Recombinant Stenoderma rufum NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What experimental systems are most suitable for studying recombinant Stenoderma rufum MT-ND4L?

For recombinant expression of Stenoderma rufum MT-ND4L, researchers should consider several expression systems based on experimental objectives. Bacterial expression systems like E. coli provide high protein yields but may require optimization for proper folding of this mitochondrial membrane protein . For functional studies, mammalian cell lines with depleted endogenous MT-ND4L (using CRISPR-Cas9) allow complementation with the recombinant bat protein to assess functionality in the native complex I environment. Respiratory-deficient yeast models also serve as valuable platforms for heterologous expression and functional rescue experiments. When designing expression constructs, researchers should incorporate appropriate purification tags that do not interfere with protein folding or complex assembly. Microscale thermophoresis or isothermal titration calorimetry can assess binding interactions between the recombinant protein and other complex I subunits, providing insights into species-specific assembly characteristics.

How does the electron transfer mechanism involving MT-ND4L differ between Na⁺-translocating and H⁺-translocating NADH:quinone oxidoreductases?

The electron transfer mechanism involving MT-ND4L presents significant differences between Na⁺-translocating and H⁺-translocating NADH:quinone oxidoreductases. In bacterial Na⁺-NQR systems, the electron transfer pathway involves a unique set of prosthetic redox groups including two covalently bound FMN residues, a [2Fe-2S] cluster, FAD, riboflavin, and a Cys4[Fe] center . The mechanism features an alternating access model where the Cys4[Fe] center is alternatively exposed to different sides of the membrane, allowing electron transfer between subunits . In contrast, mitochondrial H⁺-translocating systems like those containing MT-ND4L operate through a different mechanism where electron transfer is coupled to the vectorial transmembrane transfer of four H⁺ ions, contributing to proton motive force generation . When investigating Stenoderma rufum MT-ND4L, researchers should design experiments to determine whether species-specific adaptations exist in coupling efficiency or proton translocation rate, particularly given the high metabolic demands of flight in bats.

What techniques can resolve the real-time kinetics of electron transfer through the MT-ND4L subunit of complex I?

Resolving real-time kinetics of electron transfer through MT-ND4L requires sophisticated biophysical approaches. Ultra-fast microfluidic stopped-flow instruments with capabilities similar to those used for bacterial Na⁺-NQR (dead time of 0.25 ms and optical path length of 1 cm) can collect visible spectra at microsecond intervals, enabling identification of fleeting intermediates in the electron transfer pathway . Researchers investigating Stenoderma rufum MT-ND4L should incorporate spectral analyses comparing reaction steps with known redox transitions of individual enzyme cofactors to delineate the sequence of electron transfer events. Advanced techniques such as pulse radiolysis, electron paramagnetic resonance (EPR) spectroscopy with freeze-quench apparatus, and time-resolved fluorescence spectroscopy can further elucidate electron transfer rates. Molecular dynamics simulations based on structural data can complement experimental findings by predicting conformational changes during electron transfer and identifying rate-limiting steps specific to the bat protein. These multimodal approaches provide comprehensive insights into species-specific kinetic parameters.

How do mutations in Stenoderma rufum MT-ND4L affect the coupling mechanism between electron transfer and proton translocation?

Mutations in MT-ND4L can significantly impact the coupling mechanism between electron transfer and proton translocation, disrupting energy conversion efficiency. To investigate this in Stenoderma rufum, researchers should first identify conserved residues likely involved in the coupling mechanism based on comparative genomics and structural analysis . Site-directed mutagenesis of these residues in recombinant constructs, followed by functional expression in suitable model systems, would allow assessment of specific amino acid contributions to coupling efficiency. Researchers should measure changes in proton translocation rates using pH-sensitive fluorescent probes or patch-clamp electrophysiology while simultaneously monitoring electron transfer rates through spectroscopic methods. The coupling ratio (H⁺/e⁻) should be determined for wild-type and mutant proteins under various substrate concentrations and membrane potential conditions. Molecular dynamics simulations can further predict how specific mutations might alter proton channels or conformational changes necessary for coupling, generating testable hypotheses about structure-function relationships in this bat species.

What are the optimal conditions for expressing and purifying recombinant Stenoderma rufum MT-ND4L for structural studies?

Expressing and purifying recombinant Stenoderma rufum MT-ND4L presents distinct challenges due to its highly hydrophobic nature and requirement for proper membrane insertion. Researchers should consider codon-optimized constructs for expression in either E. coli or insect cell systems, with the latter often providing superior folding for mitochondrial membrane proteins . For E. coli expression, fusion partners such as maltose-binding protein or SUMO can improve solubility, while inclusion of a C-terminal His-tag facilitates purification without interfering with N-terminal membrane insertion. Extraction requires careful optimization of detergent selection—mild detergents like n-dodecyl-β-D-maltoside or digitonin better preserve native structure compared to harsher alternatives. Purification should incorporate multiple chromatography steps, typically beginning with immobilized metal affinity chromatography followed by size exclusion chromatography to isolate protein in its monomeric form or in complex with other subunits. For structural studies, researchers should assess protein stability through thermal shift assays and circular dichroism before proceeding to crystallization trials or cryo-EM sample preparation.

What protocols can be used to investigate the interaction between MT-ND4L and other subunits of complex I?

Investigating interactions between MT-ND4L and other complex I subunits requires multiple complementary approaches to capture both stable and transient associations. Chemical cross-linking combined with mass spectrometry provides a powerful method to map protein-protein interactions within the intact complex, identifying residues in close proximity between MT-ND4L and neighboring subunits . Researchers should optimize crosslinker selection based on spacer arm length and reactive groups to capture different types of interactions. Co-immunoprecipitation studies using antibodies against recombinant Stenoderma rufum MT-ND4L can identify stable interaction partners, while label-transfer approaches help detect more transient associations. Proximity ligation assays or FRET-based methods in intact mitochondria can verify interactions in the native cellular environment. Hydrogen-deuterium exchange mass spectrometry provides additional insights by revealing conformational changes and solvent-accessible regions at protein interfaces. For functional relevance of specific interactions, site-directed mutagenesis of putative interface residues followed by assembly assays can determine which interactions are essential for complex I biogenesis versus catalytic function.

How does the proton-pumping efficiency of complexes containing Stenoderma rufum MT-ND4L compare to those of other mammalian species?

Proton-pumping efficiency of complex I containing Stenoderma rufum MT-ND4L may exhibit adaptations related to the high metabolic demands of flight in bats. Researchers should use reconstituted proteoliposomes containing purified complex I with either native or recombinant MT-ND4L to directly measure H⁺/e⁻ stoichiometry under controlled conditions . Comparative analysis with complex I from non-flying mammals can reveal whether bat MT-ND4L contributes to enhanced coupling efficiency. Proton translocation can be measured using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor internal acidification of proteoliposomes upon NADH addition. Simultaneous tracking of NADH oxidation spectrophotometrically (340 nm) allows calculation of the H⁺/NADH ratio. Researchers should perform these measurements across different membrane potentials using valinomycin/K⁺ gradients to assess how potential impacts coupling efficiency. Kinetic analyses determining Vmax and Km values for NADH oxidation under varying ubiquinone concentrations can further reveal species-specific adaptations in catalytic efficiency that might correlate with ecological factors such as flight patterns or feeding strategies in this bat species.

What is the role of MT-ND4L in the assembly and stability of complex I, and how might this differ in Stenoderma rufum?

MT-ND4L plays a critical role in complex I assembly and stability through specific interactions with other membrane-embedded subunits. To investigate this in Stenoderma rufum, researchers should utilize siRNA knockdown of endogenous MT-ND4L in mammalian cell lines followed by complementation with the bat protein to assess rescue of complex I assembly . BN-PAGE combined with in-gel activity staining can reveal whether the bat protein supports formation of fully assembled, catalytically active complex I. Pulse-chase experiments with radiolabeled amino acids can track the kinetics of complex I assembly when incorporating the recombinant protein. Thermal stability assays using differential scanning fluorimetry can determine whether complexes containing bat MT-ND4L exhibit altered stability profiles compared to those with human MT-ND4L. Researchers should also examine MT-ND4L interaction with assembly factors using proximity-dependent biotin identification (BioID) or related approaches. These experiments may reveal adaptations in assembly pathways that could reflect evolutionary responses to metabolic demands or environmental stressors encountered by Stenoderma rufum in its natural habitat.

How does MT-ND4L contribute to ROS production in complex I, and what implications might this have for Stenoderma rufum's exceptional longevity?

MT-ND4L's position within complex I places it in proximity to sites of reactive oxygen species (ROS) production, potentially influencing electron leakage to oxygen. This question is particularly relevant for Stenoderma rufum given the exceptional longevity of many bat species despite high metabolic rates, which contradicts traditional aging theories . Researchers should measure ROS production in isolated mitochondria or purified complex I containing either bat or human MT-ND4L under various substrate conditions and inhibitor treatments. Amplex Red assays for H₂O₂ detection combined with specific superoxide detection using EPR spin-trapping can quantify ROS production rates. Site-directed mutagenesis of conserved residues unique to bat MT-ND4L can help identify amino acids that might contribute to altered ROS production. Researchers should compare ROS production during forward electron transport versus reverse electron transport, as these pathways generate different amounts of superoxide at distinct sites. Measurements under varying oxygen tensions can further reveal whether bat MT-ND4L contributes to oxygen affinity differences that might impact ROS generation under physiological conditions. These experiments may uncover adaptations that contribute to the unusual longevity-to-body mass ratio observed in bat species.

What mutations in MT-ND4L are associated with mitochondrial disorders, and how might studying Stenoderma rufum inform therapeutic approaches?

Mutations in MT-ND4L have been implicated in several mitochondrial disorders, including Leber hereditary optic neuropathy (LHON). A specific mutation, T10663C (Val65Ala), has been identified in several families with LHON, changing a single amino acid in the NADH dehydrogenase 4L protein . Comparing disease-associated residues between human and Stenoderma rufum MT-ND4L may reveal naturally occurring compensatory mechanisms in the bat protein that confer resilience against pathogenic effects. Researchers should introduce human disease mutations into recombinant bat MT-ND4L and assess their impact on complex I assembly, stability, and function compared to the same mutations in human MT-ND4L. Studies using cybrid cell lines containing mitochondria with either human or chimeric bat-human MT-ND4L can evaluate whether bat-specific sequence elements mitigate pathological phenotypes. Structural analysis may identify stabilizing interactions present in bat MT-ND4L that could inform protein engineering approaches for therapeutic interventions. These comparative studies could potentially reveal evolutionary adaptations in bat mitochondrial proteins that counteract the deleterious effects of mutations associated with human disease.

How does the A/D transition of complex I differ when incorporating Stenoderma rufum MT-ND4L, and what are the implications for ischemia-reperfusion injury models?

The active/deactive (A/D) transition represents a conformational change in complex I during prolonged ischemia, with the enzyme adopting a dormant state that slowly reactivates upon reoxygenation . This transition has significant implications for ischemia-reperfusion injury, as the deactive form produces more ROS upon reactivation. Researchers investigating Stenoderma rufum MT-ND4L should compare A/D transition kinetics between complexes containing bat versus human subunits using thermal inactivation protocols followed by activity recovery measurements. The impact of specific inhibitors like rotenone on the equilibrium between active and deactive forms should be assessed, as rotenone has been shown to shift the equilibrium toward the active complex I . Structural changes during the A/D transition can be monitored using hydrogen-deuterium exchange mass spectrometry or limited proteolysis approaches to identify regions with altered solvent accessibility. These experiments may reveal whether bat MT-ND4L contributes to different A/D transition properties that could correlate with resistance to ischemia-reperfusion injury, potentially reflecting adaptations to intermittent hypoxia experienced during diving or torpor in bat species.

How do post-translational modifications of MT-ND4L affect complex I function in normal versus pathological conditions?

Post-translational modifications (PTMs) of MT-ND4L can significantly impact complex I assembly, stability, and activity under both physiological and pathological conditions. Researchers should employ mass spectrometry-based proteomics to identify and quantify PTMs on Stenoderma rufum MT-ND4L compared to human MT-ND4L under normal conditions and oxidative stress . Phosphorylation, acetylation, and oxidative modifications are particularly relevant to investigate as they may regulate complex I activity in response to metabolic demands or stress. Site-directed mutagenesis of modified residues to either prevent modification (e.g., Ser to Ala for phosphorylation sites) or mimic constitutive modification (e.g., Ser to Asp) can reveal the functional significance of specific PTMs. Researchers should examine whether stress-induced modifications differ between bat and human proteins, potentially identifying protective mechanisms in the bat protein. Time-course experiments during cellular stress responses can track dynamic changes in PTM patterns. These studies may uncover regulatory mechanisms that contribute to the remarkable stress resistance observed in bat species and potentially inform therapeutic approaches targeting mitochondrial dysfunction in human disease.