND4L is encoded by the mitochondrial genome and shows considerable conservation across species, reflecting its essential role in cellular respiration. Phylogenetic analyses comparing ND4L sequences have been used to establish evolutionary relationships between different populations and species . For instance, research on Khorasan native chickens found no haplotype differences in their ND4L sequences, suggesting genetic homogeneity in this region. The lowest genetic distance was observed between Khorasan native chickens and other Asian chickens like Jiangbian, Lvenwv, and Red jungle fowl for both ND4 and ND4L genes, indicating their close evolutionary relationship . The conservation of key residues involved in proton translocation, such as Glu32 and Tyr59 (equivalent to Glu34 and Tyr157 in human numbering), across species like T. thermophilus, E. coli, and humans further emphasizes the evolutionary importance of this protein .

What are the optimal methods for expressing and purifying recombinant Y. lipolytica ND4L protein?

For optimal expression and purification of recombinant Y. lipolytica ND4L protein, the recommended approach involves heterologous expression in E. coli with an N-terminal His-tag . This system allows for efficient production and subsequent purification using metal affinity chromatography. The full-length ND4L protein (amino acids 1-89) should be cloned into an appropriate expression vector with the His-tag sequence, and expression can be induced in E. coli under standard conditions .

For purification, the following methodological approach is recommended:

• Harvest bacterial cells and lyse using appropriate buffer systems

• Purify using nickel or cobalt affinity chromatography

• Perform size-exclusion chromatography to enhance purity

• Verify purity using SDS-PAGE (target >90% purity)

• Lyophilize the purified protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0

For reconstitution of the lyophilized protein, centrifuge the vial briefly before opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol (recommended final concentration 50%) allows for long-term storage at -20°C/-80°C .

How can molecular dynamics simulations be applied to study ND4L function and mutations?

Molecular dynamics (MD) simulations provide powerful tools for investigating the effects of mutations on ND4L function and proton translocation mechanisms. A methodological framework for such studies includes:

• Mutation mapping: Determine amino acid changes caused by specific mutations of interest in the ND4L gene .

• Homology modeling: Create a structural model of ND4L (and binding partners like ND6) using appropriate templates. For example, respiratory complex I structures from organisms like Y. lipolytica can serve as templates with high sequence identity (>98%) .

• Model evaluation: Validate the model using tools like Ramachandran plots, QMEAN scores, and DOPE profile comparisons to ensure stereochemical quality .

• Transmembrane system building: Construct a realistic membrane system using tools like Membrane Builder in CHARMM-GUI, including lipid bilayers, explicit solvents (TIP3P water molecules), and physiological ion concentrations (K+ and Cl- at 150 mM) .

• Simulation setup: Perform MD simulations using appropriate software (e.g., AMBER) with a timestep of 2 fs, employing particle mesh Ewald techniques for long-range electrostatics .

• Analysis: Analyze trajectories using RMSD and RMSF calculations to assess structural stability and flexibility. Visualize results using software like VMD to observe potential proton translocation pathways and the effects of mutations .

• Hydrogen bond calculations: Quantify changes in hydrogen bonding patterns that may affect proton translocation .

This comprehensive approach allows researchers to visualize and quantify how mutations like T10609C and C10676G might disrupt normal proton translocation by affecting critical interactions between key residues such as Glu34 and Tyr157 .

What techniques are recommended for genetic analysis of ND4L in mitochondrial genome studies?

For genetic analysis of ND4L in mitochondrial genome studies, researchers should employ the following methodological approach:

• Sample collection and DNA extraction:

  • Collect appropriate biological samples (e.g., blood)

  • Store samples in EDTA-containing tubes at -20°C

  • Extract DNA using commercial kits or standard extraction protocols

  • Assess DNA quality via spectrophotometry (A260/A280 ratio) and agarose gel electrophoresis

• PCR amplification:

  • Design specific primers targeting the ND4L region

  • Example primers: Forward: 5'-TTCACATTCAGCAGCCTAGGACT-3'; Reverse: 5'-GCTTTAGGCAGTCATAGGTGTAGTC-3'

  • Optimize PCR conditions (example program: 94°C for 30s, 54°C for 35s, 72°C for 30s, 35 cycles, with initial denaturation at 94°C for 10 min and final extension at 72°C for 10 min)

  • Verify amplicon size by agarose gel electrophoresis (expected size for ND4L fragment: ~802 bp)

• Sequencing and analysis:

  • Purify PCR products

  • Perform Sanger sequencing or next-generation sequencing

  • Analyze sequences using bioinformatics tools:

    • Assess sequence quality and trim low-quality regions

    • Align sequences using tools like Clustal W or MUSCLE

    • Calculate nucleotide composition (e.g., for ND4L: A 30%, C 36%, G 10%, T 24%; with G+C frequency of 46% and A+T frequency of 54%)

    • Identify variants and mutations by comparison to reference sequences

• Phylogenetic analysis:

  • Construct phylogenetic trees using methods like UPGMA or Maximum Likelihood

  • Determine genetic distances between populations

  • Calculate haplotype diversity

This methodological framework enables effective genetic characterization of ND4L for evolutionary studies, population genetics, and identification of potentially pathogenic mutations.

How do specific mutations in ND4L affect proton translocation and respiratory complex function?

Mutations in ND4L can significantly disrupt proton translocation pathways and respiratory complex function through specific molecular mechanisms. Molecular dynamics studies of mutations such as T10609C (resulting in M47T amino acid change) and C10676G (resulting in C69W) have revealed several key effects:

• Disruption of water-mediated proton pathways: Both mutations cause interruption of the proton translocation pathway by inducing hydrogen bond formation between critical residues Glu34 and Tyr157. This altered interaction restricts the passage of water molecules through the transmembrane region that normally facilitates proton movement .

• Altered conformational dynamics: In native ND4L, the Glu34 residue adopts a downward conformation that allows water molecules to pass through and mediate proton translocation. Mutations can alter this conformation, disrupting the functional water channel .

• Changes in reactive oxygen species (ROS) production: Experimental studies using human cybrid cells have shown that mutations like T10609C affect ROS production, particularly under hypoxic conditions. Wild-type cells (ND4L:47Met) produced approximately 1.5-fold more H₂O₂ compared to mutant cells under 3% oxygen levels, indicating altered respiratory chain function .

• Impact on energy metabolism: Disruption of proton translocation directly affects the proton gradient across the inner mitochondrial membrane, which is essential for ATP synthesis. This can lead to reduced energy production and metabolic dysfunction in affected tissues .

These molecular effects help explain how ND4L mutations may contribute to mitochondrial disorders and conditions like high-altitude polycythemia, where alterations in complex I function and ROS production play important pathophysiological roles .

What is the association between ND4L mutations and neurodegenerative disorders?

While the search results don't directly address ND4L mutations in neurodegenerative disorders, they provide insights into the related ND4 gene, which belongs to the same respiratory complex. Multiple studies have identified novel mutations in the mitochondrial ND4 gene in patients with multiple sclerosis (MS), suggesting potential roles for complex I components in neurodegenerative processes .

Four significant missense mutations have been identified in the ND4 gene in MS patients:

• m.11150G>A causing p.A131T (alanine to threonine)

• m.11519A>C causing p.T254P (threonine to proline)

• m.11523A>C causing p.K255T (lysine to threonine)

• m.11527C>T causing p.H256L (histidine to leucine)

These mutations were found exclusively in MS patients and absent in healthy controls, suggesting a potential pathogenic role . Given the functional relationship between ND4 and ND4L in complex I, it is reasonable to hypothesize that ND4L mutations might similarly contribute to neurodegenerative conditions through disruption of mitochondrial energy metabolism, increased oxidative stress, or altered cellular respiration.

Research methodologies for investigating such associations should include:

• Comprehensive sequencing of mitochondrial DNA in patient cohorts

• Functional characterization of identified mutations using cellular and molecular models

• Assessment of bioenergetic consequences using respirometry and ATP production assays

• Evaluation of ROS production and oxidative damage markers

• Construction of cybrid cell lines to isolate effects of mitochondrial mutations

What experimental approaches can best determine the pathogenicity of novel ND4L variants?

To determine the pathogenicity of novel ND4L variants, researchers should implement a multi-faceted experimental approach:

• Genetic and population studies:

  • Sequence ND4L in large cohorts of patients and healthy controls

  • Calculate variant frequencies in different populations

  • Perform segregation analysis in families with suspected mitochondrial disorders

  • Apply bioinformatics tools to predict functional consequences of amino acid changes

• In silico structural and functional analysis:

  • Perform molecular dynamics simulations to predict effects on protein structure and function

  • Analyze conservation of affected residues across species

  • Model potential impacts on proton translocation pathways

• Cellular models:

  • Create cybrid cell lines harboring the variant of interest

  • Measure complex I activity and respiration rates

  • Assess ROS production using fluorescent probes (e.g., DCFDA)

  • Evaluate mitochondrial membrane potential

  • Measure ATP production under different conditions

  • Challenge cells with metabolic stress to unmask potential defects

• Biochemical characterization:

  • Express recombinant protein (wild-type and variant) in heterologous systems

  • Perform in vitro reconstitution experiments

  • Assess protein stability and interaction with other complex I components

  • Measure proton pumping activity in reconstituted proteoliposomes

• Animal models:

  • Create transgenic models expressing the variant

  • Characterize phenotypes across multiple systems

  • Perform tissue-specific assays of mitochondrial function

This comprehensive approach provides multiple lines of evidence to classify variants as pathogenic, likely pathogenic, or benign, following the American College of Medical Genetics guidelines for variant interpretation.

How can recombinant ND4L protein be used to study complex I assembly and function?

Recombinant ND4L protein serves as a valuable tool for investigating complex I assembly and function through several advanced research applications:

• Reconstitution studies: Purified recombinant ND4L protein can be combined with other complex I subunits to study the assembly process in vitro. His-tagged Y. lipolytica ND4L is particularly useful for these studies, as the tag allows for monitoring the incorporation of ND4L into the complex using pull-down assays or immunodetection methods.

• Interaction mapping: The recombinant protein can be used in cross-linking experiments or co-immunoprecipitation studies to identify and characterize specific interactions with other complex I subunits, particularly ND6, with which it forms a functional interface for proton translocation .

• Site-directed mutagenesis: Strategic mutations can be introduced into the recombinant ND4L to study the functional importance of specific residues. For example, mutations in the highly conserved Glu34 residue can help elucidate its role in proton translocation .

• Structural studies: Purified ND4L protein can contribute to structural determination efforts using techniques like cryo-electron microscopy or X-ray crystallography, especially when co-crystallized with interacting partners.

• Antibody development: The recombinant protein can be used to develop specific antibodies against ND4L, which are valuable tools for studying complex I assembly and localization in various cellular contexts.

• Functional reconstitution in proteoliposomes: Incorporating recombinant ND4L into artificial membrane systems allows for direct measurement of its contribution to proton translocation activities. This approach can be particularly useful for comparing wild-type and mutant forms of the protein .

For these applications, researchers should consider reconstituting the lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL and adding glycerol (final concentration 50%) for optimal stability during experimental procedures .

What are the most effective approaches for studying ND4L-mediated proton translocation in vitro?

To effectively study ND4L-mediated proton translocation in vitro, researchers should employ the following methodological approaches:

• Proteoliposome reconstitution system:

  • Reconstitute purified recombinant ND4L (alone or with ND6) into phospholipid vesicles with defined composition

  • Establish a pH gradient across the membrane using different buffer systems

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

  • Quantify proton flux rates under various conditions (substrate concentrations, inhibitors, membrane potential)

• Planar lipid bilayer electrophysiology:

  • Incorporate purified ND4L into planar lipid bilayers

  • Measure ion conductance using patch-clamp techniques

  • Characterize channel properties (selectivity, gating, voltage dependence)

  • Test effects of specific mutations on channel function

• Hydrogen/deuterium exchange mass spectrometry:

  • Monitor structural dynamics and solvent accessibility of ND4L

  • Identify regions involved in conformational changes during proton translocation

  • Compare exchange patterns between active and inhibited states

• Real-time monitoring using microfluidic devices:

  • Design microfluidic platforms with integrated sensors

  • Allow for rapid changes in experimental conditions

  • Provide continuous monitoring of proton translocation events

• Advanced spectroscopy techniques:

  • Employ FTIR difference spectroscopy to detect protonation/deprotonation events

  • Use time-resolved fluorescence to monitor conformational changes

  • Apply solid-state NMR to study dynamics of specific residues

• Combined computational-experimental approaches:

  • Integrate molecular dynamics simulations with experimental data

  • Visualize water molecule pathways through the transmembrane region

  • Correlate simulation predictions with experimental measurements of proton translocation efficiency

These methodologies provide complementary approaches to elucidate the molecular mechanisms of ND4L-mediated proton translocation, particularly focusing on the critical role of residues like Glu34 and their interactions with water molecules in the transmembrane region .

How can next-generation sequencing technologies enhance the study of ND4L genetic variants?

Next-generation sequencing (NGS) technologies significantly enhance the study of ND4L genetic variants through multiple advanced approaches:

These advanced NGS applications dramatically enhance our ability to detect, quantify, and functionally characterize ND4L variants, accelerating both basic research and clinical applications in the field of mitochondrial genetics.

What are the emerging therapeutic strategies targeting ND4L dysfunction in mitochondrial diseases?

While the search results don't directly address therapeutic strategies for ND4L dysfunction, we can extrapolate from current approaches to mitochondrial complex I disorders:

• Gene therapy approaches:

  • Mitochondrial gene replacement using adeno-associated virus (AAV) vectors

  • Allotopic expression (expressing mitochondrial genes from the nucleus)

  • CRISPR-based approaches for heteroplasmy shifting to reduce mutant mtDNA burden

  • RNA-based therapies to modulate expression or processing of ND4L

• Small molecule interventions:

  • Complex I bypass strategies using alternative electron carriers

  • Mitochondria-targeted antioxidants to mitigate ROS production resulting from ND4L dysfunction

  • Compounds that enhance residual complex I activity or stabilize partially assembled complexes

  • Molecules that specifically modulate proton translocation pathways

• Metabolic modulation:

  • Ketogenic diets to provide alternative energy substrates

  • Specific amino acid supplementation strategies

  • NAD+ precursors to enhance electron transport chain function

  • Inhibitors of mitochondrial fission to preserve network integrity in the presence of dysfunctional components

• Mitochondrial transplantation:

  • Direct replacement of damaged mitochondria with healthy organelles

  • Intercellular mitochondrial transfer enhancement

• Exercise intervention protocols:

  • Specialized training regimens to increase mitochondrial biogenesis

  • Adaptations to improve metabolic flexibility in tissues affected by ND4L dysfunction

Future research should focus on developing targeted approaches that specifically address the unique molecular consequences of ND4L mutations, particularly disruptions to the fourth proton translocation pathway at the ND4L-ND6 interface .

How might structural biology advances improve our understanding of ND4L function in respiratory complex I?

Recent and future advances in structural biology technologies promise to significantly enhance our understanding of ND4L function within respiratory complex I:

• Cryo-electron microscopy (cryo-EM) at atomic resolution:

  • Enables visualization of ND4L within the intact complex I at resolutions approaching 2Å

  • Allows identification of water molecules in the proton translocation pathway

  • Reveals dynamic interactions between ND4L and neighboring subunits

  • Captures different conformational states associated with the catalytic cycle

• Time-resolved structural methods:

  • X-ray free-electron lasers (XFELs) to capture ultrafast structural changes

  • Time-resolved cryo-EM to visualize conformational changes during proton pumping

  • Combining these approaches with rapid mixing or photocaging techniques to trigger specific steps in the proton translocation process

• Integrative structural biology approaches:

  • Combining multiple experimental techniques (cryo-EM, NMR, mass spectrometry)

  • Incorporating molecular dynamics simulations to model water dynamics

  • Using artificial intelligence to predict structural features and dynamic behaviors

  • Cross-linking mass spectrometry to map protein-protein interactions within complex I

• In situ structural studies:

  • Cryo-electron tomography of ND4L and complex I within native mitochondrial membranes

  • Correlative light and electron microscopy to link structural features with functional states

  • Visualizing complex I supercomplexes and their dynamic assembly/disassembly

• Single-molecule approaches:

  • FRET-based methods to monitor conformational changes in individual complex I molecules

  • High-speed atomic force microscopy to observe structural dynamics in real-time

  • Optical tweezers to measure force generation associated with proton pumping

These advanced structural approaches will provide unprecedented insights into how ND4L contributes to the fourth proton translocation pathway, particularly focusing on the critical roles of conserved residues like Glu34 and their interactions with water molecules and other subunits like ND6 .

What interdisciplinary approaches could advance our understanding of ND4L's role in cellular bioenergetics?

Advancing our understanding of ND4L's role in cellular bioenergetics requires innovative interdisciplinary approaches that bridge multiple scientific fields:

These interdisciplinary approaches provide complementary perspectives that together can create a more comprehensive understanding of ND4L's complex role in cellular bioenergetics.