Recombinant Pantholops hodgsonii NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the biological function of MT-ND4L in Pantholops hodgsonii?

MT-ND4L (mitochondrially encoded NADH:ubiquinone oxidoreductase chain 4L) enables NADH dehydrogenase (ubiquinone) activity in the mitochondrial respiratory chain. It plays a critical role in mitochondrial electron transport from NADH to ubiquinone and is involved in proton motive force-driven mitochondrial ATP synthesis . In Pantholops hodgsonii (Chiru), this protein is localized to the mitochondrial inner membrane and functions as part of respiratory chain complex I . The protein's amino acid sequence consists of 98 amino acids with a sequence of MSLVYMNIMTAFAVSLGLLMYRSHLMSSLLCLEGMMLSLFVMATLMILNSHFTLASMMP IILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC .

How is recombinant Pantholops hodgsonii MT-ND4L typically expressed and purified for research use?

Recombinant Pantholops hodgsonii MT-ND4L protein can be expressed in bacterial expression systems such as E. coli, similar to homologous proteins from other species . For optimal expression, the coding sequence is typically cloned into appropriate expression vectors like pcDNA3.1+/C-(K)DYK . The protein can be tagged with affinity tags such as His-tag to facilitate purification . The purification process typically involves cell lysis followed by affinity chromatography, with the final product often formulated in Tris-based buffer with glycerol for stability . The purified protein is generally available as a lyophilized powder or in solution with recommended storage at -20°C/-80°C . For long-term storage, aliquoting is necessary to avoid repeated freeze-thaw cycles that can compromise protein integrity .

What are the structural characteristics of MT-ND4L from Pantholops hodgsonii?

MT-ND4L from Pantholops hodgsonii is a small hydrophobic protein comprising 98 amino acids . It contains multiple transmembrane domains characteristic of mitochondrial inner membrane proteins . The protein's structure features several highly conserved regions that are essential for interaction with other subunits of the respiratory chain complex I. As a membrane-integrated protein, MT-ND4L contains primarily hydrophobic amino acid residues arranged in an alpha-helical conformation to span the lipid bilayer. The tertiary structure allows for proper positioning within the respiratory complex, facilitating electron transfer and proton pumping activities essential for oxidative phosphorylation.

What are the optimal conditions for working with recombinant Pantholops hodgsonii MT-ND4L in functional assays?

When conducting functional assays with recombinant Pantholops hodgsonii MT-ND4L, researchers should maintain protein stability by avoiding repeated freeze-thaw cycles . For reconstitution, the lyophilized protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage stability .

For functional assays measuring NADH dehydrogenase activity, optimal conditions typically include:

• Buffer composition: 50 mM phosphate buffer (pH 7.4-7.8) containing 0.1-0.2 mM NADH and appropriate ubiquinone analogs (such as CoQ1 or decylubiquinone)

• Temperature: 30-37°C depending on the specific assay

• Detergent: Low concentrations (0.01-0.05%) of mild detergents such as n-dodecyl-β-D-maltoside to maintain protein solubility without denaturing

• Incubation time: Short incubation periods (5-15 minutes) to minimize protein degradation

• Protective agents: Addition of protease inhibitors and reducing agents (such as DTT or β-mercaptoethanol) to prevent oxidative damage

For activity measurements, spectrophotometric monitoring of NADH oxidation at 340 nm or coupling to electron acceptors such as cytochrome c can provide reliable quantification of enzyme function.

How can I verify the identity and purity of recombinant Pantholops hodgsonii MT-ND4L?

To verify the identity and purity of recombinant Pantholops hodgsonii MT-ND4L, researchers should employ a combination of analytical techniques:

• SDS-PAGE analysis: The protein should appear as a single band at the expected molecular weight (~10-11 kDa), with purity greater than 90% as determined by densitometric analysis .

• Western blotting: Using antibodies specific to MT-ND4L or to the affinity tag (such as His-tag), researchers can confirm protein identity through immunodetection.

• Mass spectrometry: Peptide mass fingerprinting or tandem mass spectrometry can provide definitive identification of the protein by matching observed peptide masses or sequences with theoretical values derived from the known amino acid sequence (MSLVYMNIMTAFAVSLGLLMYRSHLMSSLLCLEGMMLSLFVMATLMILNSHFTLASMMP IILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) .

• N-terminal sequencing: Edman degradation can verify the first 10-15 amino acids of the protein, confirming proper expression and processing.

• Functional assays: NADH dehydrogenase activity measurements can provide functional verification of properly folded protein.

A typical verification protocol should include at minimum SDS-PAGE analysis followed by either Western blotting or mass spectrometry, with functional assays providing additional confirmation of protein integrity.

What are the recommended storage conditions for maintaining MT-ND4L stability and activity?

For optimal stability and activity maintenance of Pantholops hodgsonii MT-ND4L, the following storage conditions are recommended:

• Long-term storage: Store at -20°C/-80°C in small aliquots to prevent repeated freeze-thaw cycles .

• Buffer composition: Tris-PBS-based buffer with 50% glycerol, pH 8.0, or Tris-based buffer with 6% trehalose .

• Working aliquots: Store at 4°C for up to one week to minimize degradation from repeated freezing and thawing .

• Reconstitution: When reconstituting lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL, adding 5-50% glycerol as a cryoprotectant before aliquoting .

• Transport conditions: For short-term transport between laboratories, maintain temperature at 4°C with ice packs or in dry ice for longer durations.

How can MT-ND4L from Pantholops hodgsonii be used to study mitochondrial disorders like Leber hereditary optic neuropathy?

MT-ND4L has been implicated in Leber hereditary optic neuropathy (LHON) and diabetes mellitus . Researchers can utilize recombinant Pantholops hodgsonii MT-ND4L for comparative studies with human variants to elucidate structure-function relationships relevant to these disorders.

Methodological approaches include:

• Site-directed mutagenesis: Introducing LHON-associated mutations into the recombinant Pantholops hodgsonii MT-ND4L to create disease models.

• Functional complementation studies: Expressing wild-type or mutant Pantholops hodgsonii MT-ND4L in cell lines lacking functional MT-ND4L to assess rescue of respiratory chain function.

• Structural analysis: Using purified recombinant protein for crystallography or cryo-EM studies to understand how mutations affect protein structure.

• Protein-protein interaction studies: Using methods such as co-immunoprecipitation or proximity labeling to identify interaction partners and how these interactions are altered in disease states.

• In vitro assembly studies: Reconstituting respiratory chain complex I with wild-type or mutant MT-ND4L to determine effects on complex formation and stability.

The high sequence conservation between species makes Pantholops hodgsonii MT-ND4L a valuable model for studying human mitochondrial disorders, with the advantage of easier recombinant expression compared to human mitochondrial proteins.

What techniques can be used to study the interaction of MT-ND4L with other components of respiratory chain complex I?

To investigate interactions between Pantholops hodgsonii MT-ND4L and other components of respiratory chain complex I, researchers can employ several complementary techniques:

• Blue Native PAGE (BN-PAGE): This technique allows for the separation of intact respiratory complexes and can be combined with second-dimension SDS-PAGE to identify individual subunits and their stoichiometry.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinkers can capture transient interactions, which are then identified through mass spectrometry to map interaction interfaces between MT-ND4L and other complex I components.

• Co-immunoprecipitation (Co-IP): Using antibodies against MT-ND4L or other complex I components to pull down intact complexes and identify interacting partners.

• Proximity labeling methods: Techniques such as BioID or APEX2 can be used by fusing these enzymes to MT-ND4L to biotinylate nearby proteins, which can then be purified and identified.

• Yeast two-hybrid or split-reporter assays: Modified versions can be used for membrane proteins to screen for binary interactions between MT-ND4L and other complex components.

• Förster Resonance Energy Transfer (FRET): Fluorescently labeled MT-ND4L and potential interaction partners can reveal proximity in intact cells or reconstituted systems.

• Cryo-electron microscopy: For structural studies of the entire complex I with specifically labeled MT-ND4L to determine its position and orientation within the complex.

What are the challenges in expressing and purifying functional MT-ND4L, and how can these be overcome?

Expressing and purifying functional mitochondrial membrane proteins like MT-ND4L presents several challenges due to their hydrophobic nature and requirement for proper membrane insertion. Specific challenges and solutions include:

• Challenge: Toxicity to expression hosts due to membrane disruption
  Solution: Use of specialized strains (such as C41/C43 E. coli) designed for membrane protein expression, or inducible expression systems with tight regulation .

• Challenge: Protein misfolding and aggregation
  Solution: Expression at reduced temperatures (16-25°C), co-expression with chaperones, or fusion with solubility-enhancing tags such as MBP or SUMO.

• Challenge: Low yield of functional protein
  Solution: Optimization of codon usage for the expression host, use of stronger promoters, and optimization of induction conditions (IPTG concentration, induction time).

• Challenge: Difficulty in extracting from membranes
  Solution: Screening of multiple detergents (DDM, LDAO, Triton X-100) at various concentrations for optimal solubilization while maintaining native structure.

• Challenge: Maintaining stability during purification
  Solution: Inclusion of lipids or lipid-like molecules during purification, use of stabilizing additives such as glycerol or trehalose .

• Challenge: Verifying functional activity
  Solution: Development of robust activity assays that can be performed in detergent solutions or after reconstitution into liposomes.

How does Pantholops hodgsonii MT-ND4L compare with homologous proteins from other species?

Comparative analysis of Pantholops hodgsonii MT-ND4L with homologous proteins from other species reveals important evolutionary patterns and functional conservation. While specific alignment data for Pantholops hodgsonii MT-ND4L isn't provided in the search results, we can infer comparisons based on available information from related species.

The amino acid sequence of Pantholops hodgsonii MT-ND4L (MSLVYMNIMTAFAVSLGLLMYRSHLMSSLLCLEGMMLSLFVMATLMILNSHFTLASMMP IILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) shows characteristic features of MT-ND4L proteins. The length (98 amino acids) is consistent with MT-ND4L proteins across mammalian species .

When compared with the MT-ND4L protein from Oryzomys albigularis (amino acid sequence: MSPIYINLMMAFIFSLLGTLLFRSHLMSTLLCLEGMMLSLFIMVTSSALNTQSMITYVIP ITMLVFGACEAAIGLALLVMISNTYGTDYVQNLNLLQC) , we can observe:

• High conservation in the transmembrane regions, particularly in the central LEGMMLSLF motif

• Conservation of the C-terminal region (TYGTDYVQNLNLLQC)

• Species-specific variations primarily in the N-terminal region and in some residues of the hydrophobic domains

These patterns of conservation suggest functional constraints on the protein structure, particularly in regions involved in:

• Interaction with other complex I subunits

• Proton pumping and electron transfer functions

• Membrane integration and complex assembly

The high sequence similarity between species reflects the essential role of MT-ND4L in mitochondrial respiration and the strong evolutionary pressure to maintain this function across diverse mammalian lineages.

What unique adaptations might be found in MT-ND4L from Pantholops hodgsonii, a high-altitude mammal?

Pantholops hodgsonii (Chiru) is a high-altitude mammal native to the Tibetan plateau, living at elevations of 3,700-5,500 meters. At such altitudes, oxygen availability is significantly reduced, creating selective pressure for adaptations in proteins involved in oxygen utilization and energy metabolism.

Although the search results don't explicitly describe high-altitude adaptations in Pantholops hodgsonii MT-ND4L, we can discuss potential adaptations based on scientific understanding of high-altitude physiology:

Methodological approaches to investigate these adaptations would include:

• Comparative sequence analysis with MT-ND4L from low-altitude bovids to identify potential high-altitude adaptive mutations

• Functional studies comparing the activity of recombinant Pantholops hodgsonii MT-ND4L with homologs from low-altitude relatives under varying oxygen concentrations

• Thermal stability assays to assess protein resilience to temperature fluctuations

• Molecular dynamics simulations to predict the functional consequences of Pantholops hodgsonii-specific amino acid substitutions

• Generation of chimeric proteins exchanging domains between Pantholops hodgsonii and low-altitude species to identify regions contributing to functional differences

Such studies could provide insights into molecular adaptations to high-altitude environments and potentially inform therapeutic approaches for hypoxia-related human diseases.

How can recombinant MT-ND4L be used to study mitochondrial dysfunction in diabetes mellitus?

MT-ND4L has been implicated in diabetes mellitus , making recombinant Pantholops hodgsonii MT-ND4L a valuable tool for studying the role of mitochondrial dysfunction in this disease. Researchers can employ several methodological approaches:

• Cell culture models: Introduce wild-type or mutated recombinant MT-ND4L into pancreatic β-cell lines to assess effects on:

  • Insulin secretion in response to glucose stimulation

  • Mitochondrial membrane potential and ATP production

  • Reactive oxygen species (ROS) generation

  • Cell viability and apoptosis pathways

• Reconstitution studies: Incorporate purified recombinant MT-ND4L into artificial membrane systems with other complex I components to measure:

  • NADH:ubiquinone oxidoreductase activity under normal and hyperglycemic conditions

  • Proton pumping efficiency

  • ROS production at different glucose concentrations

• Proteomic interaction studies: Use tagged recombinant MT-ND4L to identify:

  • Changes in protein-protein interactions under diabetic conditions

  • Post-translational modifications that may occur in diabetic states

  • Altered assembly of respiratory complexes

• Comparative studies with diabetes-associated mutations:

  • Generate recombinant MT-ND4L proteins with mutations identified in diabetic patients

  • Compare functional properties with wild-type protein

  • Assess impacts on complex I assembly and function

These approaches can help elucidate the molecular mechanisms by which MT-ND4L dysfunction contributes to diabetes pathogenesis, potentially identifying novel therapeutic targets for mitochondrial-targeted interventions in diabetes treatment.

What experimental protocols are most effective for studying MT-ND4L mutations associated with Leber hereditary optic neuropathy?

Leber hereditary optic neuropathy (LHON) is a mitochondrial disorder associated with mutations in complex I genes, including MT-ND4L . To study LHON-associated MT-ND4L mutations, the following experimental protocols are particularly effective:

• Site-directed mutagenesis and heterologous expression:

  • Introduction of LHON-associated mutations into recombinant Pantholops hodgsonii MT-ND4L using overlap extension PCR

  • Expression in bacterial or mammalian systems with careful optimization of expression conditions

  • Purification using affinity chromatography with His-tag or other fusion tags

• Functional characterization:

  • Complex I activity assays measuring NADH:ubiquinone oxidoreductase activity

  • Oxygen consumption measurements in reconstituted systems

  • ROS production quantification using fluorescent probes

  • Membrane potential assessments using potentiometric dyes

• Structural impact assessment:

  • Circular dichroism spectroscopy to determine secondary structure changes

  • Limited proteolysis to assess conformational alterations

  • Thermal stability assays to determine effects on protein stability

  • Molecular dynamics simulations to predict structural consequences of mutations

• Cellular models:

  • Cybrid cell lines incorporating mitochondria with MT-ND4L mutations

  • Evaluation of neuron-specific effects using differentiated neuronal cell models

  • Assessment of retinal ganglion cell vulnerability to MT-ND4L mutations

• Therapeutic screening platforms:

  • High-throughput screening for compounds that rescue complex I function

  • Testing of mitochondrial-targeted antioxidants on mutant MT-ND4L function

  • Evaluation of gene therapy approaches using wild-type MT-ND4L

What are the emerging techniques that could advance our understanding of MT-ND4L function?

Several emerging techniques show promise for advancing our understanding of MT-ND4L function and its role in mitochondrial disorders:

• Cryo-electron microscopy (Cryo-EM): Recent advances in cryo-EM technology allow for near-atomic resolution of membrane protein complexes, enabling detailed structural analysis of MT-ND4L within the context of the complete respiratory chain complex I.

• Single-molecule techniques: Methods such as single-molecule FRET or atomic force microscopy can provide insights into the dynamics and conformational changes of MT-ND4L during the catalytic cycle.

• Nanoscale respirometry: Microfluidic devices capable of measuring oxygen consumption in small samples can enable high-throughput functional analysis of MT-ND4L variants.

• CRISPR-based mitochondrial genome editing: Recent developments in mitochondrial DNA editing technologies could allow for precise manipulation of MT-ND4L in cellular and animal models.

• In situ structural analysis: Techniques such as proximity labeling combined with mass spectrometry can map the interaction network of MT-ND4L within intact mitochondria.

• Computational approaches: Advanced molecular dynamics simulations and machine learning algorithms can predict the functional consequences of MT-ND4L mutations and identify potential therapeutic molecules.

• Mitochondrial proteomics: Quantitative proteomics approaches can identify changes in the mitochondrial proteome resulting from MT-ND4L mutations or alterations in expression levels.

These emerging technologies will enable researchers to bridge current knowledge gaps and develop more effective therapeutic strategies for mitochondrial disorders involving MT-ND4L dysfunction.

What experimental design considerations are most important when comparing wild-type and mutant forms of MT-ND4L?

When designing experiments to compare wild-type and mutant forms of MT-ND4L, researchers should consider the following critical factors:

• Expression system selection:

  • Bacterial systems offer high yield but may lack post-translational modifications

  • Mammalian expression systems provide more native-like protein but with lower yield

  • Cell-free systems allow for the incorporation of unnatural amino acids for specialized studies

• Mutation selection strategy:

  • Include known disease-associated mutations

  • Consider evolutionary conserved residues

  • Include control mutations in non-conserved regions

  • Create alanine-scanning libraries for comprehensive functional mapping

• Protein purification considerations:

  • Standardize purification protocols across all variants

  • Verify equal purity by SDS-PAGE and other analytical methods

  • Quantify protein concentration using multiple methods to ensure accuracy

  • Assess protein folding and integrity before functional studies

• Functional assay design:

  • Measure multiple parameters (activity, stability, interaction)

  • Include appropriate positive and negative controls

  • Perform dose-response studies

  • Account for potential differences in protein stability

• Statistical considerations:

  • Perform power analysis to determine appropriate sample sizes

  • Use biological replicates (different protein preparations)

  • Include technical replicates to assess method variability

  • Apply appropriate statistical tests for data analysis

• Environmental variables:

  • Test function under varying pH conditions

  • Assess temperature sensitivity

  • Evaluate performance under different oxygen tensions

  • Consider physiologically relevant ion concentrations