Recombinant Chiroderma villosum NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its role in mitochondrial function?

MT-ND4L (Mitochondrially encoded NADH:ubiquinone oxidoreductase chain 4L) provides instructions for making the NADH dehydrogenase 4L protein, which is a critical component of Complex I in the mitochondrial respiratory chain. This protein functions as part of the large enzyme complex active in mitochondria that converts energy from food into adenosine triphosphate (ATP), the cell's main energy source. MT-ND4L is specifically involved in the process of oxidative phosphorylation, which creates ATP through a series of chemical reactions involving oxygen and simple sugars .

Complex I, of which MT-ND4L is a subunit, is responsible for the initial step in the electron transport process - transferring electrons from NADH to ubiquinone. This electron transfer creates an unequal electrical charge across the inner mitochondrial membrane, establishing the electrochemical gradient necessary for ATP production .

What is the structural composition of Complex I and where does MT-ND4L fit?

Complex I (NADH:ubiquinone oxidoreductase) is one of the most intricate membrane-bound enzymes in the mitochondrial respiratory chain, comprising more than 40 subunits in most eukaryotes. The enzyme has a characteristic L-shaped structure with:

• A hydrophilic domain extending into the mitochondrial matrix

• A hydrophobic domain embedded in the inner mitochondrial membrane

What is known about the specific characteristics of Chiroderma villosum MT-ND4L?

Chiroderma villosum (Hairy big-eyed bat) MT-ND4L has been characterized as a mitochondrially encoded protein. The recombinant version has the following properties:

• Amino acid sequence: MSLTYMMNFMAFTISLLGLLMYRSHMMSSLLCLEGMMLSLFVMMTMTILNTHLTLASMIPIILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC

• Protein length: 98 amino acids

• UniProt accession number: Q1HV38

• Enzymatic activity: Part of Complex I (NADH dehydrogenase) with EC number 1.6.5.3

The bat mitochondrial MT-ND4L shows evolutionary conservation with other mammalian species while maintaining species-specific variations that may offer insights into adaptive mitochondrial function across different taxonomic groups.

How does the amino acid sequence of Chiroderma villosum MT-ND4L compare to other species?

Comparing the amino acid sequences of MT-ND4L across species reveals both conservation in functional domains and species-specific adaptations. For example:

The Canis lupus MT-ND4L sequence (MSMVYINIFLAFILSLMGMLVYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHLTLASMMPI VLLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) shows similarities to the Chiroderma villosum sequence but with species-specific variations, particularly in the N-terminal region .

These sequence differences may reflect evolutionary adaptations to different metabolic demands and environmental conditions across species, while maintaining the core functional domains required for electron transport.

What are recommended approaches for expressing and purifying recombinant MT-ND4L?

Expressing and purifying hydrophobic membrane proteins like MT-ND4L presents significant challenges. Based on established protocols, researchers should consider:

Expression Systems:

• E. coli-based expression systems have been successful for recombinant MT-ND4L production, as evidenced by the Canis lupus MT-ND4L recombinant protein

• Use of fusion tags (particularly His-tags) facilitates purification while minimizing impact on protein structure

• Codon optimization may be necessary for efficient heterologous expression

Purification Protocol:

• Cell lysis under conditions that preserve membrane protein integrity

• Solubilization using appropriate detergents (typically mild non-ionic detergents)

• Affinity chromatography utilizing the fusion tag

• Size exclusion chromatography to enhance purity

• Concentration and storage in buffer containing stabilizing agents

Storage Recommendations:

• Store at -20°C or -80°C for extended periods

• Avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

• Consider adding glycerol (30-50%) to prevent freeze damage

The use of specialized buffer systems (e.g., Tris-based buffers with glycerol) has been shown to improve stability of recombinant MT-ND4L proteins during storage .

How can researchers effectively study the assembly and function of Complex I using recombinant MT-ND4L?

Studying Complex I assembly and function using recombinant MT-ND4L can be approached through several complementary techniques:

Reconstitution Assays:

• Incorporate purified recombinant MT-ND4L into liposomes or nanodiscs

• Add other purified Complex I components sequentially

• Monitor assembly using biochemical and biophysical techniques

Functional Analysis:

• NADH:ubiquinone oxidoreductase activity assays using spectrophotometric methods

• Electrochemical measurements of electron transfer

• Membrane potential measurements using fluorescent probes

Interaction Studies:

• Cross-linking followed by mass spectrometry to identify interaction partners

• Blue Native PAGE to assess complex formation

• Cryo-EM to visualize structural integration

Research has demonstrated that the absence of ND4L polypeptides prevents the assembly of the 950-kDa whole Complex I and suppresses enzyme activity, highlighting the critical role of this subunit in complex formation and function .

How does nuclear-encoded MT-ND4L differ from mitochondrially-encoded versions?

The evolutionary transfer of MT-ND4L from the mitochondrial to the nuclear genome in certain organisms (e.g., Chlamydomonas reinhardtii) has resulted in several notable adaptations that facilitate expression and mitochondrial import:

Key Differences:

Research in Chlamydomonas has identified the nuclear NUO11 gene as the homolog of mitochondrial ND4L coding sequences, demonstrating modifications that have occurred following gene transfer to the nucleus .

What experimental approaches can be used to study MT-ND4L mutations and their impact on mitochondrial function?

Several experimental approaches can effectively study MT-ND4L mutations and their functional consequences:

RNA Interference (RNAi):

• Design RNAi constructs targeting MT-ND4L transcripts

• Transform cells with RNAi constructs

• Confirm knockdown efficiency using qRT-PCR or Western blotting

• Assess effects on Complex I assembly and activity

This approach has been successfully implemented in Chlamydomonas, where researchers constructed plasmids (e.g., pND4L-RNAi) for RNA inactivation of the NUO11 gene (encoding ND4L) .

CRISPR-Cas9 Gene Editing:

• Design guide RNAs targeting MT-ND4L

• Introduce specific mutations of interest

• Screen for successful editing events

• Characterize phenotypic consequences

Phenotypic Characterization of Mutants:

• Respiration rate measurements using oxygen electrodes

• Blue Native PAGE to assess Complex I assembly

• Enzymatic activity assays for Complex I

• Reactive oxygen species (ROS) measurement

• Mitochondrial membrane potential assessment using fluorescent probes

Studies have shown that mutations affecting MT-ND4L can lead to health conditions such as Leber hereditary optic neuropathy, with the T10663C (Val65Ala) mutation identified in several affected families .

What are the implications of MT-ND4L research for understanding mitochondrial diseases?

MT-ND4L research has significant implications for understanding mitochondrial diseases:

• Molecular Basis of Pathogenesis: Studies of MT-ND4L mutations help elucidate how specific amino acid changes disrupt Complex I assembly or function, leading to disease phenotypes such as Leber hereditary optic neuropathy

• Heteroplasmy Effects: Research on MT-ND4L contributes to understanding how the ratio of mutant to wild-type mitochondrial DNA affects disease severity and progression

• Tissue-Specific Effects: Investigating why MT-ND4L mutations affect certain tissues (particularly high-energy demanding tissues like the optic nerve) more severely than others

• Therapeutic Development: Understanding the molecular consequences of MT-ND4L mutations may inform development of targeted therapies for mitochondrial disorders

What techniques are recommended for comparative evolutionary analysis of MT-ND4L across species?

For evolutionary analysis of MT-ND4L across species, researchers should consider:

Sequence-Based Approaches:

• Multiple sequence alignment of MT-ND4L proteins across diverse species

• Phylogenetic tree construction to visualize evolutionary relationships

• Calculation of selection pressures (dN/dS ratios) to identify conserved functional domains

• Identification of co-evolving residues that may indicate functional constraints

Structural Bioinformatics:

• Homology modeling of MT-ND4L from different species

• Structural superimposition to identify conserved structural features

• Analysis of transmembrane domain conservation

• Prediction of species-specific structural adaptations

Experimental Validation:

• Functional complementation studies across species

• Creation of chimeric proteins to identify functionally important domains

• Site-directed mutagenesis of conserved residues to assess functional significance

Such comparative approaches may provide insights into the evolutionary trajectory of MT-ND4L, particularly in cases where gene transfer from mitochondria to the nucleus has occurred.