Recombinant Platyrrhinus brachycephalus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the basic structure and function of MT-ND4L in Platyrrhinus brachycephalus?

MT-ND4L (NADH-ubiquinone oxidoreductase chain 4L) is a protein component of mitochondrial Complex I, encoded by the mitochondrial genome. In Platyrrhinus brachycephalus (Short-headed broad-nosed bat), the protein consists of 98 amino acids with the sequence: MSLTYMNMFMAFTISLLGLLLYRSHMMSSLLCLEGMMLSLFVMMTMIILNTHLTLASMIP IILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC . Functionally, MT-ND4L contributes to the first step of the electron transport process in oxidative phosphorylation, specifically the transfer of electrons from NADH to ubiquinone . This protein is embedded in the inner mitochondrial membrane, where it helps establish the electrochemical gradient necessary for ATP production . As part of Complex I, MT-ND4L plays a crucial role in cellular energy metabolism across various tissues.

How does the amino acid sequence of Platyrrhinus brachycephalus MT-ND4L compare to human MT-ND4L?

The human MT-ND4L protein shares significant homology with the Platyrrhinus brachycephalus variant, though with several key differences in amino acid composition. While the human sequence contains residues like LLVSISNTYGLDYVHNLNLLQ as shown in recombinant fragments , the bat sequence has the full sequence as detailed above . These differences primarily occur in the transmembrane domains and may reflect evolutionary adaptations to different metabolic demands. When analyzing structural conservation between species, researchers should focus on the functional domains involved in electron transport and protein-protein interactions within Complex I. The conservation pattern suggests evolutionary pressure to maintain function while allowing for species-specific adaptations, making comparative studies valuable for understanding fundamental mitochondrial processes across mammals.

What are the recommended protocols for expression and purification of recombinant Platyrrhinus brachycephalus MT-ND4L?

For optimal expression and purification of recombinant Platyrrhinus brachycephalus MT-ND4L, researchers should employ the following methodological approach:

• Expression System: Utilize E. coli as the expression host with a His-tag fusion for efficient purification . The His-tag placement at the N-terminus has demonstrated superior protein stability and yield compared to C-terminal tagging.

• Culture Conditions: Grow transformed E. coli at 37°C until OD600 reaches 0.6-0.8, then induce with IPTG (0.5-1mM) at a reduced temperature of 18-25°C for 16-18 hours to maximize properly folded protein yield.

• Lysis and Extraction: Due to the hydrophobic nature of this mitochondrial protein, employ specialized lysis buffers containing 1% mild detergent (such as n-dodecyl β-D-maltoside) along with standard components (50mM Tris-HCl pH 8.0, 300mM NaCl, 10% glycerol, and protease inhibitors).

• Purification Process: Apply IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA resin with a gradient elution protocol (20-250mM imidazole) . Follow with size exclusion chromatography to achieve >90% purity as assessed by SDS-PAGE.

• Storage Conditions: Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . For long-term storage, add glycerol to a final concentration of 50% and store at -80°C in small aliquots to prevent repeated freeze-thaw cycles .

This protocol typically yields 2-5mg of purified protein per liter of bacterial culture, with biological activity retained for up to 12 months when properly stored.

What are the most effective antibody validation strategies when working with MT-ND4L proteins?

When validating antibodies against MT-ND4L, researchers should implement a multi-faceted approach to ensure specificity and reliability:

• Recombinant Protein Competition Assay: Utilize purified recombinant MT-ND4L protein antigen as a competitive blocker to confirm antibody specificity . This involves pre-incubating the antibody with excess recombinant protein before application to the experimental sample.

• Western Blot Analysis: Perform western blotting using both recombinant protein and native mitochondrial extracts, looking for a single band at the predicted molecular weight (~10-11 kDa). Include appropriate positive and negative controls, such as Complex I-deficient cell lines.

• Immunoprecipitation Followed by Mass Spectrometry: Confirm antibody specificity by immunoprecipitation followed by mass spectrometry identification of captured proteins.

• Genetic Knockdown/Knockout Validation: Where possible, validate antibodies using samples from MT-ND4L knockdown/knockout models or patient samples with known MT-ND4L mutations.

• Cross-Reactivity Assessment: Test for cross-reactivity against other NADH dehydrogenase subunits, particularly those with similar molecular weights or structural domains.

These validation steps are critical for ensuring experimental reproducibility and avoiding misinterpretation of data in Complex I research. Antibodies validated through these methods should be documented with detailed protocols and validation data prior to use in advanced applications like immunohistochemistry or chromatin immunoprecipitation.

How can Platyrrhinus brachycephalus MT-ND4L be used to study mitochondrial complex I dysfunction in human disease models?

Platyrrhinus brachycephalus MT-ND4L offers a valuable comparative model for investigating human mitochondrial disorders, particularly those involving Complex I dysfunction. Researchers can leverage this evolutionary comparison through several methodological approaches:

• Mutation Modeling: Engineer recombinant Platyrrhinus brachycephalus MT-ND4L proteins carrying equivalent human pathogenic mutations, such as the T10663C (Val65Ala) mutation associated with Leber hereditary optic neuropathy (LHON) . This allows assessment of how conserved or divergent the functional consequences of specific mutations are across species.

• Complementation Studies: Introduce bat MT-ND4L variants into human cell lines harboring MT-ND4L mutations using specialized mitochondrial transfection techniques. Measure rescue effects on Complex I activity, ROS production, and ATP synthesis to identify functionally important domains.

• Structural Analysis: Use the bat protein in comparative structural biology approaches, including cryo-EM studies of reconstituted Complex I, to identify species-specific structural adaptations that may confer differential sensitivity to mutations or environmental stressors.

• Bioenergetic Profiling: Employ Seahorse XF analyzers to measure oxygen consumption rates in cells expressing either human or bat MT-ND4L variants under various metabolic conditions. The following table illustrates typical comparative measurements:

These approaches provide insight into both fundamental evolutionary adaptations in mitochondrial function and potential therapeutic strategies for human mitochondrial disorders.

What experimental approaches are recommended for studying the interaction between MT-ND4L and other Complex I subunits?

Investigating protein-protein interactions between MT-ND4L and other Complex I components requires specialized techniques that overcome challenges associated with hydrophobic membrane proteins. The following methodological approaches are recommended:

• Crosslinking Mass Spectrometry (XL-MS): Employ chemical crosslinkers like DSS (disuccinimidyl suberate) or photo-activatable reagents to capture transient interactions, followed by digestion and LC-MS/MS analysis to identify crosslinked peptides between MT-ND4L and partner proteins.

• Co-immunoprecipitation with Specialized Detergents: Use digitonin or n-dodecyl β-D-maltoside for membrane solubilization, followed by immunoprecipitation with anti-MT-ND4L antibodies and immunoblotting or mass spectrometry to identify interaction partners .

• Proximity Labeling Techniques: Apply BioID or APEX2 proximity labeling by fusing these enzymes to MT-ND4L, allowing biotinylation of neighboring proteins within the assembled Complex I structure for subsequent purification and identification.

• Förster Resonance Energy Transfer (FRET): Utilize specifically labeled antibodies or recombinant fluorescent fusion proteins to measure physical proximity between MT-ND4L and other subunits in reconstituted systems or within mitochondrial membranes.

• Molecular Dynamics Simulations: Complement experimental data with in silico modeling of protein-protein interactions based on available structural data, focusing on transmembrane domain interactions and conformational changes during electron transport.

These approaches should be applied in combination, as each provides complementary information about the spatial and functional relationships between MT-ND4L and other Complex I components. Special attention should be paid to interactions with ND1, ND3, and ND6, which are functionally and spatially related to MT-ND4L within the membrane domain of Complex I.

How do mutations in MT-ND4L contribute to mitochondrial pathologies, and what experimental models best represent these conditions?

Mutations in MT-ND4L contribute to mitochondrial pathologies through several mechanistic pathways that can be investigated using specific experimental models:

• Bioenergetic Dysfunction: MT-ND4L mutations, such as the T10663C (Val65Ala) variant implicated in Leber hereditary optic neuropathy (LHON) , disrupt Complex I assembly or function, reducing ATP production. This manifests as tissue-specific energy deficiency, particularly affecting high-energy tissues like neural retina and optic nerve.

• Oxidative Stress Induction: Dysfunctional MT-ND4L increases electron leakage from the electron transport chain, generating excess reactive oxygen species (ROS). This oxidative stress damages mitochondrial proteins, lipids, and mtDNA, creating a vicious cycle of increasing mitochondrial dysfunction.

• Altered Mitochondrial Dynamics: MT-ND4L mutations can trigger changes in mitochondrial fusion, fission, and mitophagy processes, disrupting the normal quality control mechanisms that maintain healthy mitochondrial networks.

The following experimental models are particularly effective for studying these mechanisms:

When designing experiments, researchers should consider the tissue specificity of mitochondrial diseases, particularly the selective vulnerability of retinal ganglion cells and cardiac tissue to Complex I defects. Combining multiple models provides the most comprehensive understanding of MT-ND4L-related pathologies.

What are the latest approaches for analyzing the impact of MT-ND4L variants on mitochondrial Complex I assembly and stability?

To analyze how MT-ND4L variants affect Complex I assembly and stability, researchers should employ the following cutting-edge methodological approaches:

• Blue Native PAGE (BN-PAGE) Combined with Western Blotting: This technique separates intact respiratory complexes under non-denaturing conditions, allowing visualization of assembly intermediates. When followed by second-dimension SDS-PAGE or western blotting with subunit-specific antibodies, it reveals which assembly stages are affected by MT-ND4L variants.

• Complexome Profiling: This mass spectrometry-based approach combines BN-PAGE with quantitative proteomics to provide comprehensive, unbiased analysis of all components within each assembly intermediate. It can reveal subtle changes in Complex I composition and assembly dynamics caused by MT-ND4L variants.

• Pulse-Chase Labeling with Heavy Isotopes: SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with time-course analysis enables monitoring of Complex I assembly kinetics and stability, revealing whether MT-ND4L variants affect assembly rate, steady-state levels, or degradation.

• Thermal Shift Assays for Membrane Proteins: Modified thermal shift assays using native PAGE or activity measurements across temperature gradients can quantify Complex I stability differences between wild-type and variant MT-ND4L-containing complexes.

• Cryo-Electron Microscopy: High-resolution structural analysis can directly visualize conformational changes caused by MT-ND4L variants, particularly when coupled with image classification approaches to identify population heterogeneity.

• Super-Resolution Microscopy: Techniques like STORM or PALM with subunit-specific labeling can reveal spatial organization of Complex I assembly intermediates within mitochondria, identifying abnormal localization patterns associated with pathogenic variants.

When interpreting results, researchers should consider that assembly defects may be primary (directly caused by structural changes in MT-ND4L) or secondary (due to altered interactions with assembly factors or other subunits). Temperature sensitivity of assembly should also be assessed, as some variants may show normal assembly at lower temperatures but defects at physiological temperatures.

How can recombinant MT-ND4L be utilized in high-throughput screening for mitochondrial therapeutics?

Recombinant MT-ND4L provides a valuable tool for high-throughput screening (HTS) of mitochondrial therapeutics through several methodological approaches:

• Binding Assays for Drug Discovery: Immobilize purified recombinant MT-ND4L protein on biosensor chips (such as surface plasmon resonance or biolayer interferometry platforms) to screen compound libraries for direct binding interactions . This approach can identify molecules that stabilize mutant forms of the protein or modulate its interactions with other Complex I components.

• In Vitro Activity Assays: Incorporate recombinant MT-ND4L into proteoliposomes or nanodiscs along with other Complex I components to reconstruct partial or complete enzyme complexes. These reconstituted systems can be used to measure electron transfer rates in the presence of candidate therapeutic compounds.

• Thermal Shift Assays for Stability Screening: Use differential scanning fluorimetry to identify compounds that enhance the stability of MT-ND4L variants carrying disease-associated mutations. Compounds that increase the melting temperature of destabilized variants may serve as pharmacological chaperones.

• Cell-Based Reporter Systems: Engineer cell lines expressing MT-ND4L fused to split luciferase or fluorescent proteins that report on protein folding, stability, or assembly into Complex I. These systems enable live-cell HTS for compounds that rescue defective variants.

• Targeted Peptide Library Screening: Design and screen peptide libraries based on interacting regions of other Complex I subunits to identify peptides that can stabilize MT-ND4L or compensate for mutation-induced defects.

What are the methodological considerations for studying post-translational modifications of MT-ND4L and their functional significance?

Investigating post-translational modifications (PTMs) of MT-ND4L requires specialized approaches due to its hydrophobic nature and mitochondrial localization:

• Enrichment Strategies: Implement two-step enrichment protocols combining mitochondrial isolation with antibody-based pulldown or chemical tagging of specific modifications (phosphorylation, acetylation, etc.) . For comprehensive PTM mapping, use complementary proteases beyond trypsin (such as chymotrypsin or Asp-N) to improve sequence coverage of hydrophobic regions.

• Mass Spectrometry Approaches: Apply high-resolution MS techniques including electron transfer dissociation (ETD) or electron capture dissociation (ECD), which better preserve labile PTMs compared to collision-induced dissociation (CID). Use targeted multiple reaction monitoring (MRM) for quantitative analysis of specific modifications.

• Site-Specific Functional Analysis: Generate recombinant MT-ND4L variants with mutation of putative modification sites to non-modifiable residues (e.g., Ser to Ala for phosphorylation sites) or to modification-mimicking residues (e.g., Ser to Asp/Glu for phosphomimetics) . Assess effects on protein stability, Complex I assembly, and enzyme activity.

• Temporal Dynamics Analysis: Implement pulse-chase labeling with modification-specific isotope tags to track the temporal dynamics of PTMs under different metabolic conditions or stress states.

• Cross-Species Comparison: Analyze conservation of modification sites across species to identify functionally important PTMs. The following table illustrates conservation of potential phosphorylation sites:

When interpreting PTM data, consider that different cell types or metabolic states may exhibit distinct modification patterns. Additionally, some apparent PTMs may represent chemical artifacts from sample preparation, necessitating careful validation with multiple technical approaches.

What are the primary challenges in expressing and purifying functional MT-ND4L, and how can they be addressed?

Working with MT-ND4L presents several technical challenges due to its hydrophobic nature and mitochondrial origin. Here are the major obstacles and methodological solutions:

• Protein Aggregation and Inclusion Body Formation:

  • Challenge: MT-ND4L's hydrophobic character leads to aggregation during recombinant expression in E. coli .

  • Solution: Express with solubility-enhancing fusion partners such as SUMO, MBP, or the albumin-binding protein (ABP) tag . Lower induction temperature to 16-18°C and reduce IPTG concentration to 0.1-0.2 mM. Consider codon optimization for E. coli expression.

• Protein Extraction and Solubilization:

  • Challenge: Conventional lysis buffers fail to extract membrane-associated MT-ND4L efficiently.

  • Solution: Use specialized detergents like n-dodecyl β-D-maltoside (DDM), digitonin, or Fos-choline-12 at concentrations 2-3× above their critical micelle concentration . Include 6M urea during initial extraction, followed by step-wise dialysis to refold the protein .

• Low Yield and Purity:

  • Challenge: Typical yields of pure MT-ND4L are often sub-milligram per liter of culture.

  • Solution: Implement multi-step purification protocols combining IMAC with ion exchange and size exclusion chromatography . Consider newer expression systems like cell-free synthesis or expression in specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane proteins.

• Protein Instability and Storage Issues:

  • Challenge: Purified MT-ND4L often aggregates during storage and loses functional properties.

  • Solution: Store in specialized buffers containing trehalose (6%) and glycerol (50%) . Avoid freeze-thaw cycles by preparing small single-use aliquots. Consider lyophilization for long-term storage with appropriate cryoprotectants.

• Functional Assessment:

  • Challenge: Verifying that recombinant MT-ND4L retains native functional properties.

  • Solution: Develop reconstitution systems with other Complex I components to assess electron transport capability. Use artificial membrane systems like nanodiscs or proteoliposomes to create a native-like environment for functional studies.

Researchers should document optimization efforts systematically, as conditions that work for wild-type MT-ND4L may need further adjustment when working with disease-associated variants.

How can researchers accurately quantify and compare activity of wild-type versus mutant MT-ND4L in experimental systems?

Accurate quantification and comparison of wild-type and mutant MT-ND4L activity requires specialized methodological approaches that address the unique challenges of studying this mitochondrial protein:

• Expression Level Normalization:

  • First, ensure equivalent expression levels between wild-type and mutant proteins using quantitative western blotting with recombinant protein standards as calibration controls .

  • For cellular studies, implement dual-reporter systems where MT-ND4L variants are co-expressed with a normalization marker.

• Complex I Activity Assays:

  • NADH:ubiquinone oxidoreductase activity: Measure the rate of NADH oxidation spectrophotometrically at 340nm in isolated mitochondria or reconstituted systems containing MT-ND4L variants.

  • Diphenyleneiodonium (DPI)-sensitive superoxide production: Quantify using fluorescent probes like MitoSOX to assess electron leakage differences between variants.

  • Oxygen consumption rate (OCR): Use high-resolution respirometry or Seahorse XF analyzers with specific Complex I substrates and inhibitors to assess integrated function.

• Structural Integration Assessment:

  • Evaluate assembly efficiency using blue native PAGE followed by western blotting with antibodies against multiple Complex I subunits .

  • Apply complexome profiling to quantitatively assess differences in assembly intermediate formation between wild-type and mutant variants.

• Single-Molecule Approaches:

  • Implement fluorescence correlation spectroscopy (FCS) with labeled MT-ND4L to detect subtle differences in protein dynamics and interaction kinetics.

  • Use atomic force microscopy to assess structural stability differences under mechanical stress.

• Statistical Analysis and Normalization:

  • Apply mixed-effects models to account for batch variation in experiments.

  • Present data as percent of wild-type activity rather than absolute values to facilitate comparison across different experimental systems.

  • Perform activity measurements across a range of substrate concentrations to generate Michaelis-Menten kinetic parameters (Km and Vmax) for more informative comparisons than single-point measurements.

The following table illustrates typical parameter changes observed in MT-ND4L variants:

When reporting results, researchers should clearly specify the experimental conditions, particularly detergent types and concentrations, which significantly impact membrane protein behavior in biochemical assays.

What are the most promising research directions for understanding MT-ND4L's role in mitochondrial disease and potential therapeutic targets?

The study of MT-ND4L presents several promising research avenues that may advance our understanding of mitochondrial diseases and lead to novel therapeutic approaches:

• Structural Biology and Protein Dynamics: Leveraging advances in cryo-electron microscopy and molecular dynamics simulations to understand how disease-associated mutations in MT-ND4L affect the conformational dynamics of Complex I . This structural information can guide rational drug design targeting specific interaction interfaces to stabilize mutant proteins.

• Tissue-Specific Expression Patterns: Investigating why mutations in the ubiquitously expressed MT-ND4L cause tissue-specific pathologies, particularly in retinal ganglion cells in Leber hereditary optic neuropathy . Single-cell transcriptomics and proteomics approaches may reveal tissue-specific interaction partners or compensatory mechanisms.

• Genetic Suppressor Screening: Identifying genetic modifiers that can suppress the pathogenic effects of MT-ND4L mutations through CRISPR-based screens or yeast genetic interaction studies with heterologous expression systems. Such modifiers could represent novel therapeutic targets.

• Allotopic Expression Strategies: Developing improved methods for expressing mitochondrially-encoded genes from the nuclear genome as a potential gene therapy approach for MT-ND4L-related disorders. This includes optimizing mitochondrial targeting sequences and protein import efficiency.

• Metabolic Bypass Strategies: Investigating alternative electron transfer pathways that can compensate for Complex I dysfunction caused by MT-ND4L mutations. This includes both natural metabolic flexibility mechanisms and engineered solutions.

• Comparative Mitochondrial Biology: Studying naturally occurring variations in MT-ND4L across species with different metabolic demands (like Platyrrhinus brachycephalus) to identify adaptive mechanisms that could inspire therapeutic approaches .

These research directions require multidisciplinary approaches combining structural biology, genetics, biochemistry, and systems biology. Collaborative efforts between basic scientists and clinicians will be essential to translate findings into therapeutic strategies for patients with mitochondrial disorders.

What emerging technologies are most likely to advance our understanding of MT-ND4L function in the next decade?

Several cutting-edge technologies are poised to revolutionize our understanding of MT-ND4L function in the coming decade:

• Cryo-Electron Tomography (cryo-ET): This technique will enable visualization of Complex I containing MT-ND4L in its native mitochondrial membrane environment at near-atomic resolution, revealing physiologically relevant conformational states and interactions not captured in isolated protein studies .

• Single-Molecule Functional Imaging: Technologies like single-molecule FRET and high-speed atomic force microscopy will allow real-time observation of MT-ND4L's dynamic behavior during electron transport, providing unprecedented insights into the coupling between structural changes and catalytic function.

• Mitochondrial-Targeted CRISPR Systems: Development of mitochondrially-targeted RNA-free CRISPR technologies will enable precise editing of mtDNA, allowing creation of model systems with endogenous MT-ND4L mutations rather than relying on cybrid or overexpression approaches.

• Microfluidic Organoid Systems: Integration of patient-derived mitochondrial disease models with organ-on-a-chip technology will recreate tissue-specific microenvironments to better understand why ubiquitous mitochondrial mutations cause selective tissue pathology.

• Computational Systems Biology: Advanced computational models integrating multi-omics data will predict how MT-ND4L variants affect mitochondrial function across different metabolic states and cell types. These models will guide experimental design and therapeutic development.

• Nanobody and Aptamer Technologies: Development of highly specific binders to conformational states of MT-ND4L will provide tools for stabilizing specific protein conformations, enabling both structural studies and potential therapeutic approaches.

• Artificial Intelligence for Protein Structure Prediction: Enhanced AI models like AlphaFold will improve prediction of membrane protein structures and interactions, accelerating understanding of how MT-ND4L variants affect Complex I assembly and function.