Recombinant Pontoporia blainvillei NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the basic structure and function of MT-ND4L in Pontoporia blainvillei?

MT-ND4L (NADH-ubiquinone oxidoreductase chain 4L) in Pontoporia blainvillei is a small hydrophobic protein consisting of 98 amino acids that functions as a subunit of respiratory chain complex I in the mitochondrial inner membrane. The amino acid sequence is: MTLVHMNLLLAFAMSLTGLLMYRSHLMSALLCLEGMVLSLFILATITTLNSHFTLANMMPIILLVFAACEAAIGLALLVKISNTYGTDHVQNLNLLQC .

This protein plays a crucial role in the electron transport chain, specifically in NADH dehydrogenase (ubiquinone) activity. It contributes to the proton translocation mechanism necessary for ATP synthesis. Like its human counterpart, the P. blainvillei MT-ND4L is likely organized in transmembrane helices that help form the membrane arm of complex I, facilitating proton pumping across the mitochondrial inner membrane .

How does recombinant P. blainvillei MT-ND4L differ from the native protein?

Recombinant P. blainvillei MT-ND4L differs from the native protein primarily in its expression system and modifications:

• Expression system: The recombinant protein is expressed in E. coli rather than in mitochondria of P. blainvillei, which may affect post-translational modifications .

• His-tag addition: The recombinant protein contains an N-terminal His-tag, which facilitates purification but is not present in the native protein .

• Buffer conditions: The recombinant protein is supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, whereas the native protein exists in the lipid environment of the mitochondrial inner membrane .

• Folding environment: Native MT-ND4L folds within the mitochondrial membrane in coordination with other complex I subunits, while recombinant protein folds in the bacterial cytoplasm, potentially affecting its tertiary structure.

When using recombinant MT-ND4L for research, these differences should be considered, especially when studying protein-protein interactions or enzymatic activity.

What reconstitution methods are recommended for lyophilized recombinant MT-ND4L?

The recommended reconstitution protocol for lyophilized recombinant P. blainvillei MT-ND4L includes the following methodological steps:

• Initial preparation: Briefly centrifuge the vial prior to opening to ensure all content is at the bottom of the tube .

• Reconstitution solution: Use deionized sterile water to reconstitute the protein to a concentration of 0.1-1.0 mg/mL .

• Glycerol addition: Add glycerol to a final concentration of 5-50% for long-term storage stability. The manufacturer's default recommendation is 50% glycerol .

• Aliquoting: Prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles.

• Storage: Store reconstituted protein at -20°C/-80°C for long-term storage; working aliquots can be kept at 4°C for up to one week .

This methodological approach ensures optimal protein stability and activity for experimental use.

How can molecular dynamics simulations be used to study MT-ND4L proton translocation mechanisms?

Molecular dynamics (MD) simulations provide valuable insights into MT-ND4L proton translocation mechanisms through several methodological approaches:

• System preparation: Create a transmembrane system by placing ND4L-ND6 subunits in a lipid bilayer membrane composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), which mimics the mitochondrial inner membrane composition .

• Water and ion integration: Include explicit TIP3P water molecules with physiological concentrations of K+ and Cl- ions (150 mM) to simulate the cellular environment .

• Simulation parameters: Conduct 100+ ns simulations with appropriate force fields, using a 2 fs timestep and particle mesh Ewald technique for long-range electrostatics .

• Analysis of water movement: Track water molecule movements through the transmembrane region as they serve as proton carriers via the Grotthuss mechanism .

• Key residue identification: Monitor specific residues such as Glu34 (ND4L) and Tyr157 (ND6), which play critical roles in forming the proton translocation pathway .

• Conformational changes assessment: Analyze RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuation) values to identify structural changes affecting proton translocation .

This approach revealed that in native proteins, water molecules cluster around Glu34 due to its downward conformation, facilitating proton translocation. Mutations can alter this conformation, restricting water passage and potentially disrupting proton translocation mechanisms .

What are the implications of comparing MT-ND4L sequences across cetacean species including Pontoporia blainvillei?

Comparative analysis of MT-ND4L sequences across cetacean species including Pontoporia blainvillei (Franciscana) offers significant insights into:

• Evolutionary adaptation: Comparing sequence conservation among marine mammals can reveal regions under selective pressure, potentially identifying functionally critical domains for adaptation to marine environments.

• Functional conservation: Highly conserved regions across cetaceans likely indicate essential functional domains for complex I activity.

• Species-specific variations: Unique amino acid substitutions in P. blainvillei MT-ND4L may correlate with specific metabolic adaptations, habitat requirements, or diving physiology of this endangered river dolphin species.

• Biomarker potential: Unique sequence characteristics can serve as genetic markers for population studies and conservation efforts for the vulnerable Franciscana dolphin.

• Structure-function relationships: Amino acid changes can be mapped to 3D structures to predict functional consequences on proton translocation efficiency.

When conducting such comparative analyses, researchers should employ phylogenetic methods that account for the unique evolutionary history of cetaceans, particularly considering the evolutionary divergence of river dolphins like P. blainvillei from marine dolphins.

How does the amino acid composition of P. blainvillei MT-ND4L influence its membrane integration properties?

The amino acid composition of P. blainvillei MT-ND4L significantly influences its membrane integration properties through several biophysical mechanisms:

• Hydrophobicity profile: The sequence MTLVHMNLLLAFAMSLTGLLMYRSHLMSALLCLEGMVLSLFILATITTLNSHFTLANMMPIILLVFAACEAAIGLALLVKISNTYGTDHVQNLNLLQC contains numerous hydrophobic residues (L, I, F, V, M), enabling stable integration into the mitochondrial inner membrane lipid bilayer .

• Transmembrane helix formation: The distribution of hydrophobic and hydrophilic residues likely creates distinct transmembrane helices that adopt specific orientations within the membrane.

• Charged residue positioning: Limited charged residues (such as K at position 69) likely position at membrane-water interfaces, anchoring the protein properly.

• Protein-lipid interactions: Specific residues may form interactions with POPC or other mitochondrial membrane lipids, influencing protein stability and function .

• Loop regions: The non-helical regions connecting transmembrane segments contain more polar residues that facilitate protein folding and interactions with adjacent subunits of complex I.

To study these properties experimentally, researchers could employ techniques such as circular dichroism spectroscopy to analyze secondary structure content, fluorescence spectroscopy with environment-sensitive probes, or membrane reconstitution assays with varying lipid compositions to assess membrane integration efficiency.

What expression systems are most suitable for producing functional recombinant P. blainvillei MT-ND4L?

Several expression systems can be considered for producing functional recombinant P. blainvillei MT-ND4L, each with specific advantages:

For initial characterization studies, E. coli expression (as used in search result ) provides a practical approach. For functional studies requiring proper membrane integration, yeast or mammalian systems might offer advantages despite lower yields. When selecting an expression system, researchers should consider their specific experimental requirements, such as yield needs, budget constraints, and whether native-like function is essential.

What are the optimal methods for assessing the functional integrity of recombinant MT-ND4L in vitro?

Assessing the functional integrity of recombinant MT-ND4L in vitro requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Fluorescence spectroscopy to assess tertiary folding

  • Size exclusion chromatography to confirm monomeric state or appropriate oligomerization

• Membrane integration analysis:

  • Liposome flotation assays to verify membrane association

  • Protease protection assays to determine correct topology

  • Lipid nanodiscs reconstitution followed by electron microscopy

• Complex I assembly potential:

  • Co-immunoprecipitation with other complex I subunits

  • Blue Native PAGE to assess integration into larger complexes

  • Cross-linking mass spectrometry to identify interaction partners

• Functional assessments:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Reconstitution with other complex I components to measure NADH:ubiquinone oxidoreductase activity

  • Electron paramagnetic resonance (EPR) spectroscopy to assess redox center integrity

• Water channel functionality:

  • Molecular dynamics simulations to track water molecule movements and potential proton pathways

  • Site-directed mutagenesis of key residues (e.g., Glu34) followed by functional assays

These methodological approaches provide complementary data on both structural integrity and functional capacity, essential for validating recombinant MT-ND4L as a research tool.

How can one design experiments to investigate the interaction between MT-ND4L and other complex I subunits?

Designing experiments to investigate interactions between MT-ND4L and other complex I subunits requires a multi-technique approach:

• Co-immunoprecipitation (Co-IP):

  • Utilize the His-tag on recombinant MT-ND4L for pull-down assays

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate with reciprocal Co-IPs using antibodies against suspected partner subunits

  • Protocol modification: Incorporate mild detergents (e.g., digitonin, DDM) to preserve membrane protein interactions

• Proximity labeling techniques:

  • Fusion of biotin ligase (BioID) or peroxidase (APEX2) to MT-ND4L

  • Expression in mitochondrial environment

  • Identification of proximal proteins via streptavidin pull-down and mass spectrometry

  • Data analysis: Apply statistical thresholds comparing experimental samples to controls

• Crosslinking mass spectrometry (XL-MS):

  • Apply membrane-permeable crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and identify peptide pairs by MS/MS

  • Map interaction sites to 3D structural models

  • Quantitative analysis: Use isotope-labeled crosslinkers for comparative studies

• Förster Resonance Energy Transfer (FRET):

  • Label MT-ND4L and potential partner proteins with fluorophore pairs

  • Measure energy transfer efficiency in membrane environments

  • Controls must include non-interacting protein pairs

  • Data interpretation: Calculate distance constraints from FRET efficiencies

• Molecular Dynamics Simulations:

  • Build models of MT-ND4L with adjacent subunits (e.g., ND6)

  • Simulate in membrane environments with explicit lipids

  • Analyze stability of key interfaces and hydrogen bond networks

  • Validation: Compare simulation predictions with experimental results

• Mutational analysis:

  • Identify conserved residues at potential interfaces

  • Generate point mutations and assess effects on complex formation

  • Measure functional consequences on proton translocation

  • Experimental design: Include both conservative and non-conservative substitutions

These complementary approaches provide structural, biochemical, and functional evidence of specific interactions between MT-ND4L and other complex I subunits.

What role does MT-ND4L play in the proton translocation mechanism of complex I, and how can this be studied?

MT-ND4L plays a critical role in the proton translocation mechanism of complex I through several key functions:

• Formation of proton channel:

  • MT-ND4L contributes to forming one of the proton translocation pathways in complex I

  • The interface between MT-ND4L and ND6 creates a channel for water molecules that facilitate proton movement

  • Key residue Glu34 of MT-ND4L is particularly important in this process

• Water molecule organization:

  • Water molecules cluster around specific residues like Glu34

  • These organized water molecules enable proton hopping via the Grotthuss mechanism

  • Proper conformation of MT-ND4L is essential for maintaining this water channel

• Coupling mechanism:

  • MT-ND4L likely participates in coupling electron transfer to proton pumping

  • Conformational changes in MT-ND4L may transmit energy from the peripheral arm to drive proton translocation

This can be studied through multiple methodological approaches:

Research has shown that mutations in MT-ND4L can disrupt this proton pathway by altering the conformation of key residues, potentially explaining how MT-ND4L mutations contribute to diseases like diabetes mellitus .

What techniques can be used to study conformational changes in MT-ND4L under different physiological conditions?

Multiple advanced techniques can be employed to study conformational changes in MT-ND4L under varying physiological conditions:

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Methodology: Expose protein to D₂O buffer under different conditions (pH, temperature, substrate presence)

  • Analysis: Monitor deuterium incorporation rates by LC-MS

  • Advantages: Maps solvent-accessible regions, detects subtle conformational changes

  • Challenges: Requires specialized equipment and membrane protein protocols

  • Data interpretation: Slower exchange indicates protected regions; differential exchange between conditions reveals conformational changes

• Site-Directed Spin Labeling with Electron Paramagnetic Resonance (SDSL-EPR):

  • Methodology: Introduce cysteine mutations at strategic positions, label with nitroxide probes

  • Analysis: Measure distances between spin labels and mobility parameters

  • Advantages: Works in membrane environments, detects nanometer-scale movements

  • Implementation: Carefully select labeling sites to avoid functional disruption

• Single-Molecule FRET (smFRET):

  • Methodology: Label protein with donor-acceptor fluorophore pairs

  • Analysis: Monitor distance changes between fluorophores over time

  • Advantages: Detects dynamic conformational changes in real-time

  • Technical considerations: Requires fluorescent labeling at specific positions

• AI-Driven Conformational Ensemble Generation:

  • Methodology: Apply advanced AI algorithms to predict alternative functional states

  • Implementation: Molecular simulations with AI-enhanced sampling

  • Analysis: Trajectory clustering to identify representative structures

  • Applications: Explore conformational space under different conditions

• Time-Resolved Cryo-EM:

  • Methodology: Rapidly freeze samples at different time points after triggering conformational change

  • Analysis: Reconstruct 3D structures from particle images

  • Advantages: Directly visualizes structural states

  • Challenges: Requires specialized equipment and expertise

• Vibrational Spectroscopy (FTIR/Raman):

  • Methodology: Measure vibrational spectra under different conditions

  • Analysis: Monitor changes in secondary structure content

  • Advantages: Works with membrane proteins, minimal sample preparation

  • Data interpretation: Shift in amide I/II bands indicates structural changes

These complementary techniques provide insights at different levels of resolution, from global conformational changes to specific residue movements, essential for understanding MT-ND4L's role in complex I function under varying physiological conditions.

How do mutations in MT-ND4L contribute to mitochondrial diseases, and can P. blainvillei MT-ND4L serve as a model?

Mutations in MT-ND4L contribute to mitochondrial diseases through several key pathological mechanisms, and P. blainvillei MT-ND4L may serve as a comparative model:

• Established disease associations:

  • T10609C mutation (M47T) is linked to type 2 diabetes mellitus (T2DM)

  • C10676G mutation (C69W) is associated with cataracts

  • MT-ND4L mutations are implicated in Leber hereditary optic neuropathy (LHON)

• Pathophysiological mechanisms:

  • Disrupted proton translocation: Mutations alter the proton channel structure, limiting water molecule passage

  • Conformational changes: M47T mutation causes loop structure changes and loss of hydrogen bonds

  • Impaired complex I activity: Reduced proton pumping capacity leads to decreased ATP production

  • Increased ROS production: Dysfunctional complex I increases reactive oxygen species generation

• P. blainvillei as a comparative model:

  • Advantages:

    • Conserved functional domains allow inference of mutation effects

    • Evolutionary distance provides perspective on essential vs. adaptable regions

  • Limitations:

    • Species-specific differences in nuclear-encoded complex I subunits

    • Different metabolic demands between humans and cetaceans

• Research approach using P. blainvillei MT-ND4L:

  • Introduce equivalent human disease mutations into P. blainvillei MT-ND4L

  • Conduct comparative molecular dynamics simulations to assess effects on water channels

  • Compare structural stability and protein-protein interactions

  • Correlate findings with clinical data to identify conserved pathological mechanisms

Molecular dynamics studies have revealed that disease-associated mutations disrupt the hydrogen bond networks essential for proton translocation, specifically showing that both M47T and C69W mutations lead to hydrogen bond formation between Glu34 and Tyr157, restricting water molecule passage through the transmembrane region . This mechanism provides insight into how MT-ND4L mutations contribute to diseases like T2DM and cataracts.

What are the effects of T10609C (M47T) and C10676G (C69W) mutations on MT-ND4L structure and function based on molecular dynamics studies?

Molecular dynamics studies have revealed significant structural and functional changes resulting from T10609C (M47T) and C10676G (C69W) mutations in MT-ND4L:

T10609C (M47T) mutation effects:

• Structural alterations:

  • Loss of hydrogen bonding: The native model has 3 hydrogen bonds involving Met47's oxygen atoms with amine groups of Thr51 and Asn50, while the M47T mutant loses one hydrogen bond

  • Conformational change: Thr51 changes conformation and no longer forms hydrogen bonds with Ser53

  • Loop structure elongation: The loop structure in the M47T mutant becomes longer than in the native protein

• Functional implications:

  • Loss of hydrophobic interactions: The mutation eliminates important hydrophobic interactions between Met47 (ND4L) and Met79 of the ND2 subunit

  • Proton pathway disruption: The conformational changes affect the bottom side loop, limiting water molecule entry

  • Water channel obstruction: Hydrogen bond formation between Glu34 and Tyr157 interrupts the translocation pathway

C10676G (C69W) mutation effects:

• Structural changes:

  • Altered interactions: Native Cys69 forms hydrogen bonds with Thr257 and hydrophobic interactions with Leu258, while mutant Trp69 forms stronger hydrophobic interactions with Val73 and Ile264

  • Increased stability: The C69W mutation results in more stabilized conformation due to stronger hydrophobic interactions

  • Helix reorganization: The three hydrophobic interactions in C69W make the helix conformation more organized compared to the native structure

• Functional consequences:

  • Proton translocation obstruction: Despite different structural changes from M47T, this mutation also leads to the formation of hydrogen bonds between Glu34 and Tyr157, blocking the proton pathway

  • Water movement restriction: The mutation restricts water molecule passage through the transmembrane region

Both mutations, despite occurring at different positions, ultimately converge on a similar pathological mechanism: disruption of the proton translocation pathway by inducing conformational changes that block water molecule movement through the protein. This mechanism explains how these mutations contribute to distinct clinical conditions (T2DM and cataracts) through mitochondrial dysfunction .

How can understanding MT-ND4L structure and function contribute to developing therapeutic approaches for mitochondrial diseases?

Understanding MT-ND4L structure and function provides several avenues for developing therapeutic approaches for mitochondrial diseases:

The molecular dynamics studies of MT-ND4L mutations provide particularly valuable insights by revealing that mutations at different positions (M47T and C69W) converge on similar functional disruptions through interruption of water-mediated proton translocation pathways . This mechanistic understanding enables the development of targeted interventions that could restore proton translocation function regardless of the specific mutation site.