Recombinant Phascogale tapoatafa NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is Phascogale tapoatafa MT-ND4L and its biological significance?

Phascogale tapoatafa (Common wambenger) is a marsupial species in which the mitochondrially-encoded NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) plays a critical role in mitochondrial function. This protein constitutes a vital component of Complex I in the electron transport chain. The MT-ND4L gene provides instructions for making the NADH dehydrogenase 4L protein, which is embedded in the inner mitochondrial membrane as part of Complex I . This complex is responsible for the first step in the electron transport process, specifically transferring electrons from NADH to ubiquinone during oxidative phosphorylation, which ultimately leads to ATP production .

The significance of studying this protein from Phascogale tapoatafa lies in comparative mitochondrial biology, as marsupial mitochondrial proteins can provide evolutionary insights into the conservation and adaptation of the electron transport chain across mammalian lineages. In mitochondria, Complex I creates an unequal electrical charge across the inner mitochondrial membrane through the step-by-step transfer of electrons, providing the energy necessary for ATP synthesis . Understanding this protein's structure and function in diverse species enables researchers to identify conserved functional domains and species-specific adaptations.

What is the amino acid sequence and structural characteristics of Phascogale tapoatafa MT-ND4L?

The complete amino acid sequence of Phascogale tapoatafa MT-ND4L consists of 98 amino acids and is as follows:
mLPIHLNLTVAFFLALAGVLIYRSHLMSTLLCLEGMmLSLFILMALMITHFQVFSVSMIP PILLVFSACEAGVGLALLVKISTTHGNDYIQNLNLLQC

This hydrophobic protein is characterized by transmembrane domains that anchor it within the inner mitochondrial membrane. Structural analysis indicates that MT-ND4L has multiple membrane-spanning regions consistent with its role in the electron transport chain. The protein contains regions critical for interaction with other Complex I subunits, ensuring proper assembly and function of the complete enzyme complex.

When comparing with human MT-ND4L sequences, particularly the segment "LLVSISNTYGLDYVHNLNLLQ" referenced in research materials for human recombinant MT-ND4L , researchers can identify conserved regions that may represent functionally critical domains. The C-terminal region containing the sequence "NLLQC" in Phascogale tapoatafa shows partial homology with the human sequence, suggesting evolutionary conservation of functional domains despite species divergence.

How should researchers design experiments using recombinant Phascogale tapoatafa MT-ND4L?

When designing experiments with recombinant Phascogale tapoatafa MT-ND4L, researchers should carefully consider several methodological aspects. First, protein handling is critical - the recombinant protein should be stored at -20°C for standard storage, or at -80°C for extended storage periods. Repeated freeze-thaw cycles should be avoided to maintain protein integrity, and working aliquots should be stored at 4°C for maximum one week of use .

For experimental applications, researchers should note that the recombinant protein contains a tag (though the specific tag type may vary depending on production process) and is supplied in a Tris-based buffer with 50% glycerol . This buffer composition should be considered when designing downstream applications to avoid buffer incompatibilities.

Experimental design should include appropriate controls, including:

• Empty vector controls to account for effects of the expression system

• Negative controls without protein addition

• Positive controls with known functional properties

• Species-comparative experiments using MT-ND4L from other organisms when investigating evolutionary aspects

For quantitative studies, standard curves should be established using known concentrations of the recombinant protein. Researchers should validate antibody specificity when conducting immunological experiments, as the protein may be used in blocking/neutralizing applications to confirm antibody specificity .

What are the recommended protocols for using MT-ND4L in functional mitochondrial assays?

For functional mitochondrial assays, researchers should incorporate recombinant MT-ND4L protein in the following protocol framework:

Complex I Activity Assay:

• Isolate intact mitochondria from target tissues using differential centrifugation

• Prepare mitochondrial fraction in appropriate assay buffer (typically containing phosphate buffer, pH 7.4, with protease inhibitors)

• Supplement with recombinant MT-ND4L at optimized concentrations (typically 0.5-5 μg/mL)

• Measure NADH oxidation spectrophotometrically at 340 nm

• Calculate enzyme activity using extinction coefficient of NADH

To study electron transport specifically, researchers should design assays that measure the transfer of electrons from NADH to ubiquinone. This is particularly relevant since MT-ND4L is part of Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) .

When investigating potential mitochondrial dysfunction, comparative assays should be performed using wild-type and mutant versions of MT-ND4L. The mutation T10663C (Val65Ala) identified in Leber hereditary optic neuropathy can serve as a model for understanding how structural changes in MT-ND4L affect Complex I function .

How can recombinant MT-ND4L be used to study mitochondrial disease mechanisms?

Recombinant MT-ND4L provides a valuable tool for investigating mitochondrial disease mechanisms, particularly in conditions associated with Complex I dysfunction. Leber hereditary optic neuropathy (LHON) represents a primary research application, as mutations in MT-ND4L (such as the T10663C mutation resulting in Val65Ala substitution) have been identified in several families with this condition .

To utilize recombinant MT-ND4L in mitochondrial disease research, researchers should:

• Perform comparative functional studies between wild-type and disease-associated mutant forms of the protein to assess functional differences

• Use the recombinant protein in reconstitution experiments with isolated mitochondria lacking functional MT-ND4L

• Develop in vitro models incorporating the recombinant protein to investigate electron transport chain dynamics

• Employ the protein in binding studies to identify interaction partners and assess how disease mutations affect these interactions

The sandwich ELISA approach described for MT-ND4L detection can be adapted to quantify protein levels in patient samples, enabling correlation between protein abundance and disease severity . By immobilizing antibodies specific to MT-ND4L on microplates, researchers can quantify the protein in biological samples and assess differences between normal and pathological states.

What techniques are available for targeting recombinant RNAs to mitochondria for MT-ND4L expression modulation?

Mitochondrial targeting of recombinant RNAs represents an advanced approach to modulate MT-ND4L expression for both research and potential therapeutic applications. Research indicates that specifically designed oligoribonucleotides can be targeted to mitochondria through specialized delivery pathways .

Researchers investigating MT-ND4L expression modulation can employ several strategies:

• Design RNA constructs with mitochondrial targeting sequences that facilitate import into the organelle

• Utilize RNA import complexes that can deliver functional RNAs into mitochondria

• Develop lipid-based nanocarriers optimized for mitochondrial targeting

• Implement peptide nucleic acid (PNA) approaches for sequence-specific targeting

These techniques can be used to study heteroplasmy levels - the coexistence of wild-type and mutant mitochondrial DNA within cells . By specifically targeting recombinant RNAs to mitochondria, researchers can potentially shift the balance of wild-type to mutant MT-ND4L, which has significant implications for mitochondrial diseases associated with MT-ND4L mutations.

The experimental protocol should include careful assessment of:

• RNA stability and integrity before and after mitochondrial delivery

• Targeting efficiency using fluorescently labeled control RNAs

• Functional consequences using Complex I activity assays

• Changes in heteroplasmy levels using quantitative PCR techniques

How does Phascogale tapoatafa MT-ND4L compare with homologs in other species?

Comparative analysis of MT-ND4L across species reveals important insights into evolutionary conservation and functional adaptation of this mitochondrial protein. The Phascogale tapoatafa MT-ND4L can be compared with homologs from other mammalian orders, including primates (human and non-human), rodents, and other marsupials, to understand evolutionary patterns.

Analysis of available sequence data indicates several conserved domains across mammalian species, particularly in regions associated with electron transport functionality. When comparing the Phascogale tapoatafa sequence with the human MT-ND4L fragment "LLVSISNTYGLDYVHNLNLLQ" , researchers can identify both conserved and variable regions that may reflect species-specific adaptations.

Conservation analysis should consider:

• Transmembrane domain structure across species

• Functional motifs involved in electron transport

• Regions that interact with other Complex I subunits

• Sites subject to positive selection during mammalian evolution

The study of marsupial mitochondrial proteins like those from Phascogale tapoatafa provides unique evolutionary perspectives on mitochondrial function. As marsupials diverged from placental mammals approximately 160 million years ago, their mitochondrial proteins may reveal alternative evolutionary solutions to energy production challenges in different ecological niches .

What is known about mitochondrial disorders related to MT-ND4L mutations across species?

Mitochondrial disorders associated with MT-ND4L mutations have been documented across several species, with the most well-characterized being Leber hereditary optic neuropathy (LHON) in humans. The T10663C mutation, resulting in a Val65Ala substitution in the MT-ND4L protein, has been identified in families with LHON . This mutation affects a critical region of the protein and likely disrupts normal electron transport function.

While specific MT-ND4L mutations have not been extensively characterized in Phascogale tapoatafa, comparative analysis with human disease-associated mutations can provide insights into potential functional consequences. Researchers investigating MT-ND4L-related disorders should consider:

• Creating recombinant proteins containing known pathogenic mutations for functional studies

• Developing animal models expressing mutant MT-ND4L to study disease progression

• Investigating tissue-specific effects of MT-ND4L mutations, particularly in high-energy tissues like retinal ganglion cells relevant to LHON

• Exploring potential therapeutic approaches using recombinant wild-type protein or RNA-based gene therapy

The experimental use of recombinant MT-ND4L proteins from different species, including Phascogale tapoatafa, can help identify whether certain species have evolved natural resistance mechanisms to mutations that cause disease in humans, potentially revealing novel therapeutic approaches.

What protein interaction studies can be performed using recombinant MT-ND4L?

Recombinant MT-ND4L provides a valuable tool for investigating protein-protein interactions within Complex I and potentially with other mitochondrial components. Researchers can employ several advanced analytical methods to characterize these interactions:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to MT-ND4L or its interaction partners to pull down protein complexes, followed by mass spectrometry identification of binding partners.

• Surface Plasmon Resonance (SPR): Measuring binding kinetics between immobilized MT-ND4L and other Complex I subunits or potential interaction partners in real-time.

• Microscale Thermophoresis (MST): Detecting interactions based on changes in thermophoretic mobility upon binding, requiring minimal protein amounts.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Identifying regions involved in protein-protein interactions by measuring deuterium uptake differences between bound and unbound states.

When conducting interaction studies, researchers should consider native-like conditions to maintain physiologically relevant interactions. The recombinant protein can be reconstituted into liposomes or nanodiscs to mimic the membrane environment where MT-ND4L naturally functions. Cross-linking approaches combined with mass spectrometry (XL-MS) can capture transient interactions within the mitochondrial membrane environment.

How can researchers assess the impact of MT-ND4L variants on Complex I assembly and function?

To assess the impact of MT-ND4L variants on Complex I assembly and function, researchers can implement a multi-faceted analytical approach:

• Blue Native PAGE: Separate intact respiratory complexes to assess whether MT-ND4L variants affect Complex I assembly or stability.

• Spectrophotometric Enzyme Assays: Measure NADH:ubiquinone oxidoreductase activity to quantify functional consequences of MT-ND4L variants.

• Oxygen Consumption Analysis: Use high-resolution respirometry to measure oxygen consumption rates in reconstituted systems or cell models expressing variant MT-ND4L.

• Supercomplex Formation Analysis: Investigate whether MT-ND4L variants affect the formation of respiratory supercomplexes, which optimize electron transfer efficiency.

• ROS Production Measurement: Quantify reactive oxygen species generation as a potential consequence of dysfunctional Complex I containing MT-ND4L variants.

For mammalian cell-based studies, researchers can use CRISPR/Cas9-mediated approaches to introduce specific mutations in MT-ND4L in cybrid cell lines, then assess functional consequences using the analytical methods described above. The recombinant protein can also be used in competition assays to determine whether exogenous wild-type MT-ND4L can rescue phenotypes associated with endogenous mutant protein.

What are the emerging applications of recombinant MT-ND4L in mitochondrial research?

Emerging applications of recombinant MT-ND4L in mitochondrial research span from basic science to potential therapeutic approaches. The protein serves as a valuable tool for understanding fundamental aspects of Complex I assembly and function, while also enabling the development of novel therapeutic strategies for mitochondrial disorders.

Current research trends include:

• Development of high-resolution structural studies incorporating recombinant MT-ND4L to better understand its positioning and function within Complex I

• Creation of biosensors utilizing MT-ND4L-based constructs to monitor mitochondrial function in real-time

• Exploration of gene therapy approaches delivering functional MT-ND4L to treat disorders associated with mutations in this gene

• Investigation of small molecules that can modulate MT-ND4L function or stabilize mutant proteins

The ability to target recombinant RNAs to mitochondria offers promising approaches for correcting MT-ND4L-related dysfunction . This strategy could potentially shift heteroplasmy levels in favor of wild-type MT-ND4L, providing therapeutic benefit in conditions like LHON.

As we continue to unravel the complexities of mitochondrial function across species, the study of Phascogale tapoatafa MT-ND4L provides unique evolutionary perspectives that complement research on human mitochondrial biology, potentially revealing novel therapeutic targets and approaches.

What methodological challenges remain in studying MT-ND4L function?

Despite advances in recombinant protein technology and mitochondrial research, significant methodological challenges remain in studying MT-ND4L function:

• Membrane Protein Challenges: As a hydrophobic membrane protein, MT-ND4L presents difficulties in expression, purification, and maintenance of native conformation outside the mitochondrial membrane environment.

• Mitochondrial Targeting Efficiency: Delivering recombinant proteins or RNAs specifically to mitochondria remains challenging, with varying efficiency across cell types and tissues.

• Functional Reconstitution: Reconstituting purified MT-ND4L into functional Complex I assemblies requires optimization of lipid composition and protein ratios to achieve physiological activity levels.

• Species-Specific Differences: Extrapolating findings from Phascogale tapoatafa MT-ND4L to human clinical applications requires careful consideration of species-specific structural and functional differences.

• Quantitative Analysis: Developing sensitive and specific assays to quantify MT-ND4L in complex biological samples remains challenging due to its hydrophobic nature and relatively low abundance.

Future methodological developments should focus on improving membrane protein handling techniques, enhancing mitochondrial targeting strategies, and developing more sensitive analytical approaches. Advances in cryo-electron microscopy may facilitate structural studies of MT-ND4L within intact Complex I, while CRISPR-based approaches could enable more precise manipulation of MT-ND4L expression in cellular and animal models.