Recombinant Mesophylla macconnelli NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the basic structure of Mesophylla macconnelli MT-ND4L?

Mesophylla macconnelli MT-ND4L is a mitochondrial-encoded protein consisting of 98 amino acids with a molecular weight of approximately 11 kDa . The complete amino acid sequence is: MSITYMN MFMAFTISLLGLLMYRSHMMSSLCLEGM mLSLFVMMTMAILNTHLTLASMIP IILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC . This highly hydrophobic protein is encoded by the mitochondrial genome and forms part of the core transmembrane region of Complex I in the electron transport chain . The protein's hydrophobic nature reflects its functional role in the mitochondrial inner membrane.

What is the cellular function of MT-ND4L in Mesophylla macconnelli?

MT-ND4L functions as a subunit of NADH dehydrogenase (ubiquinone), also known as Complex I, which is located in the mitochondrial inner membrane . As part of the largest complex in the electron transport chain, MT-ND4L contributes to the process of oxidative phosphorylation by helping to transfer electrons from NADH to ubiquinone (Coenzyme Q10) . The protein participates in establishing the proton gradient across the mitochondrial inner membrane, which is essential for ATP synthesis . In Mesophylla macconnelli, MT-ND4L likely plays a similar role as in other mammals, contributing to cellular energy production through its involvement in the respiratory chain.

How does MT-ND4L interact with other subunits within Complex I?

MT-ND4L works in conjunction with six other mitochondrially encoded subunits (MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, and MT-ND6) to form the core of Complex I's membrane domain . These mitochondrially encoded subunits are the most hydrophobic components of Complex I. Within the L-shaped structure of Complex I, MT-ND4L is positioned in the transmembrane domain, while the hydrophilic peripheral arm contains the redox centers and NADH binding site . An interesting feature in the human homolog (and potentially conserved in Mesophylla macconnelli) is the 7-nucleotide gene overlap between MT-ND4L and MT-ND4, suggesting tight co-regulation of these two components . This close association indicates functional interdependence between these subunits in the proton-pumping mechanism.

What are the optimal conditions for expressing recombinant Mesophylla macconnelli MT-ND4L in experimental systems?

When expressing recombinant Mesophylla macconnelli MT-ND4L, researchers should consider several critical factors. Due to its highly hydrophobic nature as a transmembrane protein, standard expression systems may yield poor results. For optimal expression, consider using specialized membrane protein expression systems such as E. coli strains C41(DE3) or C43(DE3) specifically designed for membrane proteins. Expression should be performed at lower temperatures (16-25°C) to allow proper folding . Including detergents like n-dodecyl β-D-maltoside (DDM) in the lysis buffer will aid in solubilization. When working with this protein, store aliquots at -20°C or -80°C in a Tris-based buffer with 50% glycerol to maintain stability, and avoid repeated freeze-thaw cycles as indicated by product guidelines .

What purification strategies are most effective for isolating recombinant MT-ND4L while maintaining its native conformation?

Purification of recombinant MT-ND4L presents significant challenges due to its hydrophobic nature and relatively small size (98 amino acids, 11 kDa) . A multi-step purification strategy is recommended. Begin with affinity chromatography using an appropriate tag (His-tag is commonly employed for recombinant MT-ND4L). Throughout purification, maintain detergent concentrations above the critical micelle concentration to prevent protein aggregation. Size exclusion chromatography as a final polishing step can separate properly folded protein from aggregates. When analyzing purification efficiency, use specialized SDS-PAGE conditions optimized for small hydrophobic proteins, such as higher percentage gels (15-20%) and modified sample preparation to prevent aggregation during electrophoresis. Western blotting with antibodies specific to MT-ND4L or to the expression tag provides confirmation of successful purification.

How can researchers effectively design functional assays to measure MT-ND4L activity in vitro?

Measuring MT-ND4L activity requires assessing its contribution to Complex I function, as the protein alone does not possess catalytic activity. Design assays that measure NADH:ubiquinone oxidoreductase activity using spectrophotometric methods to track NADH oxidation at 340 nm . For more detailed functional analysis, researchers should consider reconstitution systems where purified MT-ND4L is incorporated into liposomes alongside other Complex I components. Measure proton-pumping activity using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine). Alternatively, employ oxygen consumption measurements using high-resolution respirometry to assess the impact of recombinant MT-ND4L on mitochondrial respiration. When designing these assays, include appropriate controls such as known inhibitors of Complex I (rotenone) and samples lacking MT-ND4L to distinguish specific effects from background activity.

How conserved is MT-ND4L across bat species compared to other mammals?

MT-ND4L shows significant evolutionary conservation, particularly at functionally critical positions. In Mesophylla macconnelli, the protein maintains the core functional domains seen across mammals, though with species-specific variations . Comparative analysis reveals that position 71 (where the A71T mutation occurs in some species) is highly conserved (86% in eukaryotes, 97% in mammals, and invariant in primates) . This high degree of conservation suggests functional importance. When analyzing conservation patterns across bat species, researchers should focus on regions involved in proton pumping and interactions with other Complex I subunits. Some positions show bat-specific conservation patterns that may reflect adaptations to the high metabolic demands associated with flight. Phylogenetic analysis using MT-ND4L sequences from multiple bat species, including Mesophylla macconnelli, can provide insights into evolutionary relationships and selection pressures on mitochondrial genes.

What specific sequence variations exist in Mesophylla macconnelli MT-ND4L compared to other bat species?

The MT-ND4L protein in Mesophylla macconnelli shows several specific variations compared to other bat species that may influence its functional properties. While maintaining the core structure necessary for Complex I function, these variations potentially reflect adaptations to the ecological niche of MacConnell's bat . Molecular analysis using the mitochondrial gene region composed of ND3, tRNA for arginine, ND4L, and ND4 has been employed to study phylogenetic relationships among bat species including Mesophylla macconnelli . When investigating these variations, researchers should employ restriction enzyme mapping techniques using enzymes such as AatII, AluI, BstUI, BstZ17I, DpnII, HaeIII, HhaI, HpaII, NdeI, NlaIII, PstI, RsaI, and TaqI to survey mtDNA variation . These analyses can help identify regions of divergence that may correlate with functional adaptations specific to Mesophylla macconnelli's metabolic requirements.

How do mutations in MT-ND4L affect Complex I function and what methodologies best detect these effects?

Mutations in MT-ND4L can significantly impact Complex I function, as demonstrated by studies of the m.10680G>A variant, which induces the amino acid change p.A71T in ND4L . To detect these effects, researchers should employ multiple complementary approaches. Begin with respiratory chain enzyme activity measurements comparing wild-type and mutant MT-ND4L using spectrophotometric assays that measure NADH oxidation rates. Cellular oxygen consumption rates (OCR) analysis using high-resolution respirometry provides crucial data on the functional impact of mutations . Research has shown that cells carrying the m.10680G>A/MT-ND4L variant displayed defective respiration with similar magnitude as Leber's Hereditary Optic Neuropathy (LHON) cells . For in-depth analysis, utilize cybrid cell technology, where patient-derived mitochondria are introduced into mtDNA-depleted recipient cells, allowing isolation of the effects of specific mtDNA mutations. This approach has successfully demonstrated that the m.10680G>A/MT-ND4L variant in isolation causes respiratory defects comparable to known pathogenic mutations .

How can researchers effectively model the impact of MT-ND4L variants on proton pumping in Complex I?

Modeling the impact of MT-ND4L variants on proton pumping requires sophisticated experimental and computational approaches. Experimental analysis should employ proteoliposome reconstitution systems where purified Complex I components including wild-type or variant MT-ND4L are incorporated into artificial membrane vesicles . Measure proton pumping efficiency using pH-sensitive fluorescent dyes while monitoring NADH oxidation rates. Computational modeling approaches should incorporate structural data on Complex I, particularly focusing on the putative E-channel that may be affected by MT-ND4L variants . Research has shown that variants affecting positions around this E-channel may alter proton pumping efficiency, potentially explaining the mild functional defect observed in patients with certain combinations of mtDNA variants . For accurate structure-function analysis, consider the position of the variant within the protein and its potential effect on interactions with other subunits or membrane lipids. Document changes in proton/electron ratios as a key measure of Complex I efficiency when altered by MT-ND4L variants.

What techniques are most effective for investigating how combinations of polymorphic MT-ND4L variants contribute to disease phenotypes?

Investigating how combinations of polymorphic MT-ND4L variants contribute to disease phenotypes requires a multi-dimensional approach. Evidence suggests that unusual combinations of otherwise polymorphic and non-pathogenic mtDNA missense mutations may be sufficient for causing conditions such as low-penetrance maternally inherited optic neuropathy . To study these effects, researchers should perform complete mtDNA sequencing to identify all potentially interacting variants. Cybrid cell studies isolate the mitochondrial contribution to cellular phenotypes and can demonstrate how variant combinations affect respiration, ATP production, and reactive oxygen species generation . Patient-derived fibroblasts or induced pluripotent stem cells (iPSCs) differentiated into relevant cell types (e.g., retinal ganglion cells for optic neuropathy models) provide physiologically relevant cellular models. When analyzing results, consider that while individual variants may appear neutral by prediction tools, their combination may disrupt critical functions such as proton pumping through the E-channel of Complex I . Document all experimental conditions meticulously, as subtle environmental factors may influence the expression of these combinatorial effects.

What specialized spectroscopic techniques can reveal structural insights about recombinant MT-ND4L?

Advanced spectroscopic techniques offer valuable structural insights into recombinant MT-ND4L despite challenges posed by its small size and hydrophobicity. Circular dichroism (CD) spectroscopy can assess secondary structure content, particularly the alpha-helical transmembrane domains characteristic of MT-ND4L . For detailed structural analysis, nuclear magnetic resonance (NMR) spectroscopy is particularly suitable for this 11 kDa protein when expressed with isotope labeling (15N, 13C). When preparing samples for these analyses, optimize detergent conditions to maintain native-like folding while minimizing spectral interference. Fourier-transform infrared spectroscopy (FTIR) provides complementary data on secondary structure elements in membrane environments. For probing structural dynamics, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify solvent-accessible regions and conformational changes upon interaction with other Complex I subunits. These techniques collectively provide a comprehensive view of MT-ND4L structure that cannot be achieved through any single method.

How can researchers effectively study the interaction between MT-ND4L and other Complex I subunits?

Studying interactions between MT-ND4L and other Complex I subunits requires specialized techniques suitable for membrane protein complexes. Co-immunoprecipitation (Co-IP) using antibodies against MT-ND4L or potential interacting partners can identify protein-protein interactions, though this requires careful optimization of detergent conditions to maintain interactions while solubilizing membrane proteins . Crosslinking mass spectrometry (XL-MS) provides detailed information about spatial relationships between subunits by covalently linking proteins in close proximity before mass spectrometric analysis. For mapping the interaction interface, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions that show altered solvent accessibility when MT-ND4L interacts with binding partners. Functional complementation assays, where mutant phenotypes are rescued by co-expression of interacting proteins, provide evidence of physiologically relevant interactions. When designing these experiments, consider the unique overlap between MT-ND4L and MT-ND4 genes, which suggests intimate functional relationships between these subunits .

What methodological approaches are most appropriate for using MT-ND4L in phylogenetic studies of Neotropical bats?

MT-ND4L serves as a valuable genetic marker for phylogenetic studies of Neotropical bats, including Mesophylla macconnelli. For comprehensive phylogenetic analysis, researchers should employ a combined gene approach using the mitochondrial gene region that includes ND3, tRNA for arginine, ND4L, and ND4 (approximately 2,400 base pairs) . DNA extraction from tissue samples must be optimized for the preservation method used (ethanol, RNAlater, or frozen tissue). For PCR amplification, use primers such as LGL 772 and 773 that target this region . Restriction enzyme mapping with multiple enzymes (AatII, AluI, BstUI, BstZ17I, DpnII, HaeIII, HhaI, HpaII, NdeI, NlaIII, PstI, RsaI, and TaqI) provides an effective method for surveying mtDNA variation . For phylogenetic analysis, employ both maximum parsimony and Bayesian inference methods, and assess branch support through bootstrap and jackknife analyses with 1,000 replications each, calculating decay indices using a converse constraints approach . Carefully select outgroups, such as Chiroderma trinitatum, C. villosum, and Ectophylla alba, to provide appropriate evolutionary context.

How do variations in MT-ND4L correlate with the evolutionary adaptations in bat echolocation systems?

The relationship between MT-ND4L variations and evolutionary adaptations in bat echolocation systems represents an intriguing area of research at the intersection of molecular evolution and sensory biology. Mitochondrial genes, including MT-ND4L, are critical for cellular energy production, which is especially important in metabolically demanding activities such as echolocation . Comparative analysis of MT-ND4L sequences across bat species with different echolocation strategies can reveal selection patterns potentially linked to energetic demands. When conducting such studies, researchers should employ phylogenetic comparative methods with echolocation call data from multiple bat families . Analysis should control for phylogenetic non-independence using methods such as phylogenetic generalized least squares (PGLS) or phylogenetic independent contrasts (PIC). Interestingly, research suggests that some of the most rapidly diversifying bat clades are those where species either do not use echolocation (Pteropodidae) or where less sensory reliance is placed on echolocation . This pattern may reflect metabolic trade-offs that could be visible at the molecular level in genes like MT-ND4L.