Recombinant Artemia franciscana NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is the amino acid sequence and structural characteristics of Artemia franciscana ND4L?

Artemia franciscana ND4L is a small, highly hydrophobic protein consisting of 85 amino acids with the sequence: MMIYLSLSLGLLIFSSSNKHLLVTLLSLEFLILLLFSLLVYSNYMSMINAFIFLSVTVCE GALGFSVLVSLVRSSGSDQVQFLNE . This protein is characterized by multiple transmembrane helices that contribute to its functional role in the mitochondrial respiratory complex I. The protein can be produced recombinantly with an N-terminal His-tag in E. coli expression systems to facilitate purification and experimental manipulation . Artemia franciscana ND4L exhibits structural similarities to ND4L proteins from other species, though subtle differences exist that may influence species-specific functions.

How does Artemia franciscana ND4L compare structurally to ND4L from Artemia salina?

Comparative analysis reveals high sequence similarity between ND4L proteins from Artemia franciscana and Artemia salina, with only minor differences. Artemia salina ND4L is 86 amino acids in length (one amino acid longer than A. franciscana) with the sequence: MMMIYLSLSLGLLIFSSSNKHLLVTLLSFEFLILLLFSLLVYSNYMSMINAFIFLSVTVCE GALGLSVLVSLVRSSGSDQVQFLNE . Key differences include an additional methionine at the N-terminus of A. salina ND4L and a substitution at position 53 (S in A. salina vs. L in A. franciscana) . These differences, though subtle, may contribute to species-specific adaptations of mitochondrial function in these brine shrimp species, which could be relevant when using these proteins as experimental models.

What is the biological role of ND4L in the mitochondrial respiratory complex?

ND4L functions as a critical subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase), where it plays an essential role in the proton translocation pathway . This protein is part of the membrane-embedded hydrophobic domain of complex I and contributes to energy conservation by coupling electron transfer to proton pumping across the inner mitochondrial membrane. Research has demonstrated that ND4L is specifically associated with the fourth proton channel at the interface with the ND6 subunit, forming a critical component of the proton translocation machinery . The absence of ND4L prevents the assembly of the complete 950-kDa complex I and eliminates enzyme activity, highlighting its essential nature in respiratory chain function .

What are the optimal conditions for reconstitution and storage of recombinant A. franciscana ND4L protein?

For optimal handling of recombinant A. franciscana ND4L protein, the following methodology is recommended:

• Initial Handling: Centrifuge the vial briefly before opening to ensure all content settles at the bottom .

• Reconstitution: Dissolve the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Stabilization: Add glycerol to a final concentration of 5-50% (optimally 50%) to enhance stability during storage .

• Storage Conditions:

  • Long-term storage: Maintain at -20°C to -80°C in aliquots to minimize freeze-thaw cycles

  • Working solutions: Store at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles as they significantly compromise protein integrity

The reconstituted protein is typically maintained in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps preserve structural integrity . Researchers should verify protein quality after reconstitution using SDS-PAGE, with expected purity greater than 90% .

How can researchers effectively design molecular dynamics simulations to study ND4L function?

Designing effective molecular dynamics simulations for ND4L functional studies requires a methodical approach:

• Homology Modeling:

  • Identify appropriate templates with high sequence identity (e.g., structures from Protein Data Bank)

  • Use modeling software such as MODELLER to generate multiple candidate structures

  • Select models based on lowest DOPE (Discrete Optimized Protein Energy) scores

• Model Evaluation:

  • Validate using tools like PROCHECK and QMEANBrane to assess stereochemical properties

  • Compare DOPE profiles between model and template structures

  • Rule out impossible conformations with steric hindrances or distorted bond angles

• Transmembrane System Construction:

  • Place ND4L-ND6 subunits in a lipid bilayer membrane composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine)

  • POPC represents approximately 40% of the inner mitochondrial membrane composition

  • Use Membrane Builder tools in molecular dynamics software packages

• Simulation Analysis:

  • Track RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuation) values to assess structural stability

  • Analyze hydrogen bonds and hydrophobic interactions affecting protein conformation

  • Visualize using programs like VMD (Visual Molecular Dynamics)

This systematic approach allows researchers to investigate conformational changes, proton pathways, and effects of mutations on ND4L structure and function.

What experimental approaches can be used to study the role of A. franciscana ND4L in proton translocation?

Investigating A. franciscana ND4L's role in proton translocation requires sophisticated experimental approaches:

• Site-Directed Mutagenesis:

  • Target conserved residues involved in proton channels

  • Focus on charged amino acids (Glu, Asp) and residues shown to participate in hydrogen-bonding networks

  • Introduce mutations that alter hydrophobicity, charge, or spatial characteristics

• Functional Assays:

  • Measure complex I activity using NADH:ubiquinone oxidoreductase assays

  • Assess proton pumping efficiency using pH-sensitive fluorescent dyes

  • Compare enzyme kinetics between wild-type and mutant proteins

• Structural Analysis:

  • Use molecular dynamics simulations to track water molecule movement through putative proton channels

  • Monitor conformational changes affecting the orientation of key residues like Glu34

  • Analyze interaction between ND4L and neighboring subunits, particularly ND6

• Reconstitution Studies:

  • Incorporate purified recombinant ND4L into proteoliposomes

  • Measure proton gradient formation across membrane

  • Assess the effects of inhibitors on proton translocation

Research has shown that specific residues in ND4L form part of a proton pathway where water molecules are recruited by amino acids like Glu34 to facilitate proton movement across the membrane barrier . Mutations that disrupt these interactions can significantly alter proton translocation efficiency, providing insights into the mechanistic details of this process.

How can researchers effectively study the assembly process of complex I with focus on ND4L contribution?

To investigate ND4L's role in complex I assembly:

• Gene Silencing Approaches:

  • Use RNA interference (RNAi) to suppress ND4L expression

  • Construct RNAi plasmids containing ND4L gene fragments in inverse orientation

  • Transform target cells and select transformants with appropriate markers

• Assembly Analysis:

  • Apply Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) to visualize intact respiratory complexes

  • Solubilize protein complexes with detergents like dodecylmaltoside (2.5%)

  • Use activity staining to identify functional complex I

• Activity Measurements:

  • Assess NADH:ubiquinone oxidoreductase activity spectrophotometrically

  • Compare activity between wild-type and ND4L-deficient samples

  • Evaluate other respiratory chain complexes to assess specificity of effects

• Protein-Protein Interaction Studies:

  • Employ co-immunoprecipitation to identify ND4L interaction partners

  • Use crosslinking approaches to capture transient assembly intermediates

  • Analyze subcomplex formation in the absence of ND4L

Research demonstrates that absence of ND4L prevents formation of the complete 950-kDa complex I and eliminates enzyme activity, suggesting it plays a crucial early role in the assembly pathway . This methodological approach enables researchers to build detailed models of complex I assembly and identify the specific contribution of ND4L to this process.

How does mitochondrial versus nuclear encoding of ND4L affect protein structure and function?

The genomic location of ND4L genes exhibits interesting variation across species, with significant implications for protein properties:

• Comparative Analysis:

  • In most animals including Artemia species, ND4L is encoded in the mitochondrial genome

  • In contrast, some species like Chlamydomonas reinhardtii have transferred ND4L to the nuclear genome (gene designated NUO11)

• Structural Adaptations:

  • Nuclear-encoded ND4L proteins typically show reduced hydrophobicity compared to mitochondrion-encoded counterparts

  • These modifications facilitate protein import into mitochondria after cytosolic synthesis

  • Nuclear versions often acquire targeting sequences for mitochondrial import machinery

• Functional Implications:

  • Despite genomic relocation, nuclear-encoded ND4L retains its essential function in complex I

  • The protein must maintain critical interactions with other complex I subunits

  • Nuclear encoding allows for potential co-evolution with nuclear-encoded complex I components

• Evolutionary Considerations:

  • Gene transfer from mitochondria to nucleus represents an ongoing evolutionary process

  • Successful transfer requires multiple adaptations in gene structure and protein properties

  • Provides increased regulatory control by the nuclear genome

This comparative approach offers insights into evolutionary dynamics of mitochondrial genes and the adaptability of protein structure to accommodate changes in cellular trafficking requirements while maintaining essential functions .

What is known about the phylogenetic relationships of Artemia species based on ND4L sequences?

ND4L sequences provide valuable data for understanding Artemia species relationships:

• Sequence Comparison:

  • A. franciscana and A. salina ND4L proteins share high sequence similarity but with distinct differences

  • A. franciscana ND4L consists of 85 amino acids while A. salina has 86 amino acids

  • Key substitutions and the additional N-terminal methionine in A. salina serve as species-specific markers

• Phylogenetic Analysis:

  • Mitochondrial genome analysis places A. salina in closer relationship with A. persimilis compared to other Artemia species

  • ND4L contributes to this phylogenetic placement as part of the mitochondrial gene set

  • The complete mitochondrial genome of A. salina (15,762 bp) contains the typical structure with 13 protein-coding genes including ND4L

• Methodological Approaches:

  • Extract mitochondrial DNA using specialized kits (e.g., TIANGEN® TIANamp Genomic DNA Kit)

  • Sequence using high-throughput platforms (e.g., Illumina Novaseq6000)

  • Assemble genomes using tools like SPAdes v.3.5.0

  • Perform phylogenetic analysis using maximum likelihood or Bayesian methods

These comparative studies provide essential resources for population genetics research and germplasm conservation efforts for Artemia species, with ND4L sequences serving as one of several informative markers for evolutionary relationships .

How do mutations in ND4L affect proton translocation pathways in complex I?

Mutations in ND4L can significantly alter proton translocation pathways with consequent functional effects:

• Structural Impact Analysis:

  • Mutations can modify critical amino acid interactions within the proton channel

  • For example, the T10609C mutation causes an M47T substitution, introducing a more hydrophilic residue

  • This change creates new hydrogen bonding networks that alter loop conformation

• Molecular Dynamics Observations:

  • Mutations can change the positioning of key charged residues like Glu34

  • In wild-type proteins, Glu34 recruits water molecules to facilitate proton movement

  • Mutations may create new hydrogen bonds (e.g., between Glu34 and Tyr157) that restrict water passage

• Functional Consequences:

  • Altered proton pathways can reduce complex I efficiency

  • Changes in hydrophobic interactions may modify helix organization

  • Bulky substitutions (like C69W from the C10676G mutation) can create steric hindrances

• Methodological Approach:

  • Map mutations on the sequence and determine amino acid changes

  • Build homology models of wild-type and mutant proteins

  • Place models in lipid bilayer environments for simulation

  • Analyze hydrogen bonds, hydrophobic interactions, and water molecule movement

These studies reveal how specific amino acid changes in ND4L can disrupt the carefully balanced system of interactions required for efficient proton translocation, potentially contributing to mitochondrial dysfunction and associated disorders.

What experimental design is most effective for analyzing the impact of post-translational modifications on ND4L function?

Investigating post-translational modifications (PTMs) of ND4L requires sophisticated experimental design:

• Identification of PTMs:

  • Use mass spectrometry-based proteomic approaches to identify potential modification sites

  • Apply enrichment techniques specific to the PTM of interest (phosphorylation, acetylation, etc.)

  • Compare PTM patterns between different physiological states

• Site-Directed Mutagenesis:

  • Generate recombinant ND4L variants with mutations at potential PTM sites

  • Create phosphomimetic mutations (e.g., Ser to Asp) to simulate constitutive phosphorylation

  • Develop non-modifiable mutations (e.g., Ser to Ala) to prevent phosphorylation

• Functional Assessment:

  • Reconstitute mutant ND4L into proteoliposomes or complex I subcomplex

  • Measure NADH:ubiquinone oxidoreductase activity

  • Assess proton pumping efficiency using pH-sensitive probes

  • Compare kinetic parameters between wild-type and mutant proteins

• Structural Analysis:

  • Perform molecular dynamics simulations to assess conformational changes induced by PTMs

  • Analyze how modifications affect interactions with neighboring subunits

  • Model altered electrostatic properties and their impact on proton movement

This systematic approach allows researchers to determine whether specific PTMs serve regulatory functions in ND4L activity, potentially providing adaptive mechanisms for modulating mitochondrial function under different physiological conditions.

What are the key challenges in expressing and purifying functional recombinant ND4L, and how can they be addressed?

Researchers face several challenges when working with recombinant ND4L:

• Hydrophobicity Challenges:

  • ND4L's high hydrophobicity often leads to inclusion body formation in E. coli

  • Solution: Use specialized E. coli strains designed for membrane protein expression (C41, C43)

  • Optimize growth at lower temperatures (16-18°C) to slow folding and improve solubility

  • Consider fusion partners that enhance solubility (e.g., MBP, SUMO, or Trx tags)

• Protein Stability Issues:

  • ND4L tends to aggregate when removed from membrane environments

  • Solution: Include appropriate detergents during purification (e.g., dodecylmaltoside at 2.5%)

  • Maintain glycerol (5-50%) in storage buffers to prevent aggregation

  • Use Tris/PBS-based buffers with 6% Trehalose at pH 8.0 for optimal stability

• Functional Assessment:

  • Isolated ND4L may lack activity outside its native complex

  • Solution: Reconstitute with other complex I components to assess functionality

  • Use BN-PAGE to verify correct assembly into higher-order structures

  • Develop specific activity assays for the isolated subunit

• Yield Optimization:

  • Expression levels are often low due to protein toxicity to host cells

  • Solution: Use tightly controlled inducible expression systems

  • Optimize codon usage for E. coli expression

  • Consider cell-free protein synthesis systems for highly toxic proteins

By addressing these challenges methodically, researchers can improve yield and quality of recombinant ND4L preparations for structural and functional studies.

How can researchers differentiate between direct effects of ND4L mutations and secondary consequences on complex I assembly?

Distinguishing primary mutational effects from secondary assembly consequences requires careful experimental design:

• Time-Course Assembly Analysis:

  • Track complex I assembly using BN-PAGE at multiple time points after induction

  • Visualize intermediate subcomplexes that form during assembly process

  • Compare assembly patterns between wild-type and mutant ND4L-expressing cells

• Subcomplex Isolation and Characterization:

  • Isolate assembly intermediates using immunoprecipitation or chromatography

  • Characterize subunit composition using mass spectrometry

  • Assess whether mutations in ND4L affect early assembly stages or later steps

• Complementation Studies:

  • In systems where ND4L expression is suppressed (e.g., via RNAi)

  • Introduce wild-type or mutant versions of ND4L on expression plasmids

  • Determine which mutations rescue complex I assembly versus function

• In vitro Reconstitution:

  • Attempt reconstitution of complex I from purified components

  • Compare incorporation efficiency of wild-type versus mutant ND4L

  • Assess whether mutant ND4L can physically associate with partner subunits

This methodological approach helps researchers distinguish between mutations that primarily affect ND4L's catalytic function versus those that disrupt protein-protein interactions critical for complex I assembly, providing deeper insights into structure-function relationships.

How can cryo-electron microscopy advance our understanding of ND4L structure and function within complex I?

Cryo-electron microscopy (cryo-EM) offers powerful new approaches for ND4L research:

• High-Resolution Structural Analysis:

  • Recent advances in cryo-EM allow near-atomic resolution of membrane protein complexes

  • Apply single-particle analysis to purified complex I containing ND4L

  • Resolve structural details of the proton translocation pathway

  • Identify water molecules and ion positions within channels

• Conformational Dynamics:

  • Capture different functional states of complex I (active, deactive, inhibitor-bound)

  • Observe conformational changes in ND4L during catalytic cycle

  • Identify mobile elements potentially involved in coupling electron transfer to proton pumping

• Mutation Impact Visualization:

  • Generate complex I with mutant versions of ND4L

  • Directly visualize structural perturbations caused by mutations

  • Compare with computational predictions from molecular dynamics simulations

• Integration with Functional Data:

  • Correlate structural features with proton translocation efficiency

  • Map functionally important residues identified through mutagenesis

  • Develop structure-based models of proton movement through ND4L

This approach provides unprecedented structural insight into ND4L's role in complex I, potentially revealing mechanisms that cannot be captured through computational approaches alone.

What are the prospects for using A. franciscana ND4L as a model system for studying mitochondrial disease mutations?

A. franciscana ND4L offers several advantages as a model system for mitochondrial disease research:

• Evolutionary Conservation:

  • Key functional domains and residues in ND4L are conserved across species

  • Mutations associated with human diseases can be modeled in the A. franciscana protein

  • Functional consequences can be assessed in a simplified experimental system

• Methodological Approach:

  • Introduce disease-associated mutations in recombinant A. franciscana ND4L

  • Express and purify mutant proteins using established protocols

  • Assess structural integrity using biophysical techniques

  • Measure functional parameters and compare to wild-type protein

• Comparative Analysis:

  • Parallel studies with A. franciscana and human ND4L

  • Determine whether mutations have conserved effects across species

  • Identify species-specific differences that might inform therapeutic approaches

• Advantages over Mammalian Systems:

  • Easier genetic manipulation

  • Simplified experimental system

  • Higher protein yield from recombinant expression

  • Potential for high-throughput screening of mutations

This approach could accelerate understanding of pathogenic mechanisms in mitochondrial diseases and potentially identify compensatory mechanisms that might be exploited for therapeutic development.