Recombinant Canis latrans NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the basic function of MT-ND4L in mitochondrial respiration?

MT-ND4L gene encodes the NADH dehydrogenase 4L protein, which serves as an essential component of the mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase). This protein participates in the first step of the electron transport process, facilitating the transfer of electrons from NADH to ubiquinone. The functional complex I creates an electrochemical gradient across the inner mitochondrial membrane by pumping protons from the mitochondrial matrix to the intermembrane space. This gradient drives ATP synthase to generate adenosine triphosphate (ATP), which serves as the primary energy currency for cellular functions. The integrity of MT-ND4L is therefore critical for efficient oxidative phosphorylation and cellular energy production in both humans and canids including Canis latrans .

How does the structure of Canis latrans MT-ND4L differ from human MT-ND4L?

While the search results don't specifically detail structural differences between human and Canis latrans MT-ND4L, comparative analysis reveals conserved functional domains. Both species' proteins are embedded in the inner mitochondrial membrane as part of complex I and maintain similar electron transport functions. Research employing AI-driven conformational ensemble generation techniques has expanded our understanding of protein structure variability across species. These techniques predict alternative functional states and conformational changes along collective coordinates, allowing researchers to identify species-specific structural variations that may affect function or drug binding. Modern molecular dynamics simulations with AI-enhanced sampling can generate statistically robust ensembles of equilibrium protein conformations that capture the full dynamic behavior of both human and Canis latrans variants, highlighting any key differences in binding pocket architecture or conformational flexibility .

What are the predominant experimental methods for expressing and purifying recombinant Canis latrans MT-ND4L?

Recombinant expression of membrane proteins like MT-ND4L presents unique challenges due to their hydrophobic nature and complex folding requirements. The preferred expression method involves using specialized bacterial expression systems with modified host strains that accommodate membrane protein production. The protocol typically includes:

• Gene optimization for the expression host (codon optimization for E. coli if using bacterial systems)

• Incorporation of purification tags (His6, FLAG, or STREP tags) that facilitate downstream purification

• Expression in membrane-protein optimized strains at reduced temperatures (18-25°C)

• Membrane solubilization using mild detergents (e.g., n-dodecyl β-D-maltoside or digitonin)

• Purification through affinity chromatography, followed by size exclusion chromatography

For structural studies, purified MT-ND4L can be reconstituted into nanodiscs or liposomes to maintain native-like membrane environments. Advanced AI-based approaches now complement these experimental techniques by predicting protein dynamics and potential binding sites even before physical expression is completed .

How can researchers effectively study MT-ND4L mutations associated with mitochondrial disorders?

Studying MT-ND4L mutations requires a multidisciplinary approach combining molecular genetics, biochemistry, and advanced imaging techniques. Researchers should:

• Develop site-directed mutagenesis protocols to introduce specific mutations identified in pathological conditions (such as the T10663C or Val65Ala mutation associated with Leber hereditary optic neuropathy)

• Establish cellular models expressing the mutant protein, preferably in cell lines with depleted endogenous mitochondrial DNA

• Assess functional consequences through measurements of:

  • Complex I assembly (using blue native PAGE)

  • NADH:ubiquinone oxidoreductase activity (spectrophotometric assays)

  • Mitochondrial membrane potential (using potential-sensitive dyes)

  • ROS production (using fluorescent indicators)

  • ATP synthesis rates

For comparative studies between human and Canis latrans MT-ND4L, researchers should introduce equivalent mutations in both orthologs to determine species-specific responses to the same molecular perturbation. Four novel mutations in the human ND4 gene (m.11150G>A, m.11519A>C, m.11523A>C, and m.11527C>T) have been identified in patients with Multiple Sclerosis, providing potential targets for comparative mutation studies across species .

What are the challenges in developing computational models for MT-ND4L structure prediction and how can they be overcome?

Computational modeling of MT-ND4L presents numerous challenges due to its membrane-embedded nature and complex interactions within the respiratory chain. Key challenges and solutions include:

Modern approaches incorporate AI algorithms to predict alternative functional states, including large-scale conformational changes. By employing molecular simulations with AI-enhanced sampling and trajectory clustering, researchers can explore the broad conformational space of the protein and identify representative structures. Diffusion-based AI models and active learning AutoML can generate statistically robust ensembles of protein conformations that more accurately represent the protein's dynamic behavior .

How can researchers effectively identify and characterize binding pockets in recombinant Canis latrans MT-ND4L?

Identifying binding pockets in MT-ND4L requires advanced computational and experimental approaches working in tandem. The most effective methodology involves:

• AI-based pocket prediction algorithms that integrate:

  • Structure-aware ensemble-based detection utilizing established protein dynamics

  • Machine learning models trained on known membrane protein binding sites

  • Detection of orthosteric, allosteric, hidden, and cryptic binding pockets

• Experimental validation through:

  • Site-directed mutagenesis of predicted pocket residues

  • Binding assays with known ligands or fragment libraries

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes upon ligand binding

• Characterization of identified pockets through:

  • Assessment of pocket druggability scores

  • Analysis of pocket conservation across species

  • Evaluation of pocket dynamics through molecular simulations

The integration of LLM-powered literature research with these structural approaches allows researchers to compare identified pockets with any previously reported interaction sites, validating computational predictions with experimental evidence from related proteins or species .

What methodologies are most effective for assessing the functional impact of MT-ND4L mutations identified in Canis latrans compared to human variants?

To effectively assess functional impacts of MT-ND4L mutations across species, researchers should implement a comprehensive workflow:

• Genetic Analysis:

  • Sequence alignment of Canis latrans and human MT-ND4L to identify conserved residues

  • Phylogenetic analysis to determine evolutionary conservation of specific mutation sites

  • Population genetics studies to determine mutation frequency in wild and domestic canids

• Structural Impact Prediction:

  • Homology modeling of both human and canid variants

  • In silico mutagenesis to predict structural perturbations

  • Molecular dynamics simulations to assess stability changes

• Functional Assessment:

  • Development of heterologous expression systems for wild-type and mutant proteins

  • Respirometry measurements to quantify complex I activity

  • ROS production assays to determine electron leakage

• Comparative Cellular Phenotyping:

  • Generation of cybrid cell lines containing either human or Canis latrans mitochondria with identical mutations

  • Cellular phenotyping through microscopy, metabolism analysis, and stress response assessment

For example, the T10663C (Val65Ala) mutation identified in human families with Leber hereditary optic neuropathy could be introduced in the equivalent position in Canis latrans MT-ND4L to assess if the functional impact is conserved across species .

How do multiple simultaneous mutations in MT-ND4L affect protein stability and complex I function?

Multiple simultaneous mutations in MT-ND4L can have cumulative or interactive effects on protein stability and complex I function. Research demonstrates that the combined effect of multiple mutations may differ significantly from the sum of individual mutation effects:

• Computational Analysis:

  • Energy calculations reveal that combined mutations often have non-additive effects on protein stability

  • Molecular dynamics simulations show altered conformational sampling in multi-mutation variants

  • Network analysis of residue interactions identifies synergistic perturbations in protein structure

• Experimental Evidence:

  • Functional analysis of three mutations (m.11519A>C, m.11523A>C, and m.11527C>T) observed in the same MS patient demonstrated cumulative destabilizing effects on the ND4 protein

  • While individual mutations may have varying impacts (some benign, others deleterious), their combination can significantly disrupt complex I function

  • Respirometry measurements in cells harboring multi-mutation variants show disproportionate reduction in complex I activity compared to single-mutation variants

A comparative analysis between species can determine whether the same mutational combinations have consistent effects across evolutionary distances, potentially revealing compensatory mechanisms that might exist in Canis latrans but not in humans or vice versa .

What is the relationship between MT-ND4L mutations and neurodegenerative diseases across mammalian species?

MT-ND4L mutations have demonstrated connections to neurodegenerative conditions across mammalian species, though with varying phenotypic presentations:

• Human Disease Associations:

  • Leber hereditary optic neuropathy (LHON): The T10663C mutation in MT-ND4L is linked to this maternally inherited blindness

  • Multiple Sclerosis connections: Novel mutations in MT-ND4L and related complex I genes have been identified in MS patients

  • Broader neurodegenerative implications: Mitochondrial dysfunction from MT-ND4L mutations contributes to energy deficiency in neuronal cells

• Comparative Disease Pathology:

  • Similar mitochondrial mutations in canids and other mammals often present with neurological symptoms, though exact phenotypes may differ

  • The higher mutation rate of mtDNA compared to nuclear DNA contributes to accumulation of potentially pathogenic variants across species

  • Loss of mitochondrial genomic integrity leads to progressive decline in energy production, particularly affecting high-energy tissues like the nervous system

• Mechanistic Commonalities:

  • Increased ROS production from dysfunctional complex I appears as a common pathological mechanism across species

  • Disrupted calcium homeostasis resulting from energy deficiency affects neuronal function similarly across mammals

  • Apoptotic sensitivity increases in neurons with compromised mitochondrial function regardless of species

The study of MT-ND4L mutations in Canis latrans provides valuable comparative insights into how similar genetic perturbations manifest across evolutionary distances, potentially revealing species-specific protective mechanisms or vulnerabilities .

What are the optimal protocols for studying MT-ND4L integration into complex I assembly?

Studying MT-ND4L integration into complex I requires specialized techniques addressing the challenges of membrane protein assembly. The optimal protocol includes:

• Expression System Selection:

  • For in vitro studies: Cell-free translation systems supplemented with artificial membranes

  • For cellular studies: Transmitochondrial cybrid cells with depleted endogenous mtDNA

• Assembly Monitoring:

  • Pulse-chase labeling with radioactive amino acids to track newly synthesized MT-ND4L

  • Blue Native PAGE combined with Western blotting to visualize assembly intermediates

  • Proximity labeling techniques (BioID or APEX) to identify transient assembly partners

• Integration Assessment:

  • Protease protection assays to determine proper membrane insertion

  • Crosslinking mass spectrometry to map interaction interfaces

  • Super-resolution microscopy with fluorescently tagged assembly factors

• Functional Verification:

  • In-gel activity assays for complex I function

  • High-resolution respirometry to measure oxygen consumption

  • Membrane potential measurements using potentiometric dyes

This comprehensive approach allows researchers to track MT-ND4L from synthesis through assembly into functional complex I, identifying critical checkpoints and potential species-specific differences in assembly pathways between human and Canis latrans systems .

How can researchers effectively perform comparative analyses of MT-ND4L across different species?

Performing robust comparative analyses of MT-ND4L across species requires an integrated approach combining bioinformatics, structural biology, and functional assessment:

• Sequence-based Analysis:

  • Multiple sequence alignment using specialized algorithms for membrane proteins

  • Calculation of evolutionary rates to identify conserved vs. rapidly evolving regions

  • Coevolution analysis to detect functionally linked residue networks

• Structural Comparison:

  • Homology modeling based on available complex I structures

  • AI-driven prediction of species-specific conformational ensembles

  • Analysis of binding pocket conservation and variability

• Functional Conservation Assessment:

  • Complementation studies in model systems lacking endogenous MT-ND4L

  • Respirometry measurements comparing activity of orthologs

  • ROS production and membrane potential comparison between species variants

• Data Integration:

  • Machine learning approaches to correlate sequence differences with functional outcomes

  • Network analysis of protein-protein interactions across species

  • Integration with broader mitochondrial genomics data

This methodology is particularly valuable for understanding how genetic differentiation among populations affects protein function. As noted in conservation genetics research, identifying genetic differentiation should trigger investigation into whether populations suffer from genetic problems and what interventions might be beneficial .

What therapeutic potentials exist for targeting recombinant Canis latrans MT-ND4L in comparative medicine?

The therapeutic potential of recombinant Canis latrans MT-ND4L in comparative medicine stems from its role in mitochondrial function and disease:

• Comparative Drug Discovery:

  • MT-ND4L's identified binding pockets serve as targets for small molecule development

  • Species differences in binding pocket architecture can inform selective therapeutic design

  • Comparative screening across human and canid variants enables identification of broadly effective compounds

• Gene Therapy Applications:

  • MT-ND4L gene replacement strategies developed in canid models can inform human applications

  • Allotopic expression (nuclear expression of mitochondrial genes) tested across species can bypass mitochondrial genetic disorders

  • CRISPR-based mitochondrial editing techniques benefit from cross-species validation

• Biomarker Development:

  • MT-ND4L mutations as diagnostic markers for mitochondrial dysfunction

  • Comparative analysis of mutation patterns between species informs evolutionary medicine

  • Identification of compensatory mechanisms in canids that might be therapeutically relevant to humans

• Model System Development:

  • Recombinant Canis latrans MT-ND4L expression systems serve as comparative platforms for mitochondrial disease modeling

  • Cross-species differences highlight potential therapeutic targets for neurodegenerative conditions

  • Conservation of complex I assembly pathways enables translational research between canid and human systems

As noted by Receptor.AI's assessment, MT-ND4L represents a protein with high therapeutic potential, and comparative studies between human and Canis latrans variants can accelerate drug discovery efforts through enhanced understanding of binding pocket characteristics and protein dynamics .

How can structural information about MT-ND4L inform the development of mitochondrial-targeted therapeutics?

Structural insights into MT-ND4L provide critical guidance for developing mitochondrial-targeted therapeutics:

• Structure-Based Drug Design:

  • AI-based pocket prediction identifies orthosteric, allosteric, hidden, and cryptic binding sites

  • Ensemble-based approaches account for protein flexibility in drug binding

  • Virtual screening against identified pockets accelerates compound discovery

• Mechanism-Based Intervention Strategies:

  • Structural understanding of MT-ND4L's role in complex I assembly guides development of assembly modulators

  • Interaction maps between MT-ND4L and other complex I components identify interfaces for stabilization

  • Conformational states of MT-ND4L inform design of state-specific modulators

• Mutation-Specific Therapeutics:

  • Structural characterization of mutation effects (like T10663C/Val65Ala) enables design of compensatory compounds

  • Computational prediction of structural perturbations from multiple mutations guides development of stabilizing molecules

  • Binding pocket alterations in mutant proteins create opportunities for selective targeting

• Cross-Species Applications:

  • Comparative structural analysis between human and Canis latrans MT-ND4L reveals conserved binding sites for broad-spectrum therapeutics

  • Species-specific structural features inform selective targeting when needed

  • Evolutionary analysis of binding pocket conservation guides prioritization of drug development efforts

Advanced AI algorithms that predict alternative functional states of MT-ND4L, including large-scale conformational changes along "soft" collective coordinates, have particular value for therapeutic development. These methods generate statistically robust ensembles of equilibrium protein conformations that better represent the dynamic behavior of the protein in vivo, providing a more accurate foundation for structure-based drug design approaches .

What are the most promising future research directions for studying recombinant Canis latrans MT-ND4L?

The most promising future research directions for recombinant Canis latrans MT-ND4L span multiple scientific disciplines:

• Evolutionary Mitochondrial Genomics:

  • Comparative analysis of MT-ND4L across canid species to trace evolutionary adaptations

  • Investigation of selection pressures on mitochondrial genes in different ecological niches

  • Exploration of hybridization effects on mitochondrial function in zones where coyotes interact with wolves or dogs

• Advanced Structural Biology:

  • Cryo-EM structures of canid complex I focusing on MT-ND4L architecture

  • Time-resolved structural studies capturing dynamic conformational changes during electron transport

  • Integration of AI-driven prediction with experimental structure determination

• Mitochondrial Medicine Applications:

  • Development of canid models for human mitochondrial diseases involving MT-ND4L

  • Exploration of species-specific differences in mitochondrial disease manifestation

  • Testing of gene therapy approaches in comparative systems

• Climate Change Adaptation Research:

  • Investigation of how MT-ND4L variants contribute to metabolic adaptations in changing environments

  • Assessment of mitochondrial function under temperature stress across species

  • Exploration of genetic management options for populations with compromised mitochondrial function

• Integrative Multi-omics:

  • Combination of proteomics, metabolomics, and transcriptomics to understand system-level effects of MT-ND4L variants

  • Machine learning integration of multi-dimensional data across species

  • Development of predictive models for mitochondrial dysfunction based on comparative data

These research directions align with broader trends in genetic management of fragmented populations, where understanding genetic differentiation serves as a trigger to investigate potential genetic problems and their solutions. Climate change particularly increases the need for such genetic management approaches, as noted in conservation genetics literature .

What are the key considerations for researchers designing experiments with recombinant Canis latrans MT-ND4L?

Researchers working with recombinant Canis latrans MT-ND4L should prioritize several crucial considerations: