Recombinant Sminthopsis crassicaudata NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the structural composition of Sminthopsis crassicaudata MT-ND4L?

MT-ND4L from Sminthopsis crassicaudata is a small, highly hydrophobic protein approximately 11 kDa in size, composed of 98 amino acids. The amino acid sequence is: mLSINLNLIVAFLLALMGVLIYRSHLMSTLLCLEGMmLSLFILMTLLITHFHMFSMSMTP PILLVFSACEAAIGLALLVKISATHGSDHIQNLNLLQC . As a multi-pass membrane protein, it spans the inner mitochondrial membrane and forms part of the core transmembrane region of Complex I, contributing to its characteristic L-shaped structure alongside other mitochondrially encoded subunits .

What is the functional role of MT-ND4L in the mitochondrial respiratory chain?

MT-ND4L serves as a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which catalyzes the first step in the electron transport process—the transfer of electrons from NADH to ubiquinone . It is embedded in the enzyme's membrane arm within the lipid bilayer and is involved in proton translocation across the inner mitochondrial membrane . This process creates an electrochemical gradient that drives ATP synthesis through oxidative phosphorylation, making MT-ND4L essential for cellular energy production .

How is the MT-ND4L gene organized in the mitochondrial genome?

In humans, the MT-ND4L gene is located in the mitochondrial DNA from base pair 10,469 to 10,766 . A notable feature of human MT-ND4L is its 7-nucleotide gene overlap with the MT-ND4 gene, where the last three codons of MT-ND4L (5'-CAA TGC TAA-3' coding for Gln, Cys, and Stop) overlap with the first three codons of MT-ND4 (5'-ATG CTA AAA-3') . The MT-ND4L gene is one of seven mitochondrially encoded subunits of Complex I, alongside MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, and MT-ND6 .

What expression systems are optimal for producing functional recombinant MT-ND4L?

Recombinant MT-ND4L can be produced using several expression systems:

For structural studies requiring large protein quantities, E. coli systems may be preferred, while functional studies might benefit from eukaryotic expression systems that better facilitate proper membrane insertion .

What are the challenges in purifying functional MT-ND4L, and how can they be overcome?

Purifying functional MT-ND4L presents several challenges due to its highly hydrophobic nature. Methodological solutions include:

• Solubilization strategies: Use of specialized detergents such as digitonin or lauryl maltose neopentyl glycol (LMNG) to extract the protein while maintaining its native conformation .

• Affinity purification optimization: Employing N-terminal or C-terminal tags (such as His-tags) for affinity chromatography, with tag position determined based on protein stability considerations .

• Membrane-mimetic systems: Reconstitution into nanodiscs, liposomes, or detergent micelles to maintain proper folding and function .

• Purity validation: Implementation of SDS-PAGE analysis to confirm >90% purity, as typically required for functional studies .

• Storage optimization: Maintaining the protein in buffer containing 50% glycerol at -20°C/-80°C, with working aliquots at 4°C for up to one week to prevent denaturation from repeated freeze-thaw cycles .

How can recombinant MT-ND4L be used to investigate mitochondrial disorders?

Recombinant Sminthopsis crassicaudata MT-ND4L can serve as a valuable tool for investigating mitochondrial disorders through several methodological approaches:

• Comparative structural analysis: Comparing wild-type and mutant forms to understand how mutations (such as the T10663C/Val65Ala mutation associated with Leber hereditary optic neuropathy) affect protein structure and function .

• In vitro reconstitution studies: Combining purified recombinant MT-ND4L with other Complex I subunits to reconstitute functional or dysfunctional complexes, allowing direct assessment of specific mutations on enzyme activity .

• Binding pocket characterization: AI-based pocket prediction and structure-aware ensemble-based detection to identify potential therapeutic targets, as demonstrated by the Receptor.AI approach .

• Antibody development: Using the recombinant protein to generate specific antibodies for immunodetection of MT-ND4L in patient samples, facilitating diagnosis of mitochondrial disorders .

• Functional complementation: Introducing recombinant MT-ND4L into cellular models lacking endogenous protein to evaluate functional rescue capabilities .

What experimental approaches are most effective for studying interactions between MT-ND4L and other Complex I subunits?

To effectively study interactions between MT-ND4L and other Complex I subunits, researchers should consider these methodological approaches:

• AI-driven conformational ensemble generation: Advanced AI algorithms can predict alternative functional states of MT-ND4L, including large-scale conformational changes, providing insight into dynamic protein behavior and interaction potential .

• Crosslinking studies: Chemical crosslinking coupled with mass spectrometry can capture protein-protein interactions within the complex, identifying specific interaction sites between MT-ND4L and other subunits .

• Molecular dynamics simulations: Computational approaches can explore the dynamic behavior of MT-ND4L within the complex structure and predict interaction interfaces .

• Cryo-electron microscopy: High-resolution structural determination can visualize the entire Complex I structure, showing spatial relationships between MT-ND4L and other subunits in a near-native environment .

• Co-expression systems: Expressing MT-ND4L together with interacting partners to study complex formation and stability .

How does the mitochondrial copy number affect evolutionary rate variation in MT-ND4L?

Recent research has demonstrated that mitochondrial genome copy number significantly impacts the evolutionary rate of mitochondrial genes like MT-ND4L:

• Studies across 60 diverse seed plant species revealed that mitochondrial genome copy number explains approximately 47% of the variation in synonymous substitution rates of mitochondrial DNA over ~300 million years of evolution .

• A negative correlation exists between copy number and substitution rates, suggesting that homologous recombinational repair, the primary repair mechanism in plant organelles, is less effective in low copy number environments .

• This relationship appears to be unique to mitochondrial DNA and was not observed in plastid DNA, indicating a mechanism specific to mitochondria .

• Copy number also negatively correlates with mitochondrial genome size, which may be either a cause or consequence of mutation rate variation .

• This finding may explain the extreme evolutionary rate variation observed in angiosperm mitogenomes, where rates have increased up to 5,000-fold in some lineages compared to others .

What role does MT-ND4L play in Leber hereditary optic neuropathy (LHON)?

MT-ND4L has been implicated in Leber hereditary optic neuropathy (LHON) through several lines of evidence:

• A specific mutation in the MT-ND4L gene (T10663C or Val65Ala) has been identified in several families with LHON, changing the valine amino acid at position 65 to alanine .

• A study using whole exome sequencing from 10,831 participants in the Alzheimer's Disease Sequencing Project found a significant association between AD and a rare MT-ND4L variant (rs28709356 C>T) as well as with MT-ND4L in a gene-based test .

• Methodological approaches to study this association include:

  • Next-generation sequencing to identify novel mutations

  • Cybrid cell models carrying patient-derived mitochondria

  • Complex I enzyme activity assays to assess functional impact

  • Oxygen consumption measurements using high-resolution respirometry

  • Mouse models with introduced MT-ND4L mutations

• Though researchers have not fully determined the mechanistic pathway from MT-ND4L mutation to vision loss in LHON, the mutation likely affects Complex I assembly, stability, or function, potentially leading to energy deficiency or increased oxidative stress in retinal ganglion cells .

How can AI-driven approaches enhance MT-ND4L research?

Recent developments in AI-driven approaches are revolutionizing MT-ND4L research:

• LLM-powered literature research: Custom-tailored Large Language Models can extract and formalize relevant information about MT-ND4L from structured and unstructured data sources, storing it in knowledge graphs to gain comprehensive insights into therapeutic significance, existing ligands, off-targets, and protein-protein interactions .

• AI-Driven Conformational Ensemble Generation: Advanced AI algorithms can predict alternative functional states of MT-ND4L, including large-scale conformational changes along "soft" collective coordinates .

• Enhanced molecular simulations: AI-enhanced sampling and trajectory clustering allow exploration of the broad conformational space of MT-ND4L, identifying representative structures for more accurate drug design .

• Binding pocket identification: AI-based pocket prediction modules can discover orthosteric, allosteric, hidden, and cryptic binding pockets on MT-ND4L's surface by integrating literature search data and structure-aware ensemble-based detection algorithms .

• Diffusion-based AI models: These models, combined with active learning AutoML, can generate statistically robust ensembles of equilibrium protein conformations that capture the full dynamic behavior of MT-ND4L .

What methods are most effective for investigating species-specific differences in MT-ND4L structure and function?

To effectively investigate species-specific differences in MT-ND4L structure and function, researchers should employ:

• Comparative sequence analysis: Multiple sequence alignment of MT-ND4L from different species (e.g., Sminthopsis crassicaudata, Presbytis melalophos, Distoechurus pennatus, Oncorhynchus tschawytscha, and human) reveals conservation patterns and species-specific adaptations .

• Recombinant protein expression of multiple orthologs: Expressing MT-ND4L from different species under identical conditions allows direct functional comparisons .

• Structure-function relationship studies: Site-directed mutagenesis to convert species-specific residues between orthologs can help identify functionally important regions .

• Evolutionary rate analysis: Calculation of synonymous and nonsynonymous substitution rates across lineages can identify branches under different selective pressures .

• 3D structural comparisons: Homology modeling and comparative structural analysis can reveal species-specific differences in protein folding and potential functional consequences .

How can recombinant MT-ND4L contribute to therapeutic development for mitochondrial disorders?

Recombinant Sminthopsis crassicaudata MT-ND4L can contribute to therapeutic development for mitochondrial disorders through several approaches:

• Target validation: Confirming the role of MT-ND4L in disease pathology through structural and functional studies of wild-type and mutant forms .

• Drug screening platforms: Utilizing purified recombinant protein for high-throughput screening of compounds that might stabilize mutant MT-ND4L or restore Complex I function .

• Binding pocket characterization: AI-based and experimental approaches to identify potential binding sites on MT-ND4L that could be targeted therapeutically .

• Protein replacement strategies: Developing methods to deliver functional recombinant MT-ND4L to affected tissues as a potential therapeutic approach .

• Biomarker development: Using recombinant protein to develop assays for detecting MT-ND4L dysfunction in patient samples, facilitating earlier diagnosis and treatment .

Through these diverse applications, recombinant Sminthopsis crassicaudata MT-ND4L serves as both a research tool for understanding mitochondrial biology and a potential platform for developing treatments for mitochondrial disorders.