Recombinant Semnopithecus entellus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L (mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4L) is a protein-coding gene that provides instructions for making the NADH dehydrogenase 4L protein. This protein constitutes an essential component of Complex I (NADH:ubiquinone oxidoreductase), which functions as an entry point to the electron transport chain in mitochondria and many aerobic bacteria. Complex I is embedded in the inner mitochondrial membrane and plays a crucial role in oxidative phosphorylation, the process by which mitochondria convert energy from food into ATP, the cell's main energy source .

Within the oxidative phosphorylation system, Complex I catalyzes the first step of electron transport, transferring electrons from NADH to ubiquinone. This electron transfer is coupled with proton pumping across the inner mitochondrial membrane, contributing to the generation of an electrochemical gradient that drives ATP synthesis. The MT-ND4L subunit is one of the core subunits essential for the catalytic function of Complex I .

How is MT-ND4L incorporated into the larger Complex I structure?

MT-ND4L is one of the core subunits of Complex I, which in mammals typically consists of 45 different subunits. In Complex I from various species like Pichia pastoris, 41 subunits have been identified, comprising 14 core (conserved) subunits and 27 supernumerary subunits . The core subunits include seven that are mitochondrially encoded, including MT-ND4L.

The incorporation of MT-ND4L occurs during the assembly of Complex I, which follows a modular pattern. MT-ND4L is integrated into the membrane domain of Complex I, which spans the inner mitochondrial membrane. Methodologically, researchers can track this incorporation using techniques such as blue native gel electrophoresis combined with Western blotting or radioactive pulse-chase experiments using labeled amino acids. These approaches allow for the visualization of assembly intermediates and can track the kinetics of MT-ND4L incorporation into the holoenzyme.

What post-translational modifications occur in MT-ND4L?

Analysis of mitochondrially encoded subunits in Complex I has revealed that these proteins (including MT-ND4L) retain their N-α-formyl methionine residues when translated using the mold mitochondrial genetic code . This retention of formylated methionine represents a significant post-translational characteristic of mitochondrially encoded proteins.

To investigate post-translational modifications in recombinant MT-ND4L, researchers should employ:

• Mass spectrometry-based approaches (peptide mass fingerprinting and tandem MS)

• Reverse-phase HPLC separation followed by ESI-MS analysis

• Site-directed mutagenesis of potential modification sites to assess functional consequences

When analyzing post-translational modifications, it is crucial to consider the translation system used for recombinant expression, as this may affect the modification pattern compared to the native mitochondrially-translated protein.

What expression systems are optimal for producing recombinant Semnopithecus entellus MT-ND4L?

For the expression of recombinant Semnopithecus entellus MT-ND4L, researchers must consider several critical factors that influence protein yield, folding, and functionality:

When expressing recombinant MT-ND4L, researchers should carefully consider the codon usage of the expression host. Additionally, since MT-ND4L is normally translated using the mitochondrial genetic code, codon optimization for the selected expression system is crucial. For example, if using P. pastoris (which has been successfully used for Complex I studies), the protein sequences should be adjusted according to the appropriate genetic code .

How can researchers effectively purify recombinant Semnopithecus entellus MT-ND4L while maintaining protein functionality?

Purification of recombinant MT-ND4L presents significant challenges due to its hydrophobic nature and integration into the mitochondrial membrane. A methodological approach involves:

• Membrane solubilization: Use mild detergents such as n-dodecyl-β-D-maltoside (DDM), digitonin, or LMNG to extract the protein while preserving its native conformation.

• Affinity chromatography: Incorporate affinity tags (His6, FLAG, or Strep-tag) at either terminus, considering potential interference with protein function. The choice of tag position should be validated experimentally.

• Size exclusion chromatography: Perform as a polishing step to separate monomeric protein from aggregates and remove remaining impurities.

• Functional validation: Assess protein functionality throughout purification using activity assays measuring electron transfer from NADH to ubiquinone analogs.

The optimal purification protocol established for Complex I from P. pastoris can serve as a reference model, where the complex was isolated and analyzed using a combination of techniques including SDS-PAGE and HPLC fractionation . Researchers should monitor the oxidation state of the protein during purification, as oxidation of critical thiols can affect functionality.

What analytical techniques are most effective for studying the structure-function relationship of recombinant MT-ND4L?

To comprehensively characterize recombinant Semnopithecus entellus MT-ND4L, multiple complementary techniques should be employed:

• Spectroscopic methods:

  • Circular dichroism (CD) to assess secondary structure and thermal stability

  • Fluorescence spectroscopy to monitor conformational changes upon substrate binding

  • NMR for structural determination of specific domains or the entire protein in detergent micelles

• Mass spectrometry approaches:

  • Peptide mass fingerprinting and tandem MS for sequence confirmation

  • Hydrogen-deuterium exchange MS to probe solvent accessibility and conformational dynamics

  • Crosslinking-MS to identify interaction interfaces with other Complex I subunits

• Functional assays:

  • NADH:ubiquinone oxidoreductase activity measurements

  • ROS production assessment using fluorescent probes

  • Membrane potential measurements using potential-sensitive dyes

For structural determination, researchers have successfully employed a combination of SDS-PAGE separation and HPLC fractionation followed by mass spectrometry analysis for Complex I subunits . This multi-faceted approach allows for comprehensive characterization of both the protein's primary structure and its post-translational modifications.

How can researchers investigate interactions between MT-ND4L and other Complex I subunits?

Understanding subunit interactions is crucial for elucidating Complex I assembly and function. Methodological approaches include:

• Crosslinking strategies:

  • Chemical crosslinking with MS detection to identify proximal residues

  • Photo-activatable crosslinkers for capturing transient interactions

  • In vivo crosslinking to preserve native interaction networks

• Co-immunoprecipitation and pull-down assays:

  • Using antibodies against MT-ND4L or its interaction partners

  • Incorporating affinity tags for specific isolation of protein complexes

  • Sequential immunoprecipitation to isolate specific subcomplexes

• Biophysical interaction analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Microscale thermophoresis for quantitative interaction analysis

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

• Computational approaches:

  • Molecular docking to predict interaction interfaces

  • Molecular dynamics simulations to assess stability of protein-protein interactions

  • Coevolution analysis to identify co-evolving residues indicative of interaction interfaces

When analyzing complex formation, it's important to consider that Complex I from various species contains different numbers of subunits. For example, P. pastoris Complex I has 39 subunits in common with Y. lipolytica Complex I, 37 in common with N. crassa, and 35 in common with bovine enzyme . This variability suggests species-specific interaction networks that should be considered when studying Semnopithecus entellus MT-ND4L.

How do mutations in MT-ND4L affect Complex I assembly and function?

Mutations in MT-ND4L can significantly impact Complex I assembly, stability, and catalytic activity. For example, the T10663C (Val65Ala) mutation in human MT-ND4L has been associated with Leber hereditary optic neuropathy . To systematically analyze the effects of mutations:

• Site-directed mutagenesis approach:

  • Generate a library of MT-ND4L variants with single amino acid substitutions

  • Focus on conserved residues identified through multiple sequence alignment

  • Include known pathogenic mutations from human MT-ND4L studies as positive controls

• Functional characterization:

  • Measure Complex I activity (NADH:ubiquinone oxidoreductase) with spectrophotometric assays

  • Assess proton pumping efficiency using pH-sensitive fluorescent probes

  • Quantify ROS production to determine if mutations increase oxidative stress

• Assembly analysis:

  • Blue native PAGE to visualize Complex I assembly intermediates

  • Western blotting to quantify fully assembled Complex I

  • Pulse-chase experiments to monitor the kinetics of Complex I assembly

• Structural impact assessment:

  • In silico modeling to predict structural perturbations

  • Hydrogen-deuterium exchange MS to detect conformational changes

  • Thermal stability assays to determine if mutations affect protein stability

When interpreting mutation effects, researchers should consider that certain residues may play species-specific roles. Therefore, comparative analysis with mutations in MT-ND4L from other species can provide valuable insights into evolutionary conservation of functionally important residues.

What are the best approaches for studying MT-ND4L mutations in cellular models?

To study the cellular consequences of MT-ND4L mutations:

• Cell model selection:

  • Cybrid cell lines (transmitochondrial cybrids) containing patient-derived mitochondria

  • CRISPR/Cas9-engineered cell lines with specific MT-ND4L mutations

  • Heterologous expression systems where endogenous MT-ND4L is replaced with the recombinant version

• Phenotypic characterization:

  • Cellular respiration measurements using oxygen electrodes or Seahorse analyzers

  • ATP production quantification using luminescence-based assays

  • Mitochondrial membrane potential assessment using potentiometric dyes

  • Cell viability and growth rate determination under various metabolic conditions

• Stress response analysis:

  • Sensitivity to oxidative stress inducers (H₂O₂, paraquat)

  • Response to Complex I inhibitors (rotenone, piericidin A)

  • Adaptation to different carbon sources requiring mitochondrial function

• Rescue experiments:

  • Complementation with wild-type MT-ND4L to verify causality

  • Alternative oxidase expression to bypass respiratory chain defects

  • Antioxidant supplementation to mitigate ROS-related phenotypes

These cellular models can provide insights into how MT-ND4L mutations affect mitochondrial function in a physiologically relevant context, bridging the gap between biochemical studies and disease mechanisms.

What controls should be included when studying recombinant Semnopithecus entellus MT-ND4L function in vitro?

Proper experimental controls are essential for robust and reproducible studies of recombinant MT-ND4L:

Additionally, researchers should include controls for potential post-translational modifications. Since mitochondrially encoded subunits retain their N-α-formyl methionine residues , comparison with recombinant proteins lacking this modification can help determine its functional significance.

How can researchers effectively measure electron transport activity of reconstituted Complex I containing recombinant MT-ND4L?

Accurate measurement of electron transport activity requires careful experimental design:

• Reconstitution approaches:

  • Proteoliposome reconstitution with defined lipid composition

  • Nanodiscs for a more controlled membrane environment

  • Direct incorporation into submitochondrial particles

• Activity measurement methods:

  • Spectrophotometric assays (340 nm) measuring NADH oxidation

  • Reduction of artificial electron acceptors (ferricyanide, DCIP)

  • Oxygen consumption measurements if coupled to subsequent respiratory chain components

  • Fluorescence-based assays for proton pumping (ACMA quenching)

• Critical parameters to optimize:

  • Detergent type and concentration during reconstitution

  • Lipid composition and protein:lipid ratio

  • Temperature and pH conditions

  • Buffer composition (ionic strength, presence of divalent cations)

• Data analysis considerations:

  • Initial rate calculations (first 30-60 seconds of reaction)

  • Correction for non-enzymatic NADH oxidation

  • Normalization to protein concentration or specific activity of reference samples

  • Statistical analysis across multiple independent reconstitutions

For comparative studies, it's important to note that different enzyme preparation methods can yield varying results. When possible, researchers should benchmark their recombinant system against native Complex I isolated from the same or closely related species.

How should researchers interpret discrepancies between in vitro and in vivo studies of recombinant MT-ND4L?

When confronted with discrepancies between in vitro biochemical data and in vivo cellular studies:

• Consider system complexity differences:

  • In vitro systems lack cellular regulatory mechanisms

  • The lipid environment differs between artificial membranes and mitochondria

  • Potential absence of interacting proteins in reconstituted systems

  • Different post-translational modification patterns

• Methodological reconciliation approaches:

  • Gradually increase system complexity (protein → proteoliposome → submitochondrial particles → isolated mitochondria → cells)

  • Parallel assays under identical conditions where possible

  • Identify environmental variables that may explain differences (pH, ion concentrations, metabolite levels)

• Validation strategies:

  • Structure-function correlation using site-directed mutagenesis

  • Complementation studies in cells lacking functional MT-ND4L

  • Cross-validation with orthogonal techniques

When analyzing Complex I function, researchers should be aware that different organisms may use different mitochondrial genetic codes. For example, the mold mitochondrial genetic code was found to better explain peptide mass and sequence data from P. pastoris Complex I subunits than the yeast mitochondrial code .

What statistical approaches are most appropriate for analyzing Complex I activity data?

Robust statistical analysis is crucial for meaningful interpretation of experimental data:

• Experimental design considerations:

  • Determine appropriate sample size through power analysis

  • Include biological replicates (independent preparations) and technical replicates

  • Randomize sample processing order to minimize systematic errors

  • Blind the analyst to sample identity when possible

• Data preprocessing steps:

  • Outlier identification and handling (Grubbs' test, Dixon's Q test)

  • Normality testing (Shapiro-Wilk, Kolmogorov-Smirnov)

  • Transformation of non-normal data (log, Box-Cox)

  • Standardization for comparative analyses

• Statistical test selection:

  • Parametric tests (t-test, ANOVA) for normally distributed data

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) when assumptions are violated

  • Repeated measures designs for time-course experiments

  • Multiple comparison correction (Bonferroni, Benjamini-Hochberg FDR)

• Advanced data analysis approaches:

  • Principal component analysis for multivariate data

  • Hierarchical clustering to identify patterns

  • Regression analysis to establish relationships between variables

  • Machine learning for complex datasets with multiple parameters

When analyzing enzyme kinetics data, non-linear regression to appropriate models (Michaelis-Menten, allosteric models) should be performed rather than linear transformations, which can distort error structure.

What emerging technologies could advance our understanding of MT-ND4L structure and function?

Several cutting-edge technologies hold promise for deeper insights into MT-ND4L biology:

• Structural biology advances:

  • Cryo-electron microscopy for high-resolution structures of intact Complex I

  • Integrative structural biology combining multiple data sources (X-ray, NMR, crosslinking-MS)

  • Computational prediction methods incorporating co-evolutionary information

• Single-molecule techniques:

  • Single-molecule FRET to track conformational changes during catalysis

  • Optical tweezers to measure force generation during proton pumping

  • Single-molecule electrophysiology to directly measure proton translocation

• Live-cell imaging approaches:

  • Super-resolution microscopy to visualize Complex I distribution and dynamics

  • FRET-based sensors to monitor Complex I activity in living cells

  • Correlative light and electron microscopy for structural-functional integration

• Genome editing technologies:

  • Mitochondrially targeted nucleases for precise MT-ND4L modification

  • Base editors for introducing specific point mutations

  • Prime editing for more complex sequence alterations in mitochondrial DNA

These technologies could help resolve long-standing questions about the molecular mechanism of proton pumping by Complex I and how MT-ND4L contributes to this process. Additionally, they may reveal species-specific features of Semnopithecus entellus MT-ND4L that have evolved in response to the primate's unique metabolic demands and environmental adaptations.