Recombinant Artemia salina NADH-ubiquinone oxidoreductase chain 3 (ND3)

What is NADH-ubiquinone oxidoreductase chain 3 and what is its function in Artemia salina?

NADH-ubiquinone oxidoreductase chain 3 (ND3) is a protein subunit of Complex I in the mitochondrial electron transport chain. In Artemia salina (brine shrimp), ND3 plays a crucial role in cellular respiration by participating in the transfer of electrons from NADH to ubiquinone, contributing to the proton gradient necessary for ATP synthesis. The protein consists of 70 amino acids with a molecular weight of approximately 8 kDa and contains hydrophobic domains that anchor it within the inner mitochondrial membrane .

The function of ND3 must be understood in the context of Artemia's remarkable adaptability to extreme environments, particularly high-salinity conditions. Unlike many other organisms, Artemia can survive in saturated brine environments where salt concentrations exceed 4M . This adaptation necessitates specialized energy metabolism mechanisms, as maintaining ion homeostasis under such conditions requires significant ATP expenditure, which directly involves the electron transport chain where ND3 functions.

To study ND3 function in experimental settings, researchers typically employ techniques such as site-directed mutagenesis, enzyme activity assays, and comparative analyses with homologous proteins from other species. When examining ND3 activity, it is essential to maintain proper experimental conditions mimicking the physiological environment of Artemia's mitochondria.

What are the optimal storage and handling conditions for recombinant ND3 protein?

Recombinant Artemia salina ND3 protein requires specific storage and handling conditions to maintain its structural integrity and functional activity. The optimal storage parameters include:

• Temperature maintenance at -20°C for routine storage, with extended storage preferably at -80°C to minimize degradation .

• Storage buffer composition of Tris-based buffer with 50% glycerol, which has been optimized specifically for this protein .

• Avoidance of repeated freeze-thaw cycles, which can significantly compromise protein structure and function .

• For working experiments, storage of aliquots at 4°C for up to one week to maintain activity while minimizing degradation .

When handling recombinant ND3, researchers should consider the hydrophobic nature of this membrane protein. The full-length protein (amino acids 1-70) contains multiple transmembrane domains, making it challenging to maintain in solution without appropriate detergents or membrane mimetics. The sequence "FYYINPREFVNKKVCLDREKSSPFECGFDPLEFLSYPLFIRFFVITLIFLIFDVEIYLLLPMVYLNMSSP" reveals the presence of multiple hydrophobic residues, which can contribute to aggregation in aqueous solutions .

For functional studies, researchers should maintain physiologically relevant conditions, considering that this protein operates in the unique ionic environment of Artemia mitochondria adapted to extreme salinity. Any experimental design should account for these specialized conditions to obtain relevant functional data.

How does recombinant ND3 differ from native ND3 in experimental applications?

Recombinant Artemia salina ND3 protein offers several advantages and limitations compared to the native form when used in experimental applications:

The recombinant ND3 is typically produced with expression tags that facilitate purification but may influence protein behavior. When using recombinant ND3, researchers should verify whether the tag has been removed and determine the tag type, as this information can be critical for experimental design . For structural studies or protein-protein interaction analyses, the presence of tags could introduce artifacts.

Another important consideration is the expression system used for recombinant production. Bacterial expression systems may not reproduce the same post-translational modifications present in Artemia, potentially affecting protein function. For studies focused on activity measurements, it may be necessary to compare results with native protein or consider using eukaryotic expression systems that better mimic the original cellular environment.

How can Artemia salina ND3 be utilized in toxicity assessment studies?

Artemia salina ND3 can serve as a valuable marker in toxicity assessment studies, particularly when examining mitochondrial function impairment. The methodological approach for utilizing ND3 in toxicity assessments involves:

• Baseline expression analysis: Establishing normal expression levels of ND3 in control Artemia populations using qPCR or western blotting.

• Exposure experiments: Subjecting Artemia to test compounds at various concentrations (typically 1.56-400 μg/mL as demonstrated in previous studies) .

• Post-exposure analysis: Measuring changes in ND3 expression or activity as indicators of mitochondrial toxicity.

• Correlation analysis: Relating ND3 alterations to mortality rates or other physiological parameters.

The Artemia lethality test offers significant advantages as a toxicity assessment model compared to cell culture-based methods. Studies have demonstrated that results from Artemia tests correlate well with MTT assays on cell lines, yet the Artemia model is more rapid, cost-effective, and ethically preferable . For instance, when evaluating nanoparticle toxicity, both the Artemia test and MTT assay produced statistically similar results (P > 0.05), supporting the validity of the Artemia model .

A standardized experimental protocol involves:

• Hatching Artemia cysts in artificial seawater (35 g/L sodium chloride) under conditions of strong aeration and continuous illumination for 36-48 hours .

• Exposing nauplii (10 per experimental unit) to test compounds at various concentrations in 96-well plates .

• Incubating for 24 hours at room temperature (28-30°C) .

• Assessing survival under stereoscopic microscope and calculating lethality using Abbott's formula: % Lethality = [(Test death - Control death) / (Control survival)] × 100 .

For specific ND3-focused toxicity studies, additional steps would include extracting mitochondria from exposed nauplii and measuring ND3 activity or expression levels as biomarkers of mitochondrial dysfunction.

What are the most effective methods for purifying recombinant ND3 protein for structural studies?

Purifying recombinant Artemia salina ND3 protein for structural studies presents significant challenges due to its hydrophobic nature and membrane localization. The most effective purification strategy involves a multi-step approach:

• Expression system selection: Membrane proteins like ND3 are best expressed in eukaryotic systems such as insect cells or yeast that provide appropriate membrane structures and post-translational modification capabilities.

• Solubilization optimization: Critical parameters include:

  • Detergent selection (typically mild non-ionic detergents like DDM or LMNG)

  • Buffer composition (Tris-based buffers with appropriate pH and ionic strength)

  • Addition of glycerol (50%) to enhance stability

  • Temperature control during solubilization (4°C to minimize degradation)

• Purification procedure:

  • Initial capture using affinity chromatography (dependent on tag type determined during production)

  • Intermediate purification via ion exchange chromatography

  • Polishing step using size exclusion chromatography

  • Quality assessment through SDS-PAGE and western blotting

• Optimization for structural studies:

  • Detergent exchange or reconstitution into nanodiscs or liposomes

  • Concentration optimization (typically 5-10 mg/mL for crystallization attempts)

  • Addition of stabilizing agents specific to membrane proteins

When working with recombinant ND3, researchers should be aware that the protein's high hydrophobicity may lead to aggregation during concentration steps. To mitigate this, maintaining the protein in a suitable membrane-mimetic environment throughout the purification process is essential. For structural studies specifically, cryo-electron microscopy (cryo-EM) has become the method of choice for membrane proteins like ND3, as it allows visualization in a more native-like environment compared to crystallography.

The purity and homogeneity of the final preparation should be rigorously assessed before structural studies, as heterogeneity can significantly impair structural determination efforts. Techniques such as analytical ultracentrifugation or multi-angle light scattering can provide valuable information about the sample's monodispersity.

How can recombinant ND3 be used to study the adaptation of Artemia to extreme environments?

Recombinant Artemia salina ND3 serves as an excellent model for investigating adaptations to extreme environments, particularly high-salinity conditions. A comprehensive methodological approach includes:

• Comparative sequence analysis: Analyzing ND3 sequences from Artemia populations adapted to different salinity levels to identify potential adaptive mutations. This can reveal structural modifications that might contribute to enhanced mitochondrial efficiency under osmotic stress.

• Functional characterization: Measuring electron transport activity of recombinant ND3 variants under varying salt concentrations (250 mM to 4 M) to mimic natural conditions . Key parameters to assess include:

  • Electron transfer rates

  • Proton pumping efficiency

  • ROS (reactive oxygen species) generation

  • Protein stability and half-life

• Expression pattern analysis: Quantifying ND3 expression levels in Artemia raised at different salinities using qPCR techniques similar to those employed for sodium pump studies . Previous research has demonstrated that other proteins show salinity-dependent expression patterns in Artemia; for example, a specific sodium pump isoform (α2 KK) is dramatically upregulated at high salinity .

• Structure-function relationship studies: Using site-directed mutagenesis to create ND3 variants that mimic adaptive changes, followed by functional assessment. This approach can help determine whether specific amino acid substitutions in ND3 contribute to Artemia's remarkable salinity tolerance.

A particularly informative experimental design would involve comparing ND3 properties across different Artemia species or populations with varying degrees of halotolerance. For instance, researchers could examine whether ND3 from Artemia populations adapted to hypersaline environments (>4 M NaCl) exhibits structural or functional differences compared to populations from less saline habitats.

Evidence from studies on other Artemia proteins suggests that adaptive changes often involve modifications to ion-binding sites or alterations in protein stoichiometry . Similar mechanisms might apply to ND3's role in maintaining mitochondrial function under extreme salinity conditions, potentially through modifications that enhance energy efficiency or reduce proton leakage.

How does ND3 interact with other components of the respiratory chain complex in Artemia salina?

The interaction of Artemia salina ND3 with other respiratory chain components represents a complex network of protein-protein contacts that enable efficient electron transfer and proton pumping. Understanding these interactions requires sophisticated experimental approaches:

• Crosslinking studies: Chemical crosslinking combined with mass spectrometry can identify direct interaction partners of ND3 within the respiratory complex. This approach can reveal both expected interactions with other Complex I subunits and potentially novel interactions specific to Artemia.

• Co-immunoprecipitation experiments: Using antibodies against ND3 to pull down interaction partners, followed by proteomic analysis to identify the complete interactome. This technique can detect both stable and transient interactions.

• Blue Native PAGE analysis: This technique preserves native protein complexes and can reveal the integration of ND3 into higher-order structures within the respiratory chain. It can also detect potential subcomplexes that might form during assembly or under stress conditions.

• Proximity labeling approaches: Methods such as BioID or APEX2 can identify proteins in close proximity to ND3 in living cells, providing spatial information about the protein's environment within the mitochondrial membrane.

Based on structural studies of Complex I from other organisms, ND3 likely occupies a critical position at the junction between the membrane arm and peripheral arm of the complex. This strategic location suggests that ND3 may play a role in coupling electron transfer to proton pumping, a fundamental aspect of energy conversion in mitochondria.

The highly hydrophobic nature of ND3, evidenced by its amino acid sequence "FYYINPREFVNKKVCLDREKSSPFECGFDPLEFLSYPLFIRFFVITLIFLIFDVEIYLLLPMVYLNMSSP" , indicates its deep embedding within the lipid bilayer. This positioning facilitates interactions with other membrane-integrated subunits and potentially with lipid components that might be specific to Artemia's adaptation to extreme environments.

Future research should focus on determining whether ND3 from Artemia exhibits unique interaction patterns compared to homologous proteins from non-extremophile organisms, as such differences could contribute to the exceptional environmental tolerance of this species.

What role does ND3 play in the bioenergetic adaptation of Artemia salina to varying salinity levels?

The bioenergetic adaptation of Artemia salina to extreme salinity environments likely involves specialized roles of mitochondrial proteins, including ND3. Comprehensive investigation of this role requires a multi-faceted experimental approach:

• Expression profiling across salinity gradients: While specific data for ND3 is not directly available in the provided search results, the methodology used for sodium pump studies provides a valuable template. In those studies, Artemia were raised at three distinct salinity levels (250 mM, 2 M, and 4 M salt), followed by RNA sequencing and qPCR analysis to quantify expression changes . Similar approaches could determine whether ND3 expression is salinity-dependent.

• Respiratory chain activity measurements: Mitochondria isolated from Artemia adapted to different salinities can be assessed for Complex I activity, with specific focus on the contribution of ND3. Key parameters to measure include:

  • NADH:ubiquinone oxidoreductase activity

  • Proton pumping efficiency

  • ROS production rates

  • ATP synthesis coupling efficiency

• Comparative structural analysis: Similar to the approach used for sodium pumps in Artemia, structural studies of ND3 across salinity conditions could reveal adaptations. For sodium pumps, salinity adaptation was associated with specific amino acid substitutions (Asn-to-Lys) that altered ion stoichiometry . Analogous modifications might exist in ND3.

The importance of energy metabolism in high-salinity adaptation is highlighted by the finding that Artemia's sodium pumps undergo significant adaptations when exposed to extreme salt concentrations. Specifically, a sodium pump variant with altered stoichiometry (2Na⁺/1K⁺ instead of the canonical 3Na⁺/2K⁺) is dramatically upregulated in high-salinity conditions . This adaptation allows Artemia to build and maintain larger electrochemical gradients with less ATP expenditure.

Given that mitochondrial electron transport chain (including ND3) is the primary source of cellular ATP, it is reasonable to hypothesize that similar adaptations might occur in Complex I components to support the increased energy demands of ion homeostasis in hypersaline environments. Investigation of such adaptations could reveal novel mechanisms of bioenergetic optimization in extremophile organisms.

How can molecular dynamics simulations enhance our understanding of ND3 structure-function relationships?

Molecular dynamics (MD) simulations offer powerful insights into the structure-function relationships of complex membrane proteins like Artemia salina ND3. A comprehensive MD simulation approach would include:

• Model construction and validation:

  • Homology modeling based on known structures of ND3 from other species

  • Incorporation of the specific amino acid sequence of Artemia ND3 (FYYINPREFVNKKVCLDREKSSPFECGFDPLEFLSYPLFIRFFVITLIFLIFDVEIYLLLPMVYLNMSSP)

  • Validation through energy minimization and comparison with experimental data

• Simulation of protein behavior in membrane environments:

  • Embedding the ND3 model in lipid bilayers with compositions mimicking Artemia mitochondrial membranes

  • Simulating behavior under varying ionic strengths to replicate different salinity conditions (from 250 mM to 4 M NaCl)

  • Analyzing protein stability, conformational changes, and lipid interactions

• Investigation of functional mechanisms:

  • Simulating electron transfer pathways through ND3 and adjacent subunits

  • Identifying potential proton translocation channels

  • Calculating energy barriers for conformational changes associated with catalytic activity

• In silico mutagenesis studies:

  • Simulating the effects of specific amino acid substitutions on protein function

  • Comparing wild-type and mutant behaviors under various environmental conditions

  • Predicting adaptive mutations that might enhance function in extreme environments

This approach is supported by evidence from studies on other Artemia proteins. For example, MD simulations were successfully used to study how lysine substitutions in sodium pumps alter ion binding and stoichiometry . When applying similar techniques to ND3, researchers should focus on regions likely involved in proton pumping or electron transfer, as these functions are critical for bioenergetic efficiency.

MD simulations can also help explain experimental observations by providing atomic-level details of mechanisms. For instance, if experimental data shows altered ND3 activity under high salt conditions, simulations could reveal whether this is due to direct effects on the protein structure, changes in substrate binding, alterations in proton pathways, or other mechanisms.

How does Artemia salina ND3 compare to homologous proteins in other extremophile organisms?

Comparative analysis of Artemia salina ND3 with homologous proteins from other extremophile organisms reveals important evolutionary adaptations to harsh environments. This comparison requires:

• Sequence alignment and phylogenetic analysis: Multiple sequence alignment of ND3 proteins from various extremophiles (halophiles, thermophiles, acidophiles) to identify conserved regions and environment-specific variations. Phylogenetic tree construction can reveal evolutionary relationships and convergent adaptations.

• Structural comparison: Homology modeling of ND3 from different extremophiles based on available structural data, followed by superimposition analysis to identify structural differences that might contribute to environmental adaptation.

• Functional domain analysis: Identification of key functional domains and how they differ across species, with particular focus on regions involved in electron transfer, proton pumping, and protein-protein interactions.

Although specific comparative data for ND3 across extremophiles is not directly available in the search results, we can infer potential patterns based on studies of other proteins. For example, the sodium pump adaptations in Artemia involve specific lysine substitutions that alter ion stoichiometry and enable survival in high-salinity environments . Similar adaptive modifications might exist in ND3 and other respiratory chain components.

Key features likely to differ between Artemia ND3 and homologs from non-extremophile organisms include:

Evolutionary analysis suggests that extremophile adaptations often involve a balance between structural stability and functional flexibility. The specific amino acid sequence of Artemia ND3 (FYYINPREFVNKKVCLDREKSSPFECGFDPLEFLSYPLFIRFFVITLIFLIFDVEIYLLLPMVYLNMSSP) contains regions that may have evolved to maintain function under the osmotic and ionic stresses of hypersaline environments, while preserving the core catalytic capabilities of the protein.

What insights can ND3 provide about mitochondrial evolution in crustaceans?

Artemia salina ND3 serves as a valuable molecular marker for investigating mitochondrial evolution in crustaceans, offering insights into both general evolutionary patterns and adaptive diversification. A comprehensive evolutionary analysis would include:

• Comparative genomics approach:

  • Sequence comparison of ND3 across diverse crustacean lineages

  • Calculation of evolutionary rates and selective pressures (dN/dS ratios)

  • Identification of lineage-specific adaptations and conserved functional domains

• Molecular clock analysis:

  • Dating of key evolutionary events in crustacean mitochondrial evolution

  • Correlation of ND3 evolutionary changes with major environmental transitions

  • Assessment of evolutionary rate heterogeneity across different crustacean groups

• Structure-based evolutionary analysis:

  • Mapping of evolutionary changes onto structural models

  • Identification of co-evolving residues that maintain structural and functional integrity

  • Analysis of how structural constraints influence evolutionary trajectories

The mitochondrial genome, including ND3, is particularly valuable for evolutionary studies due to its maternal inheritance, relatively rapid evolutionary rate, and essential function in energy metabolism. In crustaceans, adaptation to diverse environments from marine to hypersaline has likely driven specific adaptations in mitochondrial genes.

Artemia represents an extreme case of environmental adaptation among crustaceans, being the only animal that can survive in saturated brine environments . This exceptional tolerance makes its mitochondrial genes, including ND3, particularly interesting for studying the molecular basis of adaptation to extreme conditions.

The evolutionary history of ND3 in Artemia likely reflects both the ancient origin of the basic mitochondrial electron transport system and more recent adaptations to hypersaline environments. By comparing the sequence and structure of Artemia ND3 with homologs from crustaceans inhabiting different environments (marine, freshwater, terrestrial), researchers can identify candidate adaptive mutations and test their functional significance through experimental approaches.

Such evolutionary studies not only enhance our understanding of crustacean phylogeny but also provide insights into the fundamental mechanisms by which essential cellular machinery can be modified to function in extreme environments without compromising vital bioenergetic functions.

What are the common challenges in expressing and purifying recombinant ND3, and how can they be addressed?

Expressing and purifying recombinant Artemia salina ND3 presents several challenges due to its nature as a small, hydrophobic membrane protein. Common difficulties and their solutions include:

• Low expression levels:

  • Challenge: Membrane proteins often express poorly in standard systems.

  • Solution: Optimize expression using specialized systems such as C41/C43 E. coli strains, Pichia pastoris, or insect cell systems that are better suited for membrane proteins. Fusion partners like MBP (maltose-binding protein) can enhance solubility and expression.

• Protein aggregation:

  • Challenge: The hydrophobic nature of ND3 promotes aggregation during expression and purification.

  • Solution: Expression at lower temperatures (16-20°C), addition of chemical chaperones to the culture medium, and careful optimization of induction conditions. During purification, maintain the protein in appropriate detergent mixtures and include 50% glycerol in storage buffers as recommended for this specific protein .

• Protein instability:

  • Challenge: Small membrane proteins can be unstable once removed from their native environment.

  • Solution: Optimize buffer conditions (pH, ionic strength), include stabilizing agents, and minimize freeze-thaw cycles. For ND3 specifically, storage at -20°C for routine use and -80°C for extended storage is recommended .

• Purification difficulties:

  • Challenge: Separating recombinant ND3 from host proteins while maintaining function.

  • Solution: Implement a multi-step purification strategy, beginning with affinity chromatography (based on the tag determined during production) , followed by size exclusion chromatography. Consider using mild detergents like DDM or LMNG that preserve membrane protein structure.

• Activity loss during purification:

  • Challenge: Maintaining the functional activity of ND3 throughout the purification process.

  • Solution: Minimize exposure to extreme conditions, maintain a consistent cold chain (4°C), and include cofactors or stabilizing ligands in purification buffers.

When working with recombinant ND3, researchers should be aware that the determination of tag type during the production process will significantly impact purification strategy. Different tags (His, GST, MBP) require specific affinity resins and elution conditions, which must be optimized for each construct.

How can researchers effectively use ND3 as a model for studying mitochondrial disorders?

Recombinant Artemia salina ND3 offers a valuable model system for studying mitochondrial disorders, particularly those involving Complex I dysfunction. An effective research approach includes:

• Disease-mimicking mutation analysis:

  • Create recombinant ND3 variants with mutations that mirror those found in human mitochondrial disorders.

  • Assess the functional consequences through activity assays, structural analysis, and interaction studies.

  • Compare wild-type and mutant proteins to identify specific mechanisms of dysfunction.

• Heterologous expression systems:

  • Express Artemia ND3 and its mutant variants in model systems lacking endogenous ND3.

  • Assess the ability of different variants to rescue function in these systems.

  • Use these complementation assays to classify mutations by severity and mechanism.

• Inhibitor studies:

  • Use specific Complex I inhibitors to probe the functional role of ND3.

  • Compare inhibitor sensitivities between wild-type and mutant proteins.

  • Identify potential sites for therapeutic intervention.

• Artemia as a whole-organism model:

  • Leverage the Artemia lethality test methodology established for toxicity studies .

  • Expose Artemia to compounds that affect mitochondrial function.

  • Measure survival rates, development, and molecular markers of mitochondrial function.

The advantages of using Artemia as a model system include:

• Rapid life cycle and ease of culture under laboratory conditions.

• Established protocols for toxicity assessment that can be adapted for mitochondrial studies .

• Evolutionary conservation of fundamental mitochondrial functions.

• Ability to survive in extreme environments, potentially providing insights into compensatory mechanisms.

What are the future research directions for Artemia salina ND3 in comparative bioenergetics?

Future research on Artemia salina ND3 offers promising avenues for advancing our understanding of comparative bioenergetics, particularly in the context of adaptation to extreme environments. Key research directions include:

The methodological approaches for these future directions should build upon established techniques such as the Artemia toxicity assay system and the experimental designs used to study sodium pump adaptations in high-salinity conditions . The remarkable adaptability of Artemia, being the only animal capable of surviving in saturated brine , makes it an exceptional model for understanding the outer limits of bioenergetic adaptation.

As technologies advance, particularly in the areas of single-cell omics, high-resolution structural determination, and computational modeling, our ability to unravel the complex role of ND3 in Artemia's bioenergetic adaptation will continue to improve. These advances will not only enhance our understanding of basic biological principles but could also inspire biomimetic approaches to addressing human challenges in energy production and environmental adaptation.

How does ND3 research contribute to our broader understanding of protein evolution in extreme environments?

Research on Artemia salina ND3 makes significant contributions to our understanding of protein evolution in extreme environments, offering insights that extend beyond this specific protein to broader evolutionary principles. These contributions include:

• Model for evolutionary trade-offs: ND3 research helps elucidate how proteins balance the competing demands of structural stability and functional flexibility in extreme environments. This balance is critical for all proteins functioning under stress conditions, making ND3 a valuable model for studying evolutionary constraints.

• Identification of convergent adaptations: By comparing ND3 with other proteins adapted to extreme conditions, such as the specialized sodium pumps in Artemia that show altered stoichiometry in high-salinity environments , researchers can identify common adaptive strategies. These patterns may reveal fundamental principles of protein adaptation that apply across diverse protein families and environmental challenges.

• Understanding of evolutionary rate heterogeneity: Studies of ND3 sequence evolution across crustacean lineages with different environmental adaptations can reveal how environmental pressures influence evolutionary rates and patterns. This contributes to our broader understanding of how natural selection shapes protein evolution at different timescales.

• Insights into structure-function constraints: The specific functional role of ND3 in electron transport imposes certain structural constraints. Research on how these constraints interact with adaptive pressures in extreme environments can inform our understanding of the limits of protein evolvability.

• Comparative framework for extremophile adaptations: ND3 research provides a comparative framework for understanding how different extremophile organisms solve similar bioenergetic challenges. For example, the adaptations seen in Artemia's sodium pumps, where specific lysine substitutions alter ion stoichiometry and enable survival in high-salinity environments , might have parallels in the respiratory chain components.

The evolutionary insights gained from ND3 research have broader implications for understanding how essential cellular machinery can adapt to extreme conditions without compromising core functions. This knowledge has potential applications in synthetic biology, where designing proteins that function under non-standard conditions is a significant challenge.