Recombinant Myxine glutinosa NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the biological function of NADH-ubiquinone oxidoreductase chain 4L in Myxine glutinosa?

NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) in Myxine glutinosa functions as a core subunit of the mitochondrial membrane respiratory chain Complex I. This complex catalyzes electron transfer from NADH through the respiratory chain, utilizing ubiquinone as an electron acceptor. MT-ND4L specifically forms part of the enzyme's membrane arm that is embedded in the lipid bilayer, where it participates in the critical process of proton translocation across the inner mitochondrial membrane . This proton gradient is essential for ATP synthesis through oxidative phosphorylation. In the evolutionary context of Myxine glutinosa (Atlantic hagfish), one of the most primitive vertebrates, this protein may exhibit unique structural adaptations that contribute to the organism's remarkable metabolic efficiency in low-oxygen environments.

How does the sequence of Myxine glutinosa MT-ND4L compare with other vertebrate species?

While the specific sequence for Myxine glutinosa MT-ND4L is not directly provided in the available data, comparative analysis with other species such as Presbytis melalophos (mitred leaf monkey) and Oncorhynchus mykiss (rainbow trout) reveals important evolutionary insights. The MT-ND4L protein is typically characterized by a short sequence (approximately 90-100 amino acids) with highly conserved transmembrane domains .

The sequence conservation table below illustrates the evolutionary relationship between MT-ND4L proteins across representative vertebrate lineages:

The most conserved regions typically correspond to functional domains involved in proton pumping and electron transfer, while the highest variability is observed in loop regions facing the mitochondrial matrix .

What are the challenges in expressing recombinant Myxine glutinosa MT-ND4L in bacterial systems?

Expressing recombinant Myxine glutinosa MT-ND4L in bacterial systems presents several technical challenges. First, as a highly hydrophobic membrane protein with multiple transmembrane domains, MT-ND4L tends to aggregate and form inclusion bodies when overexpressed in E. coli systems . Second, differences in codon usage between Myxine glutinosa and E. coli may lead to translational pausing and truncated products. Third, the absence of post-translational modification machinery in bacterial hosts can affect protein folding and stability.

To address these challenges, researchers typically employ specialized expression strategies:

• Use of specialized E. coli strains optimized for membrane protein expression

• Fusion with solubility-enhancing tags (such as SUMO or MBP)

• Controlled expression at lower temperatures (16-18°C)

• Addition of specific detergents during cell lysis and protein purification

• Codon optimization of the MT-ND4L gene sequence for bacterial expression

These methodological adaptations significantly improve the yield and quality of recombinant Myxine glutinosa MT-ND4L for subsequent structural and functional studies.

How do conformational changes in recombinant MT-ND4L affect its interaction with other Complex I subunits?

The key conformational states of MT-ND4L and their functional implications include:

The transition between these conformational states is regulated by the redox state of the NADH/NAD+ pool and interactions with adjacent subunits . Using advanced AI algorithms and molecular simulations with enhanced sampling techniques, researchers can now predict these alternative functional states of MT-ND4L, providing insights into the molecular mechanisms of Complex I dysfunction in pathological conditions .

What is the role of recombinant MT-ND4L in superoxide production during Complex I dysfunction?

Recombinant MT-ND4L provides a valuable tool for investigating the molecular mechanisms of superoxide production during Complex I dysfunction. Studies with isolated Complex I from bovine heart mitochondria have demonstrated that the enzyme predominantly produces superoxide rather than hydrogen peroxide . The mechanism involves the transfer of a single electron from fully reduced flavin to molecular oxygen (O₂), with the rate of superoxide production determined by a bimolecular reaction between O₂ and reduced flavin in an empty active site .

MT-ND4L's contribution to this process appears to be indirect but crucial. While MT-ND4L itself is not the primary site of superoxide generation, alterations in its structure or function can affect:

• The conformational stability of adjacent subunits

• The proton translocation efficiency of the membrane domain

• The coupling between electron transfer and proton pumping

Experimental data using EPR spectroscopy and redox titrations have excluded iron-sulfur clusters and flavin radicals as direct sources of superoxide . Instead, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation through a preequilibrium mechanism. This mechanism provides a foundation for understanding how specific mutations or structural alterations in MT-ND4L can contribute to pathological oxidative stress through disrupted Complex I function .

How can AI-driven pocket prediction enhance targeted drug development for MT-ND4L-associated disorders?

AI-driven pocket prediction represents a revolutionary approach for identifying potential therapeutic targets on MT-ND4L. Using ensemble-based pocket detection algorithms that incorporate protein dynamics data, researchers can discover orthosteric, allosteric, hidden, and cryptic binding pockets on the protein's surface . This approach is particularly valuable for MT-ND4L, as traditional structure-based drug design methods have been limited by the protein's hydrophobic nature and conformational flexibility.

The AI-based pocket prediction workflow for MT-ND4L typically involves:

• LLM-powered literature research to identify known interaction sites and functional regions

• Structure-aware ensemble-based pocket detection utilizing established protein dynamics data

• AI scoring and ranking of tentative pockets with simultaneous detection of favorable interaction sites

This integrative approach has revealed previously unidentified binding pockets in MT-ND4L that could be targeted to modulate Complex I activity. For example, allosteric sites that influence proton translocation efficiency without affecting electron transfer could potentially alleviate oxidative stress in mitochondrial disorders without compromising energy production .

Potential therapeutic applications include:

• Small molecule modulators of MT-ND4L conformation to reduce superoxide production

• Peptide-based stabilizers of dysfunctional MT-ND4L variants

• Targeted antioxidants that accumulate near MT-ND4L binding sites

What are the optimal conditions for expressing and purifying recombinant Myxine glutinosa MT-ND4L?

The optimal expression and purification conditions for recombinant Myxine glutinosa MT-ND4L require careful optimization of multiple parameters. Based on protocols developed for similar proteins, the following methodology is recommended:

Expression System Selection:

• E. coli C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression

• Alternatively, insect cell expression systems (Sf9 or Hi5) for improved folding

Expression Conditions:

• Induction with 0.1-0.5 mM IPTG at OD₆₀₀ = 0.6-0.8

• Post-induction growth at 18°C for 16-20 hours to minimize inclusion body formation

• Supplementation with 5% glycerol in the growth medium to stabilize membrane proteins

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, and protease inhibitors

• Membrane fraction isolation by ultracentrifugation (100,000 × g, 1 hour)

• Solubilization using 1% n-dodecyl-β-D-maltoside (DDM) or 1% lauryl maltose neopentyl glycol (LMNG)

• Affinity purification using Ni-NTA for His-tagged protein

• Size exclusion chromatography in buffer containing 0.05% DDM or LMNG

Storage in a liquid solution containing glycerol at -20°C or -80°C is recommended for extended stability, with repeated freeze-thaw cycles being avoided . These conditions yield typically 1-3 mg of purified protein per liter of bacterial culture, suitable for structural and functional characterization.

How can structural studies be performed on recombinant MT-ND4L to understand its role in Complex I assembly?

Structural studies of recombinant MT-ND4L present unique challenges due to its hydrophobic nature and small size. Several complementary approaches can be employed to elucidate its structure and role in Complex I assembly:

Cryo-Electron Microscopy (Cryo-EM):

• Sample preparation using amphipol A8-35 or nanodiscs to maintain native-like lipid environment

• Grid optimization with added factors to prevent preferential orientation

• Data collection at high magnification with energy filters to enhance contrast

• 3D reconstruction with specialized software for membrane proteins

NMR Spectroscopy:

• Uniform ¹⁵N and ¹³C labeling of recombinant MT-ND4L

• Solution NMR in detergent micelles for structure determination

• Solid-state NMR for studying protein-lipid interactions

Integrative Structural Biology:

• Combining low-resolution structural data with AI-enhanced modeling

• Cross-linking mass spectrometry to identify interaction interfaces

• Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

Molecular Dynamics Simulations:

• AI-enhanced sampling to explore conformational space

• Trajectory clustering to identify representative structures

• Diffusion-based AI models to generate equilibrium protein conformations

Using these methods, researchers can generate a statistically robust ensemble of MT-ND4L conformations that capture its full dynamic behavior, providing insights into its role in Complex I assembly and function . The SWISS-MODEL Repository provides structural templates that can serve as starting points for these analyses .

What analytical techniques are most effective for studying the interaction between recombinant MT-ND4L and ubiquinone?

Studying the interaction between recombinant MT-ND4L and ubiquinone requires specialized analytical techniques that can detect and characterize these interactions in a membrane or membrane-mimetic environment. The most effective approaches include:

Biochemical Assays:

• Enzyme Kinetics: Measuring NADH oxidation rates in the presence of varying ubiquinone concentrations

• Competition Assays: Using ubiquinone analogs to determine binding specificity

• Site-Directed Mutagenesis: Systematic modification of predicted ubiquinone-binding residues

Biophysical Techniques:

• Microscale Thermophoresis (MST): Detecting binding-induced changes in thermal mobility

• Surface Plasmon Resonance (SPR): Real-time monitoring of binding kinetics

• Isothermal Titration Calorimetry (ITC): Quantifying thermodynamic parameters of binding

Spectroscopic Methods:

• Electron Paramagnetic Resonance (EPR): Examining electron transfer dynamics

• Fluorescence Quenching: Using ubiquinone's ability to quench intrinsic protein fluorescence

• FRET Analysis: With labeled ubiquinone analogs to determine binding proximity

Computational Approaches:

• AI-Based Pocket Prediction: Identifying potential ubiquinone binding sites

• Molecular Docking: In silico prediction of binding modes

• Quantum Mechanics/Molecular Mechanics (QM/MM): Modeling electron transfer reactions

The integration of these techniques provides a comprehensive understanding of MT-ND4L-ubiquinone interactions. Particularly valuable is the combination of experimental data with AI-driven molecular simulations, which can elucidate the conformational changes that occur during ubiquinone binding and reduction .

How does the function of Myxine glutinosa MT-ND4L compare with its homologs in higher vertebrates?

The functional comparison between Myxine glutinosa MT-ND4L and its homologs in higher vertebrates reveals both conservation of core functions and specialized adaptations. As one of the most primitive vertebrates, Myxine glutinosa (Atlantic hagfish) provides valuable insights into the evolution of mitochondrial function.

Analysis of the mitochondrial genomes across vertebrate lineages suggests that MT-ND4L maintains its core function in proton translocation while exhibiting lineage-specific adaptations in its regulatory mechanisms . The Atlantic hagfish's ability to survive in oxygen-poor environments may be partly attributed to specialized adaptations in its MT-ND4L that optimize energy production under these conditions. These adaptations could involve modifications in the proton-pumping efficiency or altered interaction with ubiquinone .

Understanding these functional differences provides insights into both the fundamental mechanisms of mitochondrial respiration and the evolution of bioenergetic systems in vertebrates.

What can comparative studies between recombinant MT-ND4L from different species reveal about its evolutionary adaptation?

Comparative studies of recombinant MT-ND4L from different species provide a powerful approach to understand evolutionary adaptations in mitochondrial function. By examining MT-ND4L from species occupying diverse ecological niches, researchers can identify specific adaptations that contribute to environmental fitness.

Key revelations from such comparative studies include:

• Conservation of Functional Domains: The core proton-translocating machinery shows remarkable conservation from hagfish to primates, indicating fundamental constraints on its evolution .

• Species-Specific Sequence Variations: Regions exposed to the lipid bilayer show higher variability, potentially reflecting adaptation to different membrane compositions and environmental temperatures.

• Thermostability Differences: MT-ND4L from species adapted to different thermal environments (like the cold-adapted Myxine glutinosa versus warm-blooded mammals) exhibits altered stability profiles reflecting their native operating temperatures.

• Oxygen Affinity Variations: Species from oxygen-variable environments may show adaptations in ubiquinone interaction sites that affect oxygen consumption efficiency.

• Regulatory Element Diversity: The evolution of regulatory elements controlling MT-ND4L expression reveals adaptation to different metabolic demands.

This comparative approach can be particularly informative when examining recombinant proteins from evolutionary distinct species like Myxine glutinosa (a basal vertebrate), Oncorhynchus mykiss (a teleost fish), and Presbytis melalophos (a primate) . The analysis of sequence conservation patterns, coupled with functional studies of recombinant proteins, illuminates how selective pressures have shaped mitochondrial function throughout vertebrate evolution.

How do therapeutic targeting strategies for MT-ND4L differ between species with varying susceptibility to mitochondrial disorders?

Therapeutic targeting strategies for MT-ND4L must account for species-specific variations in protein structure, function, and pathological implications. This is particularly relevant when developing therapies for mitochondrial disorders that may affect MT-ND4L function.

The AI-based pocket prediction and characterization outlined in search result provides a sophisticated framework for identifying species-specific therapeutic targets. For Myxine glutinosa MT-ND4L, such analysis might reveal unique binding pockets that could be exploited for selective modulation of its function .

Several factors influence species-specific targeting approaches:

By understanding these species-specific differences, researchers can develop more precise and effective therapeutic interventions for mitochondrial disorders involving MT-ND4L dysfunction.