Recombinant Vampyressa brocki NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) and what is its functional significance?

MT-ND4L is a protein component of the mitochondrial respiratory chain Complex I, playing a critical role in cellular energy production. The gene provides instructions for making NADH dehydrogenase 4L protein, which participates in the first step of the electron transport process during oxidative phosphorylation. Specifically, MT-ND4L contributes to the transfer of electrons from NADH to ubiquinone across the inner mitochondrial membrane, helping to create the electrochemical gradient necessary for ATP synthesis . In Vampyressa brocki, a phyllostomid bat species, this protein has a sequence of 98 amino acids and is encoded by the mitochondrial genome .

The MT-ND4L protein is located within the mitochondrial membrane as a multi-pass membrane protein, where it functions as part of the core machinery of Complex I. Its immediate electron acceptor is believed to be ubiquinone, making it integral to the initial stages of the electron transport chain . Understanding this protein's structure and function provides critical insights into mitochondrial energy metabolism across species.

What characterizes Vampyressa brocki as a species and why is its MT-ND4L of particular research interest?

Vampyressa brocki, commonly known as Brock's yellow-eared bat, is a phyllostomatid bat species from the Neotropical region. Specimens have been documented in Colombian rainforests, specifically in mature tropical rainforest at Leticia, Amazonas, expanding the known geographical distribution of this species . Morphologically, V. brocki exhibits distinctive features including variations in dorsal striping and facial markings that help distinguish it from related species .

The MT-ND4L from V. brocki has garnered research interest due to several factors. First, its genetic structure provides insights into evolutionary relationships between bat species. Karyotype analyses have revealed close genetic relationships between V. brocki and V. nymphaea, while also highlighting distinct chromosomal characteristics that differentiate V. brocki from other related species . Additionally, studying MT-ND4L from diverse species like V. brocki can illuminate evolutionary adaptations in mitochondrial function, potentially revealing how different organisms have optimized energy production to meet their unique metabolic demands.

How does the structure of MT-ND4L relate to its function in mitochondrial metabolism?

The MT-ND4L protein from Vampyressa brocki consists of 98 amino acids with a specific sequence that determines its structural and functional properties: MSLTYMMNFMAFTISLLGLLMYRAHMMSSLLCLEGMMLSLFVMMTMTILNTHTLTASMIPILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC . This sequence reveals hydrophobic regions consistent with a membrane-embedded protein, allowing it to function within the lipid bilayer of the inner mitochondrial membrane.

The protein's structure features multiple transmembrane domains that anchor it within the inner mitochondrial membrane, positioning it optimally for its role in electron transfer. These structural characteristics enable MT-ND4L to participate in the formation of proton channels within Complex I, contributing to proton pumping across the membrane . The conserved amino acid residues within the protein sequence are particularly critical for maintaining proper electron transfer functionality and interactions with other Complex I subunits.

The relationship between structure and function becomes evident when examining mutations. For example, in human MT-ND4L, the T10663C mutation (resulting in a Val65Ala substitution) has been associated with Leber hereditary optic neuropathy, demonstrating how structural alterations can disrupt mitochondrial function with pathological consequences .

What experimental approaches are most effective for studying recombinant Vampyressa brocki MT-ND4L?

Effective experimental approaches for studying recombinant V. brocki MT-ND4L typically employ a multi-faceted strategy combining molecular biology, biochemistry, and structural biology techniques. The recombinant protein can be produced in expression systems optimized for membrane proteins, with purification methods designed to maintain structural integrity. Based on established protocols for similar mitochondrial proteins, researchers should consider:

• Expression System Selection: Bacterial (E. coli), yeast (P. pastoris), or insect cell systems optimized for membrane protein expression.

• Purification Strategy: Two-step purification using affinity chromatography followed by size exclusion chromatography in the presence of appropriate detergents.

• Validation Techniques: Western blotting with specific antibodies, mass spectrometry for identity confirmation, and circular dichroism for secondary structure verification .

For functional analyses, researchers can employ spectrophotometric assays measuring NADH oxidation activity, oxygen consumption measurements, and electron paramagnetic resonance (EPR) spectroscopy to analyze electron transfer capabilities. Additionally, reconstitution into liposomes or nanodiscs can provide a native-like environment for functional studies. Blue native PAGE can be used to assess integration into Complex I and interaction with other subunits .

How can recombinant V. brocki MT-ND4L be utilized in comparative evolutionary studies?

Recombinant V. brocki MT-ND4L offers unique opportunities for comparative evolutionary studies across bat species and broader mammalian lineages. Researchers can employ the following methodological approaches:

• Sequence Alignment Analysis: Compare the amino acid sequence of V. brocki MT-ND4L with homologs from related species to identify conserved domains and species-specific variations. This approach has already revealed informative relationships between V. brocki and V. nymphaea .

• Functional Comparative Assays: Measure electron transfer rates, NADH oxidation kinetics, and proton pumping efficiency across recombinant MT-ND4L proteins from multiple species under identical experimental conditions.

• Structural Comparative Analysis: Apply comparative modeling and, where possible, experimental structure determination to analyze structural differences that may correlate with functional adaptations.

• Evolutionary Rate Analysis: Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS) to identify regions under selective pressure, which can reveal functionally critical domains across species .

These comparative approaches can illuminate how mitochondrial proteins have evolved in response to different metabolic demands, particularly in species like bats that have unique energetic requirements for flight. For example, the genetic relationships already established between V. brocki and other phyllostomatid bats provide a foundation for understanding how mitochondrial gene evolution correlates with ecological adaptations .

What are the implications of MT-ND4L mutations for understanding mitochondrial disorders?

MT-ND4L mutations provide critical insights into mitochondrial disorders, as evidenced by the documented association between human MT-ND4L mutations and Leber hereditary optic neuropathy (LHON). The T10663C mutation, which causes a valine-to-alanine substitution at position 65 (Val65Ala), has been identified in several families with LHON . This association demonstrates how single amino acid changes in MT-ND4L can disrupt mitochondrial function with tissue-specific consequences.

Research methodologies for investigating MT-ND4L mutation effects include:

• Site-Directed Mutagenesis: Generate recombinant MT-ND4L with specific mutations observed in clinical settings or predicted to affect function.

• Cybrid Cell Models: Transfer mitochondria containing MT-ND4L mutations into cells lacking mitochondrial DNA to isolate the effects of the mutation.

• Biochemical Characterization: Measure changes in Complex I assembly, stability, and activity resulting from mutations.

• Computational Prediction: Apply molecular dynamics simulations to predict structural and functional consequences of mutations.

The study of MT-ND4L mutations across species, including V. brocki, can provide evolutionary context for understanding human mitochondrial disorders. For instance, comparing naturally occurring variations in MT-ND4L across species with known pathogenic mutations in humans can help identify critical functional regions of the protein and potentially reveal compensatory mechanisms that might inform therapeutic approaches .

What are the optimal conditions for expressing and purifying recombinant V. brocki MT-ND4L?

The expression and purification of recombinant V. brocki MT-ND4L requires careful optimization due to its hydrophobic nature as a membrane protein. Based on established protocols for similar mitochondrial proteins, the following methodological approach is recommended:

Expression System Selection:

• Bacterial systems (E. coli C41/C43 strains) with specialized vectors containing fusion partners (MBP, SUMO) to enhance solubility

• Yeast systems (P. pastoris) for eukaryotic post-translational modifications

• Insect cell expression systems for complex membrane proteins requiring eukaryotic processing

Expression Conditions:

• Temperature: Typically lowered to 16-18°C after induction to slow expression and improve folding

• Induction: Gradual induction with low concentrations of inducer (0.1-0.5 mM IPTG for bacterial systems)

• Media supplements: Addition of specific phospholipids or membrane-mimicking compounds

Purification Strategy:

• Cell lysis using gentle detergents (DDM, LMNG, or digitonin) at concentrations just above CMC

• Affinity chromatography using the appropriate tag (typically His6 or other tags determined during production)

• Size exclusion chromatography in buffer containing 50% glycerol and Tris-based buffer as described for the commercial protein

• Storage at -20°C for short-term or -80°C for extended storage to maintain stability

Validation of proper folding and function is essential, typically performed using circular dichroism spectroscopy for secondary structure confirmation and functional assays measuring electron transfer activity.

How can researchers validate antibody specificity for studies involving V. brocki MT-ND4L?

Validating antibody specificity for V. brocki MT-ND4L is crucial for ensuring reliable experimental outcomes. A methodical approach should include:

Cross-Reactivity Testing:

• Test antibodies on tissues or cell lines known to express MT-ND4L positively and negatively, as described in commercial antibody validation processes

• Compare reactivity across related species with varying degrees of sequence homology to V. brocki MT-ND4L

• Include recombinant MT-ND4L proteins from different species as controls

Validation Techniques:

• Western Blotting: Confirm single band of appropriate molecular weight (approximately 10.7 kDa, similar to human MT-ND4L)

• Immunoprecipitation followed by mass spectrometry to confirm identity

• Immunocytochemistry with subcellular fractionation to verify mitochondrial localization

• Competitive binding assays with purified recombinant protein

• CRISPR knockout controls where applicable in model systems

Documentation Parameters:

• Record antibody source, catalog number, and lot number

• Document all validation experiments with appropriate positive and negative controls

• Standardize protocols for consistent results across experiments

Researchers should prioritize antibodies validated against multiple species or those with demonstrated cross-reactivity to related bat species. Custom antibody development may be necessary if commercial options lack sufficient specificity for V. brocki MT-ND4L, with validation using the recombinant protein as a reference standard .

What techniques are most effective for assessing MT-ND4L interactions with other Complex I components?

Investigating the interactions between MT-ND4L and other Complex I components requires specialized approaches that can capture transient and stable protein-protein interactions within the membrane environment. The following methodological strategies are recommended:

Biochemical Approaches:

• Blue Native PAGE: Separates intact protein complexes to analyze MT-ND4L incorporation into assembled Complex I

• Chemical Cross-linking coupled with Mass Spectrometry: Identifies interaction interfaces between MT-ND4L and neighboring subunits

• Co-immunoprecipitation: Pulls down MT-ND4L along with interacting partners

• FRET/BRET Analyses: Measures proximity between tagged protein pairs in reconstituted systems

Structural Biology Methods:

• Cryo-electron Microscopy: Provides structural context of MT-ND4L within the larger Complex I architecture

• Hydrogen-Deuterium Exchange Mass Spectrometry: Maps interaction surfaces by measuring solvent accessibility changes

• Native Mass Spectrometry: Analyzes intact complexes and subcomplexes containing MT-ND4L

Computational Approaches:

• Molecular Docking: Predicts interaction interfaces based on structural models

• Molecular Dynamics Simulations: Examines dynamic interactions in a membrane environment

• Evolutionary Coupling Analysis: Identifies co-evolving residues likely to be at interaction interfaces

These approaches should be applied in complementary fashion, as each method has distinct strengths and limitations. For example, biochemical methods provide functional evidence of interactions, while structural approaches offer spatial context. Computational methods can generate hypotheses that guide experimental design and help interpret experimental results .

How should researchers interpret variations in MT-ND4L sequence across species?

Interpreting variations in MT-ND4L sequences across species requires a systematic analytical approach that distinguishes between functionally significant changes and neutral variations. Researchers should employ the following methodological framework:

Sequence Analysis Pipeline:

• Multiple Sequence Alignment: Align MT-ND4L sequences from diverse species, including V. brocki and related bat species, using algorithms optimized for membrane proteins

• Conservation Analysis: Calculate per-residue conservation scores to identify highly conserved regions likely critical for function

• Variability Mapping: Map variable regions onto structural models to determine their location relative to functional domains

Evolutionary Analysis Methods:

• Selective Pressure Analysis: Calculate dN/dS ratios across the sequence to identify regions under purifying or positive selection

• Ancestral Sequence Reconstruction: Infer evolutionary trajectories of specific amino acid positions

• Phylogenetic Analysis: Construct trees based on MT-ND4L to examine how sequence variations correlate with taxonomic relationships, as demonstrated in studies of V. brocki and V. nymphaea

Functional Correlation Approaches:

• Structure-Function Mapping: Correlate sequence variations with known functional data across species

• Clinical Variant Analysis: Compare natural variations with known pathogenic mutations in humans (e.g., the T10663C mutation associated with LHON)

• Biochemical Property Analysis: Evaluate how amino acid substitutions affect properties such as hydrophobicity, charge, or size

When interpreting data from V. brocki specifically, researchers should consider the ecological and physiological context of this species. Bats have unique energetic demands due to flight, and variations in MT-ND4L might reflect adaptations to these demands. Comparative analysis with other bat species with different ecological niches can be particularly informative .

What bioinformatic tools are most useful for analyzing MT-ND4L structural and functional data?

Researchers studying MT-ND4L can leverage a variety of bioinformatic tools specifically suited for analyzing membrane proteins involved in electron transport. The following methodological approach to tool selection is recommended:

Sequence Analysis Tools:

• HMMER/Pfam: Identify conserved domains and family relationships

• ConSurf: Map conservation onto structural models

• PROVEAN/SIFT/PolyPhen: Predict functional effects of amino acid substitutions

Structural Prediction and Analysis:

• AlphaFold2/RoseTTAFold: Generate structural models of MT-ND4L

• TMHMM/TOPCONS: Predict transmembrane topology

• CAVER/MOLE: Analyze channels and cavities that may be relevant for proton or electron transport

• PyMOL/Chimera: Visualize and analyze structural features

Molecular Dynamics Simulation Platforms:

• GROMACS/NAMD: Simulate protein behavior in membrane environments

• CHARMM-GUI: Set up membrane protein simulations

• VMD: Analyze simulation trajectories

Evolutionary Analysis Tools:

• PAML/HyPhy: Detect signatures of selection

• IQ-TREE/MrBayes: Construct phylogenetic trees

• Datamonkey: Web-based platform for selection analysis

Integrated Platforms:

• Galaxy: Workflow management for complex analyses

• Jalview: Integrated environment for sequence and structure analysis

• InterPro: Integrated analysis of protein domains and functions

For V. brocki MT-ND4L specifically, researchers should prioritize tools that handle mitochondrial proteins well and account for the specific challenges of membrane protein analysis. When comparing across species, tools that can integrate phylogenetic information with structural analysis are particularly valuable for interpreting variations in an evolutionary context .

How can recombinant V. brocki MT-ND4L contribute to understanding mitochondrial genome recombination?

Recombinant V. brocki MT-ND4L represents an interesting model for investigating mitochondrial genome recombination, a phenomenon that challenges traditional understanding of mitochondrial inheritance. Although mitochondrial recombination is not typically observed in mammals, studies in other organisms have identified recombination events in mitochondrial genes, including those encoding complex I components .

Methodological approaches to investigate this include:

• Comparative Sequencing Analysis: Compare MT-ND4L sequences across V. brocki populations to identify potential recombination events, using methods similar to those applied in studies of recombinant mitochondrial genomes in fish species

• PCR-Based Recombination Detection: Employ specialized PCR techniques to amplify potential recombination junctions in the MT-ND4L gene region

• Next-Generation Sequencing Analysis: Use deep sequencing to identify low-frequency recombinant molecules that might exist in heteroplasmic states

• Sliding Window Analysis: Apply computational methods to detect non-uniform distribution of sequence differences that might indicate recombination events, similar to approaches that revealed recombination in salangid fishes

• Experimental Recombination Models: Develop systems to study the mechanisms and consequences of induced recombination in mitochondrial genes

Studies of fish species have shown that mitochondrial recombination can result from interspecific hybridization, often facilitated by human activities like species translocation and habitat modification . Similar investigations in bat species like V. brocki could reveal whether comparable processes occur in mammals, particularly in regions where different bat species have overlapping ranges.

What is the potential role of V. brocki MT-ND4L in comparative studies of mitochondrial disease models?

Vampyressa brocki MT-ND4L offers unique opportunities for developing comparative models of mitochondrial diseases, particularly those involving Complex I dysfunction. The following methodological framework can guide such research:

Comparative Disease Modeling Approaches:

• Cross-Species Mutation Analysis: Introduce known pathogenic mutations (such as the human T10663C mutation associated with LHON) into recombinant V. brocki MT-ND4L to compare functional consequences across species

• Resistance Mechanism Identification: Investigate whether V. brocki MT-ND4L contains natural variations that might confer resistance to dysfunction at positions where mutations cause disease in humans

• Compensatory Mechanism Studies: Identify sequence or structural features in V. brocki MT-ND4L that might compensate for potentially deleterious variations through:

  • Biochemical assays measuring electron transfer activity

  • Structural analysis of protein stability and interaction surfaces

  • Molecular dynamics simulations comparing human and bat proteins

• Evolutionary Medicine Applications: Analyze patterns of selection in MT-ND4L across species to identify regions critical for function that might inform therapeutic target selection

This research direction holds particular promise because bats have unique mitochondrial adaptations related to their high metabolic demands for flight. Understanding how these adaptations might confer resistance to dysfunction could provide insights into protective mechanisms that could be translated to human mitochondrial disease therapies .