Recombinant Phoca hispida NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its normal function in cellular metabolism?

MT-ND4L (mitochondrially encoded NADH:ubiquinone oxidoreductase chain 4L) is a protein encoded by the mitochondrial genome that serves as a core subunit of Complex I in the electron transport chain. This protein is essential for the first step in electron transport during oxidative phosphorylation, facilitating the transfer of electrons from NADH to ubiquinone .

The protein is embedded in the inner mitochondrial membrane where it contributes to generating the electrochemical gradient necessary for ATP production. Complex I, which includes MT-ND4L, creates an unequal electrical charge across the inner mitochondrial membrane through the step-by-step transfer of electrons, providing the energy required for ATP synthesis .

In Phoca hispida specifically, the MT-ND4L protein consists of 98 amino acids with the sequence: MSMVYANIFLAFIMSIMGLLMYRSHLMSSLLCLEGMMLSLFVMMTVTILNNHFTLANMAPIILLVFAACEAALGLLLLVMVSNTYGTDYVQNLNLLQC .

How is MT-ND4L evolutionarily conserved across different species?

MT-ND4L shows significant conservation across mammalian species, reflecting its essential role in energy metabolism. Comparative analysis of amino acid sequences reveals high similarity between Phoca hispida (ringed seal) and Canis lupus (wolf/dog) , with both containing 98 amino acids in their full-length proteins.

The Canis lupus MT-ND4L sequence (MSMVYINIFLAFILSLMGMLVYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHLTLASMMPIVLLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) shares approximately 90% identity with the Phoca hispida sequence, with most variations occurring in regions not critical for electron transport function.

This high degree of conservation suggests strong evolutionary pressure to maintain the structure and function of this protein, which is consistent with its fundamental role in cellular energy production across diverse mammalian lineages.

What are the structural features of recombinant Phoca hispida MT-ND4L?

Recombinant Phoca hispida MT-ND4L is a full-length protein (amino acids 1-98) that maintains the structural features necessary for its function in Complex I. The protein contains:

• A highly hydrophobic transmembrane domain structure

• Conserved regions critical for electron transport

• The complete amino acid sequence: MSMVYANIFLAFIMSIMGLLMYRSHLMSSLLCLEGMMLSLFVMMTVTILNNHFTLANMAPIILLVFAACEAALGLLLLVMVSNTYGTDYVQNLNLLQC

When produced recombinantly, the protein typically includes a tag (commonly His-tag) to facilitate purification. This tag may be positioned at either the N-terminus or C-terminus, depending on the expression system and experimental requirements .

The recombinant protein's secondary structure contains multiple transmembrane alpha-helices that are critical for its proper integration into membranes during functional studies.

What are the optimal storage and handling conditions for recombinant MT-ND4L?

Based on manufacturer recommendations for similar recombinant proteins, the following storage and handling conditions are optimal for maintaining MT-ND4L stability and activity:

• Storage temperature: -20°C to -80°C for long-term storage

• Storage buffer: Tris-based buffer with 50% glycerol, pH approximately 8.0

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

• Avoid repeated freeze-thaw cycles to prevent protein degradation and loss of activity

For reconstitution of lyophilized protein:

• Briefly centrifuge the vial before opening

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

• Add glycerol to a final concentration of 5-50% for stabilization

• Aliquot for long-term storage to minimize freeze-thaw cycles

How can researchers use MT-ND4L in metabolomic studies?

MT-ND4L has significant applications in metabolomic research, particularly in understanding mitochondrial function and related metabolic pathways. Approaches include:

• Association studies with metabolite ratios: Research has revealed important associations between MT-ND4L variants and specific metabolite patterns. For example, the variant mt10689 G>A in MT-ND4L is associated with multiple metabolite ratios, particularly those involving phosphatidylcholine diacyl C36:6 (PC aa C36:6) .

• Glycerophospholipid metabolism analysis: MT-ND4L variants show strong associations with glycerophospholipid metabolism. Researchers can use recombinant MT-ND4L to investigate how structural changes in this protein affect lipid metabolism .

What experimental approaches are most effective for studying MT-ND4L function in mitochondrial Complex I?

Several experimental approaches have proven effective for studying MT-ND4L function:

• Reconstitution assays: Incorporating recombinant MT-ND4L into artificial membrane systems to assess electron transport capability.

• Complex I activity measurements: Using recombinant MT-ND4L in NADH:ubiquinone oxidoreductase activity assays to measure electron transfer rates.

• Site-directed mutagenesis: Creating specific mutations in recombinant MT-ND4L to study structure-function relationships, particularly those mutations observed in mitochondrial diseases .

• Comparative analysis of wild-type and mutant proteins: Evaluating protein stability and complex I assembly efficiency between normal and disease-associated variants .

• Protein-protein interaction studies: Using tagged recombinant MT-ND4L to identify binding partners within Complex I and potential regulatory proteins.

How are mutations in MT-ND4L associated with mitochondrial diseases?

MT-ND4L mutations have been implicated in several mitochondrial diseases:

• Leber hereditary optic neuropathy (LHON): A specific mutation in MT-ND4L, T10663C (Val65Ala), has been identified in families with LHON. This mutation changes valine to alanine at position 65 of the protein, potentially affecting Complex I function and leading to vision loss .

• Leigh disease: MT-ND4L mutations have been associated with Leigh disease, a severe neurological disorder characterized by progressive brain abnormalities. These mutations likely disrupt Complex I function, leading to energy deficiency in high-energy-demanding neural tissues .

• Multiple sclerosis (MS): While not directly mentioned in the search results for MT-ND4L, mutations in other ND genes including ND4 have been implicated in MS, suggesting potential roles for mitochondrial dysfunction in neuroinflammatory diseases .

• Other mitochondrial disorders: MT-ND4L mutations have been associated with several other conditions including cerebellar ataxia, dilated cardiomyopathy, and mitochondrial metabolism diseases .

How can recombinant MT-ND4L be used to study potential therapeutic approaches for mitochondrial diseases?

Recombinant MT-ND4L provides several advantages for therapeutic research:

• Drug screening platforms: Recombinant MT-ND4L can be used to establish high-throughput screening systems for compounds that might stabilize mutant protein or enhance residual Complex I activity.

• Structure-based drug design: With purified recombinant protein, researchers can perform structural studies to design molecules that specifically target disease-causing mutations.

• Gene therapy model development: Recombinant MT-ND4L can serve as a control in gene therapy approaches aimed at replacing mutant mitochondrial genes.

• Validation of mitochondrial targeted therapeutics: Using functional assays with recombinant MT-ND4L allows for validation of compounds designed to overcome mitochondrial dysfunction.

• Protein replacement strategies: Studying the feasibility of delivering functional recombinant MT-ND4L to mitochondria as a potential therapeutic approach.

What are the most effective expression systems for producing functional recombinant MT-ND4L?

Several expression systems have been used for MT-ND4L production, each with specific advantages:

• E. coli expression system: Most commonly used for MT-ND4L expression due to high yield and ease of manipulation. For example, Canis lupus MT-ND4L has been successfully expressed in E. coli with an N-terminal His-tag .

• Yeast expression systems: Advantageous for expressing mitochondrial proteins due to the presence of eukaryotic post-translational modification machinery.

• Cell-free expression systems: Useful for hydrophobic mitochondrial proteins like MT-ND4L that may be toxic to host cells.

• Mammalian cell expression: Provides the most native-like environment for proper folding and post-translational modifications, though typically with lower yields.

For optimal expression of hydrophobic mitochondrial proteins like MT-ND4L, considerations include:

• Using specialized E. coli strains designed for membrane proteins

• Including solubilizing tags (such as His, GST, or MBP)

• Optimizing codon usage for the expression host

• Controlling expression rate through reduced temperature or inducible promoters

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

Verifying the functional integrity of recombinant MT-ND4L can be accomplished through multiple complementary approaches:

• Structural assessment:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Limited proteolysis to evaluate proper folding

  • Size exclusion chromatography to assess oligomeric state

• Functional assays:

  • NADH:ubiquinone oxidoreductase activity measurements

  • Membrane integration assays

  • Electron transport chain functional reconstitution

• Interaction studies:

  • Co-immunoprecipitation with other Complex I subunits

  • Liposome incorporation efficiency

  • Blue native PAGE to assess complex formation

• Comparative analysis:

  • Comparing properties with native mitochondrial preparations

  • Evaluation against known functional parameters of Complex I

Functional recombinant MT-ND4L should demonstrate appropriate membrane integration, ability to participate in electron transport, and correct interaction with other Complex I components.

What are the emerging research areas involving MT-ND4L beyond traditional mitochondrial studies?

Several emerging research areas are expanding our understanding of MT-ND4L beyond its classical role:

• Metabolomic profiling: Recent research has revealed associations between MT-ND4L variants and specific metabolite patterns, particularly involving glycerophospholipids. This connection opens new avenues for understanding how mitochondrial genes influence broader metabolic networks .

• Connection to complex diseases: Beyond classical mitochondrial disorders, MT-ND4L may play roles in more common conditions. Research has linked mitochondrial dysfunction to neurodegenerative diseases, cancer, and metabolic disorders .

• Inter-organelle communication: Exploring how MT-ND4L and Complex I function influences communication between mitochondria and other cellular compartments.

• Mitochondrial-nuclear crosstalk: Investigating how MT-ND4L, as a mitochondrially-encoded protein, participates in the complex regulatory relationship between mitochondrial and nuclear genomes.

• Environmental adaptation: Studying MT-ND4L variations across species adapted to different environments (like Phoca hispida's adaptation to cold marine environments) may reveal insights into mitochondrial evolution and adaptation.

How might the study of species-specific MT-ND4L variants contribute to understanding evolutionary adaptation of mitochondrial function?

The study of species-specific MT-ND4L variants, such as those in Phoca hispida, offers unique insights into mitochondrial adaptation:

• Cold adaptation: Phoca hispida (ringed seal) has evolved to survive in extreme cold environments, which likely required adaptations in mitochondrial energy production. Comparative studies of MT-ND4L between cold-adapted species and others may reveal functional adaptations in Complex I.

• Diving physiology: Marine mammals like Phoca hispida experience prolonged hypoxia during dives, potentially leading to adaptive changes in electron transport chain components including MT-ND4L.

• Metabolic adaptation: Different ecological niches require specific metabolic adaptations. Comparing MT-ND4L sequences and functions across species can illuminate how mitochondrial adaptations support diverse metabolic demands.

• Evolutionary rate analysis: Studying the rate of MT-ND4L evolution across lineages can identify regions under selective pressure versus those that tolerate variation.

• Correlation with environmental factors: Associating MT-ND4L variants with environmental parameters (temperature, oxygen availability, dietary patterns) across species can reveal mechanisms of mitochondrial adaptation.