Recombinant Nasalis larvatus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the functional role of MT-ND4L in mitochondrial metabolism?

MT-ND4L (mitochondrially encoded NADH dehydrogenase 4L) serves as a critical component of Complex I in the respiratory chain, enabling NADH dehydrogenase (ubiquinone) activity. The protein participates in the first step of the electron transport process by transferring electrons from NADH to ubiquinone . This electron transfer creates an unequal electrical charge on either side of the inner mitochondrial membrane, establishing the proton motive force necessary for ATP synthesis . The protein is embedded in the inner mitochondrial membrane as part of the larger Complex I assembly, which is essential for oxidative phosphorylation . Research methodologies to study its function typically involve mitochondrial isolation techniques, membrane potential assays, and oxygen consumption measurements.

How is MT-ND4L conserved across primate species?

Mitochondrial genes such as MT-ND4L typically show conservation across closely related species but with sufficient variation to be useful in evolutionary studies. Comparative analysis of mitochondrial sequences has been used to determine divergence times between primate species. For example:

Researchers investigating MT-ND4L conservation should employ multiple sequence alignment tools, calculate Ka/Ks ratios to assess selective pressure, and conduct phylogenetic analyses using maximum likelihood or Bayesian methods .

What experimental systems are available for studying recombinant Nasalis larvatus MT-ND4L?

The recombinant Nasalis larvatus MT-ND4L protein is commercially available with the following specifications:

For researchers developing their own expression systems, consideration should be given to the hydrophobic nature of this membrane protein. Expression systems might include bacterial systems with membrane-targeting sequences, insect cell systems for eukaryotic post-translational modifications, or cell-free systems optimized for membrane proteins.

How do mutations in MT-ND4L contribute to Leber hereditary optic neuropathy pathogenesis?

A specific mutation in MT-ND4L (T10663C or Val65Ala) has been identified in several families with Leber hereditary optic neuropathy (LHON) . This mutation changes a single amino acid in the protein, replacing valine with alanine at position 65 . While the exact mechanism linking this mutation to LHON remains unclear, researchers investigating this connection should consider:

• Mitochondrial respiratory chain activity assays to measure Complex I function

• ROS (reactive oxygen species) production measurements in cells carrying the mutation

• ATP synthesis capacity assessments

• In silico modeling of protein structure changes caused by the mutation

• Patient-derived cellular models including fibroblasts or induced pluripotent stem cells differentiated into retinal ganglion cells

The relationship between MT-ND4L mutations and LHON represents an important area for future research, as the pathophysiological mechanisms remain incompletely understood .

What methodological considerations are important for expressing and purifying recombinant Nasalis larvatus MT-ND4L?

When expressing and purifying recombinant Nasalis larvatus MT-ND4L, researchers should consider:

• Expression system selection: Membrane proteins like MT-ND4L typically require specialized expression systems. Options include:

  • E. coli with special membrane protein-friendly strains (C41, C43)

  • Insect cell systems (Sf9, High Five)

  • Mammalian cell systems for proper folding and post-translational modifications

• Tag selection: The commercial recombinant protein notes that "tag type will be determined during production process" , suggesting optimization is needed. Consider:

  • N-terminal vs. C-terminal tags based on protein topology

  • His-tag for IMAC purification

  • Fusion partners (MBP, GST) to enhance solubility

• Extraction and purification:

  • Detergent screening is critical (DDM, LMNG, digitonin)

  • Purification under native conditions to maintain structure

  • Consideration of lipid nanodiscs or amphipols for stabilization

• Storage considerations:

  • The commercial protein is stored in 50% glycerol in a Tris-based buffer

  • Avoid repeated freeze-thaw cycles

  • Consider aliquoting and storing at -80°C for long-term stability

How can researchers effectively analyze MT-ND4L integration into Complex I assemblies?

Analyzing MT-ND4L integration into Complex I requires specialized approaches:

• Blue Native PAGE (BN-PAGE) for intact complex isolation and analysis

• Two-dimensional BN-PAGE/SDS-PAGE to resolve individual subunits

• Immunoprecipitation with antibodies against other Complex I components

• Crosslinking mass spectrometry (XL-MS) to identify protein-protein interactions

• Cryo-electron microscopy for structural determination

Researchers should consider the following experimental workflow:

Since Complex I contains approximately 45 subunits in mammals, comprehensive analysis requires multiple complementary approaches to fully characterize MT-ND4L's role in the assembly and function of the complex.

What approaches can be used to study the evolutionary rate of MT-ND4L in primates?

Studying the evolutionary rate of MT-ND4L in primates requires both computational and experimental approaches:

• Sequence acquisition and alignment:

  • Collect MT-ND4L sequences from multiple primate species

  • Perform multiple sequence alignment using MUSCLE or MAFFT

  • Ensure proper codon alignment for coding sequence analysis

• Evolutionary rate analysis:

  • Calculate nucleotide substitution rates using maximum likelihood methods

  • Determine Ka/Ks (dN/dS) ratios to assess selective pressure

  • Apply RelTime method for dating evolutionary events

• Phylogenetic reconstruction:

  • Use Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods

  • Implement models such as the Tamura-Nei model

  • Perform bootstrap analysis (1,000 replicates) to assess branch support

• Calibration approaches:

  • Use established divergence times as calibration points

  • For example, researchers have used the 6.57 MYA divergence between SG R. roxellana and P. nigripes as a calibration point

Such analyses have revealed valuable insights into primate evolution, such as the divergence of Rhinopithecus genus species between 2.23 and 2.64 Ma .

How can researchers investigate the impact of MT-ND4L variants on mitochondrial function?

Investigating MT-ND4L variants requires a multi-faceted approach:

• In vitro functional assays:

  • Complex I enzyme activity measurements using standardized spectrophotometric methods

  • ROS production assessment using fluorescent probes

  • Membrane potential measurements using potentiometric dyes

  • Oxygen consumption rate analysis using respirometry

• Cell-based models:

  • Generation of cybrid cell lines with specific MT-ND4L variants

  • CRISPR-based approaches for introducing variants in model systems

  • Patient-derived cells harboring natural variants

• Analysis techniques:

  • Seahorse XF analysis for real-time measurement of cellular respiration

  • Fluorescence lifetime imaging for NAD(P)H redox state assessment

  • In-gel activity assays for Complex I function

  • Proteomic analysis of Complex I assembly states

• Clinical correlation:

  • Association studies between specific variants and disease phenotypes

  • For example, the Val65Ala variant has been associated with Leber hereditary optic neuropathy

What quality control methods should be employed when working with recombinant MT-ND4L?

When working with recombinant Nasalis larvatus MT-ND4L, researchers should implement the following quality control measures:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining

  • Western blot with specific antibodies

  • Mass spectrometry for identity confirmation

• Functional validation:

  • NADH:ubiquinone oxidoreductase activity assays

  • Reconstitution into proteoliposomes for functional studies

• Storage stability monitoring:

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • For extended storage, keep at -20°C or -80°C

• Structural integrity:

  • Circular dichroism to assess secondary structure

  • Thermal stability assays

  • Limited proteolysis to verify proper folding

How should researchers design experiments to compare MT-ND4L function across different primate species?

Comparative analysis of MT-ND4L across primate species requires careful experimental design:

• Sequence-based considerations:

  • Construct a comprehensive phylogenetic tree using complete mitochondrial sequences

  • Identify conserved and variable regions for targeted functional studies

  • Calculate evolutionary rates using appropriate models (e.g., Tamura-Nei model)

• Functional assays:

  • Use standardized biochemical assays to enable direct comparison

  • Implement identical expression systems for all species variants

  • Control for cell background effects using identical host cells

• Structural biology approaches:

  • Compare predicted protein structures using homology modeling

  • Identify species-specific differences in critical functional domains

  • Consider co-evolution with interacting proteins

• Evolutionary context:

  • Correlate functional differences with speciation events and habitat adaptations

  • Consider the divergence time between species (e.g., Rhinopithecus species diverged approximately 2.23-2.64 Ma)

This experimental framework allows for meaningful cross-species comparisons that can provide insights into both functional conservation and species-specific adaptations.

What emerging technologies might enhance MT-ND4L research?

Several emerging technologies show promise for advancing MT-ND4L research:

• Single-particle cryo-electron microscopy for high-resolution structural studies of Complex I

• AlphaFold and other AI-based protein structure prediction tools for modeling MT-ND4L interactions

• CRISPR-based mitochondrial genome editing for creating precise mutations

• Nanopore sequencing for rapid mitochondrial genome analysis

• Microfluidic approaches for high-throughput functional screening of variants

• In-cell NMR for studying protein-protein interactions in their native environment

These technological advances will enable researchers to address previously intractable questions about MT-ND4L structure, function, and role in disease.

What are the potential therapeutic applications of MT-ND4L research?

Research on MT-ND4L has significant therapeutic implications:

• Gene therapy approaches for mitochondrial diseases:

  • Targeted treatments for Leber hereditary optic neuropathy associated with MT-ND4L mutations

  • Development of allotopic expression strategies to express mitochondrial genes from the nucleus

• Drug discovery opportunities:

  • Identification of compounds that can modulate Complex I activity

  • Development of therapies that can bypass Complex I defects

  • Mitochondrial-targeted antioxidants to address ROS production in mutant cells

• Biomarker development:

  • Use of MT-ND4L variants as diagnostic or prognostic markers for mitochondrial diseases

  • Application in personalized medicine approaches for patients with mitochondrial dysfunction

As our understanding of MT-ND4L function expands, so too will the potential therapeutic applications of this research.