Recombinant Artemia salina NADH-ubiquinone oxidoreductase chain 4 (ND4)

What is NADH-ubiquinone oxidoreductase chain 4 (ND4) in Artemia salina?

NADH-ubiquinone oxidoreductase chain 4 (ND4) is a mitochondrial membrane protein component of Complex I in the electron transport chain of Artemia salina (brine shrimp). This 71-amino acid protein (UniProt ID: P19046) plays a crucial role in cellular respiration by transferring electrons from NADH to ubiquinone . The protein has the amino acid sequence: LFVLLWLTFTTQSFILFYVFFECSLIPTIILILGWGYQPERLPASYYFLFYTLLSSLPLLFIIMLTRVFIR, which contributes to its hydrophobic nature and membrane localization . As part of the mitochondrial respiratory complex, ND4 is essential for energy production in this extremophile organism that can withstand prolonged periods of metabolic dormancy.

How does recombinant ND4 differ from native ND4 in Artemia salina?

Recombinant ND4 from Artemia salina is typically produced with modifications that facilitate its isolation and study. The most common modification is the addition of an N-terminal histidine tag (His-tag), which allows for simplified purification using metal affinity chromatography . While the primary sequence of the protein remains intact (amino acids 1-71), the addition of the His-tag can potentially affect protein folding, solubility, and interaction dynamics. When expressing recombinant ND4, researchers must consider that the hydrophobic nature of the native protein may present challenges in heterologous expression systems, potentially affecting its conformation and function compared to the native form present in Artemia salina mitochondria .

What is the significance of studying Artemia salina ND4 in broader research contexts?

Studying Artemia salina ND4 provides valuable insights into several research areas. First, Artemia salina serves as an excellent model organism due to its resilience in extreme conditions and its unique developmental biology, where encysted gastrulae remain metabolically dormant until rehydration . The study of ND4 and other mitochondrial proteins helps elucidate how energy metabolism is regulated during dormancy and subsequent activation. Second, Artemia salina has emerged as a useful model for toxicity assessments, with high correlation to mammalian cell-based assays (such as MTT) . Understanding the function of mitochondrial proteins like ND4 can provide insights into mechanistic aspects of toxicity. Finally, comparative studies between ND4 and other related proteins (such as ND6, P19048) contribute to our broader understanding of mitochondrial evolution and function across species .

What expression systems are most effective for producing recombinant Artemia salina ND4?

The most effective expression system for recombinant Artemia salina ND4 is E. coli, which provides high yields with relatively short turnaround times . When expressing this hydrophobic membrane protein, the following methodological considerations are critical:

What purification strategies overcome the challenges associated with the hydrophobic nature of ND4?

Purification of recombinant His-tagged ND4 requires specialized approaches to address its hydrophobic properties. The following methodological workflow has proven effective:

• Initial Lysis and Solubilization: Use detergent-based buffers (typically containing 1-2% Triton X-100 or n-dodecyl β-D-maltoside) to extract the protein from membranes or inclusion bodies .

• Affinity Chromatography: Utilize the N-terminal His-tag for immobilized metal affinity chromatography (IMAC) using Ni-NTA resin. Gradual reduction of imidazole concentration in washing buffers (20-50 mM) helps maintain protein solubility while removing contaminants .

• Buffer Optimization: Maintaining 0.1-0.5% detergent in all buffers prevents protein aggregation. Addition of 5-10% glycerol further enhances stability .

• Storage Considerations: The purified protein is typically stored in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 to maintain stability during freeze-thaw cycles . For long-term storage, addition of 5-50% glycerol (with 50% being optimal) and aliquoting before storage at -20°C/-80°C is recommended .

The purity achieved using these methods typically exceeds 90% as determined by SDS-PAGE analysis, which is sufficient for most research applications .

How can researchers assess the functional integrity of purified recombinant ND4?

Assessing the functional integrity of purified recombinant ND4 requires multiple complementary approaches:

• Spectroscopic Activity Assays: Measure NADH oxidation rates by monitoring absorbance decrease at 340 nm in the presence of artificial electron acceptors such as ferricyanide or ubiquinone analogs.

• Reconstitution Experiments: Incorporate purified ND4 into liposomes or nanodiscs to assess membrane integration and potential contribution to proton pumping activity.

• Binding Assays: Evaluate interaction with known partners such as other complex I subunits or ubiquinone using microscale thermophoresis or surface plasmon resonance.

• Structural Integrity Assessment: Use circular dichroism spectroscopy to confirm secondary structure elements expected for this predominantly hydrophobic membrane protein.

• Comparative Analysis: Compare activity metrics with native Complex I preparations from Artemia salina mitochondria as a benchmark for functional assessment.

It's important to note that isolated ND4 may not display full enzymatic activity as it normally functions as part of the larger Complex I assembly. Therefore, functional assessment often requires reconstitution with other subunits or comparative analysis with established activity parameters .

How can Artemia salina ND4 be used in toxicity assessment research?

Artemia salina has been established as a valuable model organism for toxicity assessment with high correlation to mammalian cell-based assays . Recombinant ND4 can be utilized in this context through several methodological approaches:

• Mechanism-Based Toxicity Screening: Since ND4 is critical for mitochondrial function, compounds that interfere with its activity can be identified by measuring changes in ND4 function after exposure to test substances. This provides insight into mitochondrial toxicity mechanisms.

• Binding Studies with Xenobiotics: Direct interaction between potential toxicants and recombinant ND4 can be assessed using biochemical binding assays, helping to identify compounds that may disrupt mitochondrial function.

• Comparative Toxicity Assessment: The Artemia salina lethality test can be performed with wild-type organisms and compared with studies using recombinant ND4 to establish structure-function relationships in toxicity mechanisms.

The advantage of using purified recombinant ND4 over whole-organism tests is the ability to determine specific molecular mechanisms of toxicity. Research has shown that the Artemia salina test results correlate well with mammalian cell-based assays (p > 0.05), making this a valuable approach for preliminary toxicity screening that is more rapid and less expensive than traditional cell culture methods .

What role does ND4 play in Artemia salina's ability to survive extreme conditions?

Artemia salina is known for its remarkable ability to survive extreme conditions, particularly through its dormant cyst stage. Research suggests ND4 and other mitochondrial proteins play key roles in this adaptation:

• Metabolic Dormancy Regulation: During encystment, Artemia embryos enter a state of metabolic dormancy in which mitochondrial activity is severely reduced. ND4 expression and activity patterns shift during this transition, contributing to the reorganization of energy metabolism.

• Rapid Reactivation Mechanism: Upon rehydration of dormant cysts, development resumes with a rapid increase in metabolic activity. Similar to observations with RNA polymerases that show activity within 1 hour of rehydration, mitochondrial proteins including ND4 appear to exist in an inactive state that can be rapidly activated rather than synthesized de novo .

• Oxidative Stress Management: During transitions between dormancy and active metabolism, Artemia experiences significant oxidative stress. ND4 and related proteins of the electron transport chain must maintain integrity during these transitions to prevent excessive reactive oxygen species generation.

Understanding the role of ND4 in these processes provides insights into fundamental mechanisms of metabolic regulation during extreme physiological transitions, with potential applications in biopreservation and stress biology research .

How does recombinant ND4 contribute to evolutionary studies of mitochondrial function?

Recombinant ND4 from Artemia salina provides a valuable tool for evolutionary studies of mitochondrial function through several research approaches:

• Comparative Sequence Analysis: The 71-amino acid sequence of Artemia salina ND4 can be compared with homologs from other species to identify conserved domains and species-specific adaptations. This approach reveals evolutionary constraints on mitochondrial proteins.

• Functional Conservation Testing: Recombinant ND4 can be tested for functional complementation in systems lacking endogenous ND4, assessing the degree to which this protein's function is conserved across evolutionary distance.

• Structure-Function Relationship Studies: Site-directed mutagenesis of recombinant ND4 can identify critical residues that have been conserved through evolution, providing insights into the protein's fundamental mechanisms.

• Adaptation Analysis: Comparison between ND4 from Artemia salina and related extremophiles can help identify adaptive changes that enable mitochondrial function under varied environmental conditions.

These approaches contribute to our understanding of mitochondrial evolution and the specific adaptations that have arisen in different lineages in response to environmental pressures .

What controls should be included when working with recombinant Artemia salina ND4?

Proper experimental controls are essential when working with recombinant Artemia salina ND4 to ensure valid and reproducible results:

Additionally, when conducting toxicity studies using the Artemia salina model, proper controls must include artificial sea water only wells for negative controls, as specified in the standardized methodology . For biochemical assays, controls should account for potential interference from buffer components, particularly detergents used to maintain membrane protein solubility .

How should researchers troubleshoot low expression yields of recombinant ND4?

Low expression yields of recombinant Artemia salina ND4 are a common challenge due to its hydrophobic nature. The following methodological troubleshooting approach is recommended:

• Codon Optimization: Analyze the codon usage in the expression host compared to Artemia salina. Synthetic genes with optimized codons often improve expression levels significantly for heterologous proteins.

• Expression Construct Modification:

  • Try different fusion tags (SUMO, MBP, GST) that may improve solubility

  • Adjust the position of the His-tag (N-terminal vs. C-terminal)

  • Include short linker sequences between the tag and ND4

• Expression Conditions Optimization:

  • Reduce induction temperature (18-20°C instead of 37°C)

  • Lower inducer concentration (e.g., 0.1 mM IPTG instead of 1 mM)

  • Extend expression time (overnight vs. 4 hours)

  • Test specialized media formulations (e.g., Terrific Broth with supplements)

• Host Strain Selection: E. coli strains specialized for membrane protein expression (C41(DE3), C43(DE3), or Lemo21(DE3)) often yield better results than standard BL21(DE3) .

• Solubilization Protocol Adjustment: If the protein is expressed but forms inclusion bodies, optimize the solubilization protocol by testing different detergents (DDM, LDAO, Triton X-100) and chaotropic agents (urea, guanidine HCl) at various concentrations .

Implementation of these strategies has been shown to increase yields from sub-milligram to several milligrams per liter of culture for challenging membrane proteins like ND4.

What are the critical factors for successful structural studies of recombinant ND4?

Structural studies of recombinant Artemia salina ND4 present significant challenges due to its hydrophobic nature and small size (71 amino acids). The following methodological considerations are crucial for success:

• Sample Preparation:

  • Achieve >95% purity through rigorous purification

  • Ensure monodispersity by size exclusion chromatography

  • Identify optimal detergent/lipid combinations that maintain native-like conformations

  • Consider nanodiscs or amphipols as alternatives to traditional detergents

• Crystallization Approaches:

  • Implement sparse matrix screening with membrane protein-specific conditions

  • Consider lipidic cubic phase (LCP) crystallization methods

  • Explore fusion partners (e.g., T4 lysozyme) to provide crystal contacts

  • Test in situ proteolytic removal of tags during crystallization

• Cryo-EM Considerations:

  • Despite the small size (below typical resolution limits for single-particle cryo-EM), ND4 can be studied in the context of reconstituted Complex I assemblies

  • Prepare samples with optimal contrast by adjusting grid types and vitrification conditions

• NMR Spectroscopy:

  • Consider solution NMR with detergent micelles for this relatively small protein

  • Implement selective isotope labeling (15N, 13C) for improved signal resolution

  • Use TROSY-based pulse sequences optimized for membrane proteins

• Computational Integration:

  • Implement molecular dynamics simulations to complement experimental structural data

  • Use homology modeling based on related structures from other species

Success in structural studies ultimately depends on optimizing sample quality, selecting appropriate structural biology techniques for this challenging protein, and integrating computational approaches to develop a comprehensive structural model .

How does the function of ND4 change during the development cycle of Artemia salina?

The function of ND4 undergoes significant changes throughout the Artemia salina life cycle, particularly during transitions between dormancy and active development:

• Dormant Cyst Stage: In encysted gastrulae, mitochondrial activity is minimal, with regulatory mechanisms keeping ND4 and other respiratory components in an inactive state. Similar to observations with RNA polymerases, which show minimal activity in dormant cysts, mitochondrial proteins appear to exist in an inactive form rather than being absent .

• Reactivation Upon Hydration: Within hours of rehydration, a dramatic increase in mitochondrial activity occurs. Research with RNA polymerases shows activity within 1 hour of rehydration, suggesting that preexisting but inactive proteins are rapidly activated rather than synthesized de novo . A similar mechanism likely applies to ND4 and other components of the electron transport chain.

• Nauplius Development: As development progresses to the nauplius larval stage, mitochondrial activity reaches its peak to support the energy demands of the free-swimming larvae. ND4 function is essential during this high-energy demand phase.

• Adult Stage: In adult Artemia, ND4 continues to function in standard mitochondrial energy production but with adaptations to the organism's unique physiological requirements.

This developmental regulation of ND4 and other mitochondrial proteins provides insights into mechanisms of metabolic control during extreme physiological transitions, with potential applications in understanding metabolic reprogramming in other contexts .

What protein-protein interactions are critical for ND4 function in the context of Complex I?

ND4 functions as part of the membrane domain of Complex I, where it engages in several critical protein-protein interactions:

Understanding these interactions is crucial for elucidating the mechanism of proton pumping and energy transduction in Complex I, with implications for understanding mitochondrial diseases and developing potential therapeutic approaches .

How can recombinant ND4 be used to study mitochondrial dysfunction mechanisms?

Recombinant Artemia salina ND4 provides a valuable tool for investigating fundamental mechanisms of mitochondrial dysfunction through several research approaches:

• Mutation Analysis: Site-directed mutagenesis of recombinant ND4 can recreate mutations associated with mitochondrial diseases in humans. By expressing these mutant forms and assessing their function, researchers can gain insights into pathological mechanisms.

• Inhibitor Binding Studies: Recombinant ND4 can be used to investigate the binding of known Complex I inhibitors, helping to elucidate inhibition mechanisms and potentially identify novel binding sites for therapeutic development.

• Oxidative Damage Models: By subjecting recombinant ND4 to controlled oxidative stress conditions and assessing resulting structural and functional changes, researchers can better understand how oxidative damage contributes to mitochondrial dysfunction.

• Protein Quality Control Interactions: Studies of how recombinant ND4 interacts with components of protein quality control systems (chaperones, proteases) can reveal mechanisms for dealing with misfolded or damaged mitochondrial proteins.

• Comparative Analysis: The relatively simple system of Artemia salina ND4 can be compared with more complex mammalian systems to identify conserved mechanisms of dysfunction that represent fundamental vulnerabilities in mitochondrial function.

These approaches contribute to our understanding of fundamental mechanisms underlying mitochondrial diseases, aging processes, and metabolic disorders, potentially leading to therapeutic strategies targeting mitochondrial dysfunction .