Recombinant Arctocephalus australis NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L (NADH-ubiquinone oxidoreductase chain 4L) is a protein encoded by the mitochondrial genome that functions as an essential component of Complex I in the electron transport chain. This protein participates in the first step of 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 creating the electrochemical gradient necessary for ATP production. Complex I creates an unequal electrical charge on either side of the inner mitochondrial membrane through the step-by-step transfer of electrons, and this difference in electrical charge provides the energy for ATP synthesis . The full amino acid sequence of the protein consists of 98 amino acids: MSMVYFNIFMAFTVSFVGLLMYRSHLMSSLLCLEGMMLSLFVMMSMTILNNHFTLASMAPIILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC .

How should recombinant MT-ND4L protein be stored and handled for optimal stability?

For optimal stability of recombinant MT-ND4L protein, the following storage and handling protocols are recommended:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being optimal in most cases) and store at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as they can degrade the protein

• Prior to opening, briefly centrifuge the vial to bring contents to the bottom

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

The protein is typically supplied in a Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain stability during storage .

What expression systems are commonly used for recombinant MT-ND4L production?

Recombinant MT-ND4L is typically expressed in prokaryotic systems, with E. coli being the predominant expression host for this mitochondrial protein. The E. coli expression system offers several advantages for producing MT-ND4L, including rapid growth rates, high protein yields, and well-established protocols for induction and purification. Researchers commonly use N-terminal His-tagging to facilitate purification through affinity chromatography techniques . While eukaryotic expression systems like yeast, insect cells, or mammalian cells might provide more native-like post-translational modifications, the relatively small size and simple structure of MT-ND4L (98 amino acids) makes bacterial expression sufficient for most research applications . The expression construct typically includes the full-length protein (residues 1-98) with appropriate fusion tags to improve solubility and enable purification .

What purification methods yield the highest purity for recombinant MT-ND4L?

To achieve high purity (>90%) recombinant MT-ND4L protein, a multi-stage purification protocol is recommended:

• Affinity Chromatography: For His-tagged MT-ND4L, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides the initial capture step with good selectivity.

• Size Exclusion Chromatography (SEC): Following IMAC, SEC helps remove aggregates and further purifies the protein based on molecular size.

• Ion Exchange Chromatography: This optional step can be employed to remove charged contaminants and improve homogeneity.

• Quality Control: SDS-PAGE analysis is routinely performed to verify purity, which should exceed 90% for research-grade applications .

The addition of mild detergents in purification buffers may improve yield and stability, as MT-ND4L is a membrane protein. Purity assessment through multiple methods (SDS-PAGE, Western blotting, and mass spectrometry) ensures the removal of host cell proteins and other contaminants before experimental use .

How do mutations in MT-ND4L affect mitochondrial function and what methodologies are used to study these effects?

Mutations in MT-ND4L can significantly impact mitochondrial function through several mechanisms that can be studied using complementary methodologies:

Impact of MT-ND4L Mutations:

• Reduction in Complex I activity and electron transport efficiency

• Altered reactive oxygen species (ROS) production

• Disrupted mitochondrial membrane potential

• Compromised ATP synthesis

• Potential triggering of mitochondrial quality control mechanisms

• Association with conditions like Leber hereditary optic neuropathy, particularly the T10663C (Val65Ala) mutation

Methodological Approaches:

• Oxygen Consumption Analysis:

  • High-resolution respirometry to measure oxygen consumption rates

  • Seahorse XF analyzers to assess basal respiration, maximal respiration, and spare respiratory capacity

• Complex I Activity Assays:

  • Spectrophotometric measurement of NADH oxidation rates

  • Diphenyleneiodonium (DPI) sensitivity assays

  • Blue native PAGE followed by in-gel activity staining

• ROS Production Measurement:

  • Fluorescent probes (DCF-DA, MitoSOX Red)

  • Electron paramagnetic resonance (EPR) spectroscopy

  • Genetically-encoded redox sensors

• Mitochondrial Membrane Potential Analysis:

  • Potential-dependent fluorescent dyes (TMRM, JC-1)

  • Potentiometric probes with confocal microscopy

• ATP Production Quantification:

  • Luciferase-based ATP assays

  • 31P-NMR spectroscopy for in vivo measurements

• Structural Analysis:

  • AI-driven conformational ensemble generation to predict alternative functional states

  • Molecular simulations with AI-enhanced sampling to explore conformational space

Changes in MT-ND4L gene expression have long-term consequences on energy metabolism and have been suggested to be a major predisposition factor for various metabolic disorders .

What metabolomic changes are associated with MT-ND4L variants and how can these be experimentally validated?

MT-ND4L variants have been linked to specific metabolomic signatures that can be experimentally investigated and validated:

Associated Metabolomic Changes:

The variant mt10689 G > A in MT-ND4L has been significantly associated with alterations in glycerophospholipid metabolism. Specifically, a large number of significant metabolite ratios were observed involving phosphatidylcholine (PC) aa C36:6 and this MT-ND4L variant, suggesting important interconnections between mitochondrial function and lipid metabolism .

Experimental Validation Methods:

• Targeted Metabolomics:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify specific phospholipids

  • Gas chromatography-mass spectrometry (GC-MS) for volatile metabolites

  • Nuclear magnetic resonance (NMR) spectroscopy for structural confirmation

• Stable Isotope Tracing:

  • 13C-labeled substrates to track carbon flux through metabolic pathways

  • Analysis of isotopologue distributions to determine pathway activities

• Lipidomics:

  • Comprehensive profiling of glycerophospholipids, especially phosphatidylcholines

  • Analysis of fatty acid composition and saturation levels

• In Vitro Functional Validation:

  • Cell models expressing wild-type versus mutant MT-ND4L

  • Measurement of lipid synthesis rates and turnover

  • Flux analysis using labeled precursors

• Integration with Genomic Data:

  • Mitochondrial genome-wide association studies (mt-GWAS)

  • Correlation of metabolite ratios with specific MT-ND4L variants

This integrative approach provides insight into how MT-ND4L variants might contribute to disease through altered metabolism, particularly in conditions associated with mitochondrial dysfunction.

What are the challenges in studying recombinant MT-ND4L interactions with other Complex I subunits and how can these be overcome?

Studying interactions between recombinant MT-ND4L and other Complex I subunits presents several challenges due to its membrane-embedded nature and dependence on the complex structural environment:

Key Challenges:

• Membrane Protein Solubility: MT-ND4L is highly hydrophobic with multiple transmembrane segments, making it difficult to maintain in a properly folded state outside its native membrane environment.

• Structural Integrity: The protein's structure depends on interactions with other Complex I subunits, and isolated MT-ND4L may not adopt its native conformation.

• Complex Assembly: Complex I contains 45 subunits in mammals, making reconstitution of functional complexes technically demanding.

• Functional Assessment: Without the complete complex, measuring functional interactions and electron transfer is challenging.

Methodological Solutions:

• Advanced Membrane Mimetics:

  • Nanodiscs composed of phospholipid bilayers encircled by scaffold proteins

  • Styrene-maleic acid lipid particles (SMALPs) that preserve native lipid environments

  • Amphipols and detergent micelles optimized for membrane protein stability

• Protein Fusion and Engineering Approaches:

  • Split fluorescent protein complementation to visualize interactions

  • Cysteine crosslinking to map proximity of interacting residues

  • Targeted mutations to probe interaction interfaces

• AI-Enhanced Structural Analysis:

  • Employing AI algorithms to predict alternative functional states

  • Molecular simulations with enhanced sampling to explore conformational space

  • Ensemble-based binding pocket detection to identify interaction sites

• Computational Methods:

  • Molecular dynamics simulations to model MT-ND4L in membrane environments

  • Protein-protein docking to predict interactions with other subunits

  • Coevolutionary analysis to identify co-varying residues that may indicate interaction sites

• Hybrid Experimental Approaches:

  • Combining cryo-EM with crosslinking mass spectrometry

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Site-directed spin labeling with electron paramagnetic resonance (EPR) to measure distances between subunits

These approaches collectively enable researchers to overcome the inherent difficulties in studying this membrane-embedded component of mitochondrial Complex I.

How can recombinant MT-ND4L be used in drug discovery for mitochondrial disorders?

Recombinant MT-ND4L offers several applications in drug discovery targeting mitochondrial disorders:

Strategic Applications in Drug Discovery:

• Target-Based Screening Platforms:

  • Development of binding assays using purified recombinant MT-ND4L

  • Fluorescence-based or surface plasmon resonance (SPR) assays to identify compounds that interact with MT-ND4L

  • Thermal shift assays to detect stabilizing compounds

• Structure-Based Drug Design:

  • Using AI-driven conformational ensemble generation to identify druggable pockets

  • Virtual screening against predicted binding sites

  • Fragment-based approaches targeting specific functional domains

• Functional Rescue Assays:

  • Cell-based assays using patient-derived cells with MT-ND4L mutations

  • Measurement of Complex I activity restoration upon compound treatment

  • Respirometry to assess functional improvement in oxidative phosphorylation

• Disease Model Development:

  • Using recombinant protein to develop antibodies for diagnostic purposes

  • Creating cellular models expressing mutant forms of MT-ND4L

  • Establishing assays to measure restoration of normal function

• Therapeutic Protein Development:

  • Engineering modified forms of MT-ND4L with enhanced stability or function

  • Developing delivery systems for protein replacement therapy

  • Testing strategies to improve mitochondrial targeting and import

Methodological Approach for Binding Site Identification:

For specific disease relevance, the T10663C (Val65Ala) mutation associated with Leber hereditary optic neuropathy provides a focused target for therapeutic development . Drug discovery efforts can be directed at either enhancing residual activity of mutant MT-ND4L or stabilizing the protein to prevent degradation.

What are the optimal conditions for enhancing solubility and stability of recombinant MT-ND4L during expression and purification?

Optimizing solubility and stability of recombinant MT-ND4L requires careful consideration of expression conditions and buffer composition:

Expression Optimization:

• Temperature Modulation: Lowering expression temperature to 16-20°C after induction slows protein synthesis, often improving folding of membrane proteins.

• Induction Protocol: Using lower IPTG concentrations (0.1-0.5 mM) and longer induction times can improve yield of properly folded protein.

• Expression Strain Selection: Strains like C41(DE3) or C43(DE3), specifically developed for membrane proteins, often yield better results than standard BL21(DE3).

• Co-expression Strategies: Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ) can significantly enhance proper folding.

Solubilization and Purification Conditions:

• Detergent Screening: A systematic approach testing multiple detergents is critical:

  • Mild detergents (DDM, LMNG, digitonin)

  • Zwitterionic detergents (CHAPS, Fos-choline)

  • Non-ionic detergents (Triton X-100, C12E8)

• Buffer Optimization:

  • pH range: 7.0-8.0 typically provides optimal stability

  • Salt concentration: 150-300 mM NaCl to reduce aggregation

  • Addition of glycerol (10-20%) to enhance stability

  • Inclusion of lipids (POPC, cardiolipin) to mimic native environment

• Additives for Stability Enhancement:

  • Trehalose (6%) for lyophilization and storage stability

  • Reducing agents (DTT, β-mercaptoethanol) to prevent oxidation

  • Protease inhibitors to prevent degradation

• Purification Strategy:

  • Two-step purification combining affinity chromatography with size exclusion

  • Maintaining detergent above critical micelle concentration throughout

  • Using shorter purification protocols to minimize exposure time

The addition of 5-50% glycerol for long-term storage and aliquoting to avoid freeze-thaw cycles also significantly contributes to maintaining protein stability . Implementation of these conditions can improve both the yield and functional quality of recombinant MT-ND4L preparations.

How can researchers verify the structural integrity and functional activity of purified recombinant MT-ND4L?

Verifying both structural integrity and functional activity of recombinant MT-ND4L requires a multi-faceted approach:

Structural Integrity Assessment:

• Biophysical Characterization:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure

  • Dynamic Light Scattering (DLS) to determine size distribution and detect aggregation

  • Thermal shift assays to measure protein stability

• Structural Analysis:

  • Negative stain electron microscopy to visualize protein particles

  • Limited proteolysis to probe folded state

  • Hydrogen-deuterium exchange mass spectrometry to map solvent-accessible regions

  • NMR spectroscopy for solution structure determination (challenging but informative)

Functional Assessment:

• Electron Transfer Activity:

  • NADH:ubiquinone oxidoreductase activity assays

  • Spectrophotometric monitoring of NADH oxidation at 340 nm

  • Artificial electron acceptor (ferricyanide) reduction assays

  • Sensitivity to known Complex I inhibitors (rotenone, piericidin A)

• Protein-Protein Interaction Assays:

  • Pull-down assays with other Complex I subunits

  • Microscale thermophoresis to measure binding affinities

  • Native PAGE to assess complex formation

  • Crosslinking followed by mass spectrometry to identify interaction partners

• Membrane Integration Assessment:

  • Liposome reconstitution and flotation assays

  • Proteoliposome activity measurements

  • Accessibility to lipophilic probes

Comparative Analysis Protocol:

The combination of these complementary approaches provides comprehensive validation of recombinant MT-ND4L quality before proceeding with further experimental applications.

What experimental designs are most effective for studying MT-ND4L interactions with mitochondrial lipids and their impact on protein function?

Studying MT-ND4L interactions with mitochondrial lipids requires specialized experimental approaches that preserve the native-like lipid environment while enabling quantitative measurements:

Experimental Design Strategies:

• Reconstitution Systems:

  • Proteoliposomes with defined lipid compositions mimicking mitochondrial inner membrane

  • Lipid nanodiscs with controlled stoichiometry of protein:lipid

  • Supported lipid bilayers for surface-sensitive techniques

  • Giant unilamellar vesicles (GUVs) for microscopy-based studies

• Lipid-Protein Interaction Analysis:

  • Fluorescence anisotropy with labeled lipids to measure binding

  • Native mass spectrometry to identify bound lipids

  • Lipid photocrosslinking to map interaction sites

  • Molecular dynamics simulations to predict lipid binding pockets

• Functional Impact Assessment:

  • Activity assays in different lipid environments

  • Electron transfer kinetics measurements

  • Proton pumping efficiency determination

  • ROS production in various lipid contexts

Critical Mitochondrial Lipids to Investigate:

Advanced Analytical Methods:

• Site-Specific Probing:

  • Site-directed spin labeling with EPR to measure lipid-protein distances

  • Tryptophan fluorescence to monitor local environment changes

  • Deuterium exchange mass spectrometry to identify lipid-protected regions

• Microscopy Approaches:

  • Single-molecule fluorescence to track protein dynamics in membranes

  • Atomic force microscopy to visualize protein organization in lipid bilayers

  • Super-resolution microscopy to map protein clusters

• Thermodynamic and Kinetic Measurements:

  • Isothermal titration calorimetry to determine binding constants

  • Stopped-flow spectroscopy to measure lipid-dependent conformational changes

  • Pressure perturbation calorimetry to assess lipid-induced volume changes

This multi-technique approach can reveal how specific lipids interact with MT-ND4L and modulate its function within Complex I, providing insights into the lipid-dependent regulation of mitochondrial energy production.

How does the structure and function of MT-ND4L differ between marine mammals and terrestrial species, and what methodologies are appropriate for comparative studies?

The comparison of MT-ND4L between marine mammals (like Arctocephalus australis and Arctocephalus forsteri) and terrestrial species reveals evolutionary adaptations related to diving physiology and energy metabolism:

Key Structural and Functional Differences:

• Amino Acid Sequence Variations:

  • Marine mammals show specific substitutions in transmembrane domains that may affect proton pumping efficiency

  • Conservation patterns differ in regions interacting with other Complex I subunits

  • The complete amino acid sequences of Arctocephalus forsteri and Arctocephalus australis MT-ND4L proteins (98 amino acids each) reveal species-specific adaptations

• Functional Adaptations:

  • Marine mammals exhibit enhanced oxidative capacity to support diving

  • Differences in electron transfer efficiency and coupling to proton pumping

  • Altered sensitivity to hypoxia and oxidative stress

• Evolutionary Selection Pressures:

  • Different selection patterns on mitochondrial genes in marine versus terrestrial mammals

  • Positive selection on specific residues related to hypoxia tolerance in diving species

Methodological Approaches for Comparative Studies:

• Sequence Analysis and Molecular Evolution:

  • Phylogenetic analysis to trace evolutionary history

  • Selection pressure analysis (dN/dS ratios) to identify adaptively evolving sites

  • Ancestral sequence reconstruction to infer evolutionary trajectories

  • Comparison of sequences between Arctocephalus species and terrestrial mammals

• Functional Comparison:

  • Recombinant expression of MT-ND4L from different species

  • Side-by-side activity assays under identical conditions

  • Oxygen consumption measurements in reconstituted systems

  • ROS production comparison under normoxic and hypoxic conditions

• Structural Biology:

  • Homology modeling based on known Complex I structures

  • Comparative molecular dynamics simulations

  • AI-driven conformational ensemble generation to compare dynamic properties

  • Analysis of species-specific binding pockets and interaction surfaces

• Experimental Validation:

  • Creation of chimeric proteins swapping domains between species

  • Site-directed mutagenesis to introduce species-specific residues

  • Functional testing in cellular systems under normal and stressed conditions

  • Expression in MT-ND4L-deficient cell lines to assess functional complementation

These comparative approaches can reveal how evolutionary adaptations in MT-ND4L contribute to the remarkable physiological capabilities of marine mammals, particularly their ability to withstand prolonged diving and hypoxic conditions.

What are the differences in biochemical properties between recombinant MT-ND4L and native protein, and how can these differences impact experimental results?

Recombinant and native MT-ND4L proteins exhibit several key differences that can significantly influence experimental outcomes:

Biochemical Property Differences:

• Post-translational Modifications (PTMs):

  • Native MT-ND4L may undergo specific PTMs absent in recombinant systems

  • Bacterial expression systems lack machinery for mammalian-type modifications

  • PTMs can affect protein stability, interactions, and function

• Lipid Environment:

  • Native protein exists in the specialized lipid composition of the inner mitochondrial membrane

  • Recombinant protein is typically purified in detergent micelles or artificial lipid systems

  • Lipid-protein interactions may differ substantially between systems

• Protein Folding and Structure:

  • Native MT-ND4L folds co-translationally in the presence of assembly factors

  • Recombinant protein folds under different cellular conditions (e.g., in E. coli)

  • Structural subtleties may differ even with identical primary sequences

• Presence of Tags and Fusion Partners:

  • Recombinant proteins typically contain affinity tags (e.g., His-tag)

  • These additions can affect protein properties and interactions

  • Cleavage of tags may not restore fully native-like properties

Impact on Experimental Results:

Mitigation Strategies:

Understanding and accounting for these differences is crucial for proper experimental design and interpretation when working with recombinant MT-ND4L.

How can advanced computational approaches and AI-driven techniques enhance our understanding of MT-ND4L structure-function relationships?

Advanced computational and AI-driven approaches offer powerful new avenues for investigating MT-ND4L:

AI-Enhanced Structural Analysis:

• Conformational Ensemble Generation:

  • AI algorithms can predict alternative functional states of MT-ND4L

  • Advanced molecular simulations with AI-enhanced sampling explore the protein's conformational space

  • Identification of representative structures from the conformational ensemble

  • Diffusion-based AI models and active learning AutoML generate statistically robust ensembles of equilibrium conformations

• Binding Pocket Identification:

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

  • Structure-aware ensemble-based detection algorithms utilize established protein dynamics

  • Integration of literature data with computational predictions provides comprehensive binding site mapping

• Molecular Dynamics Innovations:

  • Enhanced sampling techniques (metadynamics, replica exchange) explore energy landscapes

  • Integration of experimental constraints to guide simulations

  • Coarse-grained models to access longer timescales relevant to protein function

  • Machine learning force fields improving accuracy of conformational predictions

Knowledge Extraction and Integration:

• LLM-Powered Literature Research:

  • Custom-tailored language learning models extract information from structured and unstructured sources

  • Knowledge graph creation capturing protein-protein interactions and functional relationships

  • Identification of therapeutic significance and existing small molecule ligands

• Multi-Omics Data Integration:

  • Integration of genomic, metabolomic, and proteomic data

  • Network analysis to position MT-ND4L in cellular pathways

  • Identification of metabolites associated with MT-ND4L variants, such as glycerophospholipids

Methodological Implementation:

These computational approaches complement experimental work by providing atomic-level insights into mechanisms that are challenging to observe directly, generating testable hypotheses, and guiding experimental design for maximum efficiency.

What role does MT-ND4L play in the pathogenesis of mitochondrial disorders beyond Leber hereditary optic neuropathy, and what research methodologies are most promising for investigating these connections?

MT-ND4L's involvement in mitochondrial disorders extends beyond Leber hereditary optic neuropathy (LHON) to potentially impact a broader spectrum of conditions:

Emerging Disease Associations:

• Metabolic Disorders:

  • Changes in MT-ND4L gene expression have long-term consequences on energy metabolism

  • MT-ND4L variants are associated with alterations in glycerophospholipid metabolism

  • The variant mt10689 G > A has been linked to significant changes in phosphatidylcholine ratios

• Neurological Conditions:

  • Complex I dysfunction is implicated in multiple neurodegenerative diseases

  • MT-ND4L mutations may contribute to disease progression through energy deficiency and oxidative stress

  • Subtle alterations in MT-ND4L function could affect vulnerable tissues with high energy demands

• Aging-Related Pathologies:

  • Mitochondrial DNA mutations accumulate with age

  • MT-ND4L variants may contribute to the aging phenotype

  • Complex I efficiency decline is a hallmark of aging tissues

Promising Research Methodologies:

• Patient-Derived Models:

  • Induced pluripotent stem cells (iPSCs) from patients with MT-ND4L mutations

  • Differentiation into affected cell types (neurons, retinal cells, muscle)

  • CRISPR-based genome editing to create isogenic controls

  • Organoid development to model tissue-specific effects

• Multi-Omics Integration:

  • Mitochondrial genome-wide association studies (mt-GWAS) linking MT-ND4L variants to metabolic phenotypes

  • Metabolomic profiling to identify biomarkers of MT-ND4L dysfunction

  • Proteomic analysis of altered mitochondrial protein expression and modifications

  • Integration of genomic, transcriptomic, proteomic, and metabolomic data

• Advanced Imaging Techniques:

  • Live-cell imaging of mitochondrial dynamics and function

  • Super-resolution microscopy to visualize Complex I distribution

  • Correlative light and electron microscopy for structural-functional analysis

  • In vivo imaging of mitochondrial function in disease models

• Systems Biology Approaches:

  • Computational modeling of Complex I function in health and disease

  • Flux analysis to measure metabolic consequences of MT-ND4L mutations

  • Network analysis to identify compensatory pathways

  • Machine learning to recognize patterns in multi-omics data

These integrative approaches can reveal how MT-ND4L dysfunction contributes to disease pathogenesis beyond LHON and identify potential therapeutic targets. The combination of patient-derived models with advanced molecular techniques and computational methods provides a comprehensive framework for investigating the role of MT-ND4L in mitochondrial disorders.