Recombinant Asterina pectinifera NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is the biological function of ND4L in mitochondrial respiration?

ND4L protein functions as a critical subunit of complex I (NADH-ubiquinone oxidoreductase) in the mitochondrial respiratory complex. It plays an essential role in the proton translocation process, contributing to the establishment of the proton gradient necessary for ATP synthesis. Current consensus indicates that respiratory complex I operates with a 4H+/2e- stoichiometry, with ND4L potentially contributing to the fourth proton translocation pathway . This function is evolutionarily conserved across diverse organisms, from bacteria to eukaryotes, consistent with the endosymbiosis theory of mitochondrial origin .

Why is Asterina pectinifera used as a model organism for studying mitochondrial proteins?

Asterina pectinifera serves as an important model organism for mitochondrial studies due to its completely sequenced mitochondrial genome and the high conservation of mitochondrial gene order compared to other echinoderms. Its mtDNA contains standard metazoan components including 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes arranged in a specific order that facilitates comparative genomic analyses . The sequence information available for A. pectinifera enables the design of oligonucleotide primers for amplifying and studying homologous genes in related species, making it a valuable reference point for evolutionary and functional studies of mitochondrial proteins including ND4L .

What is the genomic organization of the ND4L gene in Asterina pectinifera mitochondrial DNA?

In Asterina pectinifera, the ND4L gene is encoded on the L strand of the mitochondrial genome along with several other mitochondrial genes. The complete organization includes 10 protein-coding genes (including ND4L), 11 tRNA genes, and the 12S rRNA gene on the L strand, while the H strand carries the remaining genes including ND1, ND2, and ND6 . This genomic arrangement is consistent with that found in other asteroid species and is important for understanding the transcriptional regulation and evolutionary relationships of mtDNA genes. The specific nucleotide sequence of the ND4L gene can be accessed through databases with the appropriate accession numbers for Asterina pectinifera (similar to NC-001627 for the complete mtDNA) .

How do mutations in the ND4L gene affect proton translocation pathways?

Molecular dynamics (MD) simulations of ND4L mutations have revealed significant disruptions to the proton translocation pathway. For example, the T10609C mutation (causing M47T amino acid change) and C10676G mutation (causing C69W change) interfere with proton translocation through specific molecular mechanisms . The native ND4L structure facilitates proton movement via a pathway involving conserved amino acid residues such as Glu34 and Tyr157. When mutations occur, hydrogen bond formation between these key residues can restrict the passage of water molecules through the transmembrane region .

What computational approaches are most effective for modeling ND4L structure and function?

Effective computational modeling of ND4L structure and function requires a multi-step approach:

• Homology Modeling: Using templates with high sequence identity (≥98%) from organisms like Thermus thermophilus (PDB ID: 5XTC) is recommended for generating reliable structural models . Multiple models should be generated (typically 50+) using software like MODELLER 9.21, with selection based on the lowest DOPE (Discrete Optimized Protein Energy) score.

• Model Evaluation: Rigorous validation through Ramachandran plot analysis (>90% residues in favorable regions), QMEANBrane assessment, and DOPE profile comparison between model and template .

• Transmembrane System Building: Creating realistic membrane systems using tools like CHARMM-GUI's Membrane Builder to incorporate lipid bilayers, explicit TIP3P water molecules, and physiological ion concentrations (150 mM K+ and Cl-) .

• Molecular Dynamics Simulation: Running simulations for at least 100 ns with timesteps of 2 fs, employing particle mesh Ewald technique for long-range electrostatics, and using appropriate force fields for protein, lipids, water, and ions .

• Analysis: Trajectory analysis through RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuation) calculations using programs like cpptraj in Amber18, with visualization through VMD (Visual Molecular Dynamics) .

This computational workflow has proven effective for studying the structural implications of mutations and predicting functional changes in proton translocation pathways.

How can researchers effectively compare ND4L sequences across different species for evolutionary studies?

For comprehensive evolutionary studies of ND4L across species, researchers should implement the following methodological approach:

Table 1: Recommended Workflow for Cross-Species ND4L Comparison

This approach reveals that while ND4L sequences may vary considerably between species (e.g., between Asterina and Acanthaster), the functional constraints often manifest as conserved amino acid sequences rather than nucleotide sequences. In some cases, as seen with the ATP8 gene, nucleotide conservation may exceed amino acid conservation, suggesting relaxed functional constraints at the protein level .

What are the optimal protocols for cloning and expressing recombinant Asterina pectinifera ND4L?

The optimal protocol for cloning and expressing recombinant Asterina pectinifera ND4L requires careful consideration of its transmembrane nature and mitochondrial origin:

• Gene Amplification:

  • Design primers based on the Asterina pectinifera mtDNA sequence (similar to accession number NC-001627)

  • Amplify using a high-fidelity DNA polymerase with GC buffer optimization

  • Recommended cycling conditions: initial denaturation at 94°C for 1 min; 40 cycles of 94°C for 30 sec, 50-55°C for 30 sec, 72°C for 7 min; final extension at 72°C for 10 min

• Vector Selection and Cloning:

  • Use specialized vectors for membrane proteins (pET series with fusion tags)

  • Alternative approach: employ the pGEM-T easy vector system or TopoXL vector for initial cloning

  • For difficult amplicons, LaTaq with GC buffer I has proven effective

• Expression System Selection:

  • E. coli strains (C41/C43) engineered for membrane protein expression

  • Consider codon optimization for eukaryotic host organisms

  • Use reduced temperature (16-20°C) to minimize inclusion body formation

• Purification Strategy:

  • Gentle detergent extraction (DDM or LMNG)

  • Affinity chromatography followed by size exclusion

  • Validate functional integrity through proton translocation assays

This methodology addresses the significant challenges in expressing functional mitochondrial membrane proteins in recombinant systems while maintaining their native conformational properties.

How can researchers accurately measure the impact of ND4L mutations on mitochondrial function?

Accurately measuring the impact of ND4L mutations on mitochondrial function requires a multi-parameter assessment approach:

• Proton Translocation Efficiency:

  • Direct measurement of proton flux using pH-sensitive fluorescent probes

  • Computational simulation of water molecule passage through transmembrane regions as a proxy for proton movement

  • Analysis of hydrogen bond networks between key residues like Glu34 and Tyr157

• ROS Production Assessment:

  • Quantification of H2O2 production using DCFDA fluorescence under varied oxygen concentrations

  • Comparative analysis between wild-type and mutant cells, with particular attention to hypoxic conditions (3% oxygen) where significant differences (1.5-fold) have been observed between wild-type and mutant forms

• ATP Synthesis Capacity:

  • Measurement of ATP production rates in isolated mitochondria

  • Assessment of the relationship between ATP synthesis and insulin secretion in pancreatic beta cells

• Complex I Assembly and Stability:

  • Blue Native PAGE for complex I integrity assessment

  • Pulse-chase experiments to determine half-life of assembled complex

• Computational Validation:

  • Molecular dynamics simulations for at least 100 ns to observe structural perturbations

  • Analysis of RMSD and RMSF values to quantify structural stability differences

This comprehensive approach enables researchers to establish clear genotype-phenotype relationships between ND4L mutations and mitochondrial dysfunction, potentially serving as computational assays for biomarker validation in conditions like T2DM.

What primer design strategies are most effective for amplifying the ND4L gene from Asterina pectinifera?

Effective primer design for amplifying the ND4L gene from Asterina pectinifera requires strategic approaches that account for the unique properties of mitochondrial DNA:

• Reference Sequence Utilization:

  • Base primer design on the published Asterina pectinifera mtDNA sequence (similar to NC-001627)

  • Consider using conserved regions flanking the ND4L gene to design primers that work across related species

• Primer Design Parameters:

  • Optimal length: 18-25 nucleotides

  • GC content: 45-55%

  • Melting temperature (Tm): 55-65°C with minimal Tm difference between pairs (<5°C)

  • Avoid self-complementarity and secondary structure formation

  • Check for specificity against the complete mitochondrial genome

• PCR Optimization:

  • Use LaTaq polymerase with GC buffer I for challenging templates

  • Optimize cycling conditions: initial denaturation (94°C for 1 min), followed by 40 cycles of denaturation (94°C for 30 sec), annealing (50-55°C for 30 sec), and extension (72°C for 7 min), with final extension at 72°C for 10 min

  • For long PCR products, extend elongation time appropriately

• Verification and Purification:

  • Electrophorese products in 1% agarose gels and stain with ethidium bromide

  • Excise bands under UV and purify using spin columns (e.g., GenElute Agarose Spin Columns)

  • Perform ethanol precipitation to concentrate DNA

• Sequencing Validation:

  • Sequence products directly or clone into appropriate vectors

  • Use modern sequencing technologies like ABI PRISM 3100 Genetic Analyzer with DYEnamic ET Terminator Cycle Sequencing Kit

This methodical approach has successfully yielded complete mtDNA sequences for related species and can be adapted specifically for the ND4L gene in Asterina pectinifera.

How should researchers design experiments to investigate the functional conservation of ND4L across different species?

To investigate functional conservation of ND4L across species, researchers should implement a comprehensive experimental design that integrates multiple levels of analysis:

• Sequence-Based Conservation Analysis:

  • Compare amino acid conservation rates across diverse taxonomic groups

  • Focus on key functional residues involved in proton translocation (e.g., Glu34, Tyr157)

  • Generate phylogenetic trees based on ND4L sequences to visualize evolutionary relationships

• Structural Conservation Assessment:

  • Develop homology models for ND4L from multiple species using appropriate templates

  • Compare hydrophobicity profiles, which often remain conserved despite sequence divergence

  • Identify structurally conserved motifs that may indicate functional importance

• Functional Complementation Studies:

  • Create chimeric proteins by swapping domains between species

  • Express these constructs in model systems lacking endogenous ND4L

  • Measure restoration of function (proton translocation, complex I activity)

• Site-Directed Mutagenesis:

  • Target evolutionarily conserved amino acids with single point mutations

  • Assess impact on proton translocation using methods described in FAQ 3.2

  • Compare effects across species to identify functionally critical residues

• Cross-Species Complex I Assembly Analysis:

  • Investigate whether ND4L from one species can incorporate into complex I of another

  • Use Blue Native PAGE and immunoprecipitation to assess assembly competence

  • Correlate with functional readouts (ATP synthesis, ROS production)

This multi-faceted approach provides robust evidence for functional conservation beyond simple sequence similarity and reveals evolutionary constraints on ND4L structure and function.

What controls should be included when studying recombinant ND4L protein function in experimental systems?

A robust experimental design for studying recombinant ND4L function requires comprehensive controls at multiple levels:

Table 2: Essential Controls for Recombinant ND4L Functional Studies

Additionally, researchers should include temporal controls (measurements at multiple time points), dosage controls (titration of protein expression levels), and system-specific controls depending on the chosen expression system. These controls collectively ensure that observed effects can be confidently attributed to the specific functional properties of recombinant ND4L.

How can researchers isolate and verify the purity of recombinant ND4L protein for biochemical studies?

Isolating and verifying pure recombinant ND4L protein requires specialized techniques due to its hydrophobic nature and membrane localization:

• Optimized Extraction Protocol:

  • Harvest recombinant expression systems during peak expression

  • Lyse cells using methods optimized for membrane proteins (sonication in buffer containing 20mM Tris-HCl pH 7.5, 150mM NaCl)

  • Solubilize membrane fraction with gentle detergents (0.5-1% n-Dodecyl β-D-maltoside or 0.1% Lauryl Maltose Neopentyl Glycol)

  • Maintain samples at 4°C throughout extraction

• Multi-Step Purification Strategy:

  • Primary purification: Affinity chromatography using tags (His-tag, FLAG-tag)

  • Secondary purification: Size exclusion chromatography

  • Tertiary purification: Ion exchange chromatography if necessary

  • Consider using specialized resins designed for membrane proteins

• Purity Verification Methods:

  • SDS-PAGE (12-15%) with Coomassie staining

  • Western blotting with antibodies against the protein or tag

  • Mass spectrometry for definitive identification and purity assessment

  • Dynamic light scattering to assess homogeneity

• Functional Verification:

  • Reconstitution into liposomes for functional assays

  • Proton translocation assays using pH-sensitive dyes

  • Structural integrity assessment via circular dichroism

  • Binding studies with known interaction partners

• Contaminant Assessment:

  • Proteomic analysis to identify co-purifying proteins

  • Endotoxin testing if preparing for cellular assays

  • Lipid analysis to quantify co-purifying membrane components

Successful purification typically yields microgram to low milligram quantities of protein per liter of expression culture, with purity exceeding 90% as determined by densitometry of stained gels. This purified protein serves as the foundation for subsequent biochemical, structural, and functional studies.

What are the most common challenges in expressing functional recombinant ND4L, and how can they be overcome?

Researchers face several significant challenges when expressing functional recombinant ND4L:

• Protein Misfolding and Aggregation:

  • Challenge: As a highly hydrophobic membrane protein, ND4L tends to aggregate when overexpressed

  • Solution: Lower expression temperature (16-20°C), use specialized expression strains (C41/C43), employ fusion partners (MBP, SUMO) to enhance solubility

• Codon Usage Bias:

  • Challenge: Mitochondrial genes use a different genetic code from standard nuclear genes

  • Solution: Optimize codons for the expression host, use specialized strains with rare tRNAs, or synthesize codon-optimized genes

• Toxicity to Host Cells:

  • Challenge: Expression of membrane proteins can disrupt host cell membrane integrity

  • Solution: Use tightly regulated inducible expression systems, titrate inducer concentration, consider cell-free expression systems

• Improper Membrane Insertion:

  • Challenge: Recombinant ND4L may not insert correctly into membranes

  • Solution: Co-express with chaperones, use specialized membrane protein expression vectors, consider native-like lipid environments

• Difficult Purification:

  • Challenge: Detergent choice can affect protein stability and function

  • Solution: Screen multiple detergents (DDM, LMNG, GDN), use lipid nanodiscs or amphipols for stabilization, purify at 4°C with protease inhibitors

• Low Expression Yields:

  • Challenge: Membrane proteins typically express at lower levels than soluble proteins

  • Solution: Scale up culture volumes, optimize media composition (e.g., supplementation with trace elements), consider expression as fusion with high-yield proteins

• Functional Assessment Limitations:

  • Challenge: Difficult to verify if recombinant protein retains native function

  • Solution: Develop miniaturized functional assays, use computational validation methods (MD simulations), and complement with in vivo functional studies

By systematically addressing these challenges, researchers can significantly improve their chances of obtaining functional recombinant ND4L protein for subsequent studies.