Recombinant Pan troglodytes NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial (NDUFB11)

What is NDUFB11 and what is its role in mitochondrial function?

NDUFB11 is a subunit of NADH dehydrogenase (complex I) in the mitochondrial respiratory chain. It provides instructions for making a protein that is part of this important enzyme, which is the largest complex in the cell energy-producing system. NADH dehydrogenase helps cells produce energy and is located in the mitochondria, the cell's energy "powerhouse." It is a critical component of the electron transport chain, assisting in the generation of ATP through oxidative phosphorylation .

The protein primarily functions as part of the membrane domain of complex I and is essential for proper complex assembly and stability. In research contexts, understanding NDUFB11's structure-function relationship is crucial for investigating mitochondrial energy production mechanisms and associated disorders.

How conserved is NDUFB11 between humans and Pan troglodytes?

NDUFB11 shows high conservation between humans and chimpanzees, reflecting the protein's essential role in mitochondrial function. Sequence alignment studies demonstrate approximately 98-99% amino acid identity between the two species, with most variations occurring in non-critical regions of the protein. This high conservation makes Pan troglodytes NDUFB11 a valuable model for understanding human mitochondrial complex I function.

When designing experiments, researchers should note that despite this high conservation, small sequence differences may affect antibody binding specificity, protein-protein interactions, or post-translational modifications. Comparative analyses of functional domains should be conducted to ensure experimental validity when translating findings between species.

What are the primary structural characteristics of NDUFB11?

NDUFB11 is a relatively small protein (approximately 122 amino acids in its mature form) characterized by:

• A transmembrane domain that anchors it to the inner mitochondrial membrane

• Specific interaction sites with other complex I subunits

• Post-translational modification sites that may regulate its function

• A mature protein sequence (in mouse) spanning amino acids 30-151 after processing of the mitochondrial targeting sequence

For experimental design purposes, researchers should consider these structural features when designing constructs, especially when adding tags that might interfere with membrane integration or protein-protein interactions within complex I.

What are the optimal conditions for expressing recombinant Pan troglodytes NDUFB11 in bacterial systems?

For bacterial expression of recombinant Pan troglodytes NDUFB11:

• Use E. coli strains optimized for membrane protein expression (such as C41(DE3) or C43(DE3))

• Express the mature protein (without mitochondrial targeting sequence) to improve solubility

• Consider fusion tags that enhance solubility (such as MBP or SUMO) in addition to affinity tags (His)

• Use lower induction temperatures (16-18°C) to slow protein production and improve folding

• Include mild detergents in lysis buffers to solubilize the membrane-associated protein

A typical expression protocol would involve:

• Transformation into appropriate E. coli strain

• Growth at 37°C to OD600 of 0.6-0.8

• Temperature reduction to 18°C before induction

• Induction with 0.1-0.5 mM IPTG

• Extended expression period (16-20 hours)

• Harvesting and lysis in detergent-containing buffer

Researchers should perform small-scale optimization experiments to determine ideal conditions for their specific construct, as membrane proteins like NDUFB11 often require customized protocols .

What are the most effective methods for purifying recombinant Pan troglodytes NDUFB11?

Purification of recombinant NDUFB11 requires special consideration due to its hydrophobic nature:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tag

  • Buffer composition: PBS-based with 0.1-0.5% mild detergent (DDM or LDAO)

  • Include 6% trehalose as a stabilizing agent

  • pH 8.0 is optimal for maintaining protein stability

• Secondary purification: Size exclusion chromatography

  • Remove aggregates and contaminants

  • Assess oligomeric state of the protein

• Quality control assessments:

  • SDS-PAGE to confirm >90% purity

  • Western blot with anti-NDUFB11 antibodies

  • Mass spectrometry for identity confirmation

For storage, add glycerol (30-50% final concentration) and store in small aliquots at -80°C to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity .

How can researchers effectively assess the quality and functionality of purified recombinant NDUFB11?

A comprehensive quality assessment should include:

Structural Integrity Assays:

• Circular dichroism to assess secondary structure

• Limited proteolysis to confirm proper folding

• Thermal shift assays to determine stability

Functional Assays:

• Complex I activity measurements using NADH oxidation assays

• Binding studies with known interaction partners

• Membrane integration assessment using liposome reconstitution

Activity Comparison Table:

Researchers should compare results with those obtained from native mitochondrial preparations to benchmark functionality of the recombinant protein .

How can researchers use recombinant Pan troglodytes NDUFB11 to study mitochondrial complex I assembly?

To investigate complex I assembly using recombinant NDUFB11:

• Reconstitution experiments:

  • Introduce labeled recombinant NDUFB11 into isolated mitochondria with depleted endogenous NDUFB11

  • Track incorporation using blue native PAGE and immunodetection

  • Assess assembly intermediate formation by time-course analysis

• Interaction studies:

  • Use pull-down assays with tagged recombinant NDUFB11 to identify binding partners

  • Compare interactomes between normal and pathogenic variants

  • Quantify binding affinities with key complex I subunits

• Structural analysis:

  • Incorporate recombinant NDUFB11 into partial complex I assemblies for cryo-EM studies

  • Map the position and orientation of NDUFB11 within the complex

  • Identify critical interfaces with other subunits

These approaches can reveal the step-wise assembly process of complex I and NDUFB11's role within it. Western blot analysis using antibodies against subunits from different complex I modules (N, Q, and P) can help determine which modules are affected by NDUFB11 dysfunction .

What are the best approaches for studying disease-associated variants of NDUFB11 using the Pan troglodytes model?

To study disease-associated variants using Pan troglodytes NDUFB11:

• Site-directed mutagenesis:

  • Introduce known pathogenic variants (e.g., those causing mitochondrial complex I deficiency or cardiomyopathy) into the Pan troglodytes NDUFB11 sequence

  • Use PCR-based methods with high-fidelity polymerases to minimize unwanted mutations

• Functional characterization:

  • Compare biochemical properties of wild-type and mutant proteins

  • Assess impacts on complex I assembly and activity

  • Measure ROS production and membrane potential in reconstituted systems

• Cell-based assays:

  • Introduce wild-type or mutant NDUFB11 into NDUFB11-knockout cell lines

  • Measure rescue effects on mitochondrial function

  • Analyze transcriptional and metabolic changes using omics approaches

• Comparative analysis between species:

  • Compare effects of equivalent mutations in human, chimpanzee, and mouse NDUFB11

  • Identify species-specific differences in pathogenicity

Researchers should pay particular attention to splicing variants, as evidenced by the case of neonatal lethal cardiomyopathy where a variant caused aberrant splicing rather than the predicted amino acid change .

How can researchers investigate the impact of NDUFB11 deficiency on mitochondrial function?

To comprehensively assess NDUFB11 deficiency effects:

• Protein expression analysis:

  • Quantify levels of NDUFB11 protein via Western blot

  • Assess impacts on other complex I subunits (NDUFA9, NDUFB8, NDUFV1)

  • Evaluate effects on other respiratory chain complexes (II-V)

• Respiratory chain analysis:

  • Measure oxygen consumption rates in intact and permeabilized cells

  • Determine complex I-specific activity using substrate-specific approaches

  • Assess electron transfer between complexes

• Mitochondrial dynamics assessment:

  • Analyze mitochondrial network morphology

  • Quantify mitochondrial membrane potential

  • Measure mitochondrial turnover and biogenesis markers

Expected Findings in NDUFB11 Deficiency:

These comprehensive analyses can help distinguish primary effects of NDUFB11 deficiency from secondary adaptations .

How does NDUFB11 expression and function differ across tissues in Pan troglodytes?

While comprehensive Pan troglodytes tissue-specific data is limited, extrapolation from human and mouse studies suggests:

• Expression patterns:

  • Highest expression in heart, brain, and skeletal muscle

  • Moderate expression in liver and kidney

  • Lower expression in other tissues

• Tissue-specific vulnerabilities:

  • Cardiac tissue shows particular sensitivity to NDUFB11 dysfunction

  • Neurological tissues demonstrate high susceptibility to complex I defects

  • Skeletal muscle exhibits biochemical abnormalities but variable clinical manifestations

Researchers investigating tissue-specific effects should consider:

• Using tissue-specific cell lines or primary cells from relevant tissues

• Comparing mitochondrial parameters across different cell types

• Correlating expression levels with functional outcomes

Tissue-specific isoforms or post-translational modifications may explain differential sensitivity to NDUFB11 dysfunction across tissues, warranting investigation of these factors in comparative studies .

How can researchers evaluate the impact of NDUFB11 variants on splicing and transcript processing?

As demonstrated in cases of NDUFB11-related disorders, variants can affect splicing rather than directly altering protein structure:

• Transcript analysis methodologies:

  • RT-PCR with primers spanning exon-exon junctions

  • Quantitative PCR to measure relative abundance of transcript variants

  • RNA-seq to identify novel splicing events and quantify isoform expression

• Minigene assays:

  • Clone genomic segments containing exons and introns of interest into expression vectors

  • Introduce variants using site-directed mutagenesis

  • Transfect into relevant cell lines and analyze transcript patterns

• In silico prediction tools:

  • Use splicing prediction algorithms to assess potential impacts of variants

  • Validate computational predictions with experimental data

Important considerations:

• Variants at exon boundaries (as in the case of c.338G>A) require particular attention

• Alternative transcripts may represent non-functional, unprocessed intermediates

• Tissue-specific splicing factors may influence variant effects

Researchers should examine both "short" and "long" transcripts, as demonstrated in the study of neonatal lethal cardiomyopathy where the canonical short transcript was essential for proper protein synthesis .

How can researchers integrate proteomic and transcriptomic approaches to study NDUFB11 function?

An integrated multi-omics approach provides comprehensive insights into NDUFB11 function:

• Coordinated experimental design:

  • Collect matched samples for both proteomic and transcriptomic analyses

  • Include time-course measurements when studying dynamic processes

  • Incorporate wild-type controls, NDUFB11-deficient samples, and rescue conditions

• Transcriptomic analyses:

  • RNA-seq to identify differentially expressed genes

  • Alternative splicing analysis to detect transcript variants

  • miRNA profiling to identify post-transcriptional regulators

• Proteomic analyses:

  • Quantitative proteomics to measure changes in protein abundance

  • Post-translational modification analysis

  • Protein-protein interaction studies using proximity labeling or co-immunoprecipitation

• Integrative analysis:

  • Correlation between transcript and protein levels for mitochondrial genes

  • Pathway enrichment analysis combining both datasets

  • Network analysis to identify regulatory hubs

Data Integration Table:

This integrated approach can reveal compensatory mechanisms and regulatory networks in response to NDUFB11 dysfunction .

What approaches can be used to investigate the evolutionary conservation of NDUFB11 function across primates?

To study evolutionary aspects of NDUFB11:

• Sequence analysis:

  • Perform multiple sequence alignment of NDUFB11 across primates and other mammals

  • Calculate evolutionary rates (dN/dS) to identify conserved functional domains

  • Map disease-associated variants onto evolutionary conservation patterns

• Structural comparisons:

  • Model species-specific NDUFB11 structures using homology modeling

  • Compare binding interfaces with other complex I subunits

  • Identify species-specific structural features

• Functional conservation testing:

  • Express NDUFB11 from different species in NDUFB11-deficient human cells

  • Measure complementation efficiency across species

  • Identify species-specific functional differences

• Regulatory element analysis:

  • Compare promoter and enhancer regions across species

  • Identify conserved transcription factor binding sites

  • Analyze species-specific regulatory mechanisms

This evolutionary perspective can provide insights into the fundamental versus adaptable aspects of NDUFB11 function and identify which regions might tolerate engineering for research applications.

What are the emerging technologies that may advance NDUFB11 research?

Several cutting-edge approaches show promise for advancing NDUFB11 research:

• Cryo-electron microscopy:

  • High-resolution structural analysis of NDUFB11 within intact complex I

  • Visualization of conformational changes during catalytic cycles

  • Structural effects of disease-associated variants

• CRISPR-based approaches:

  • Precise genome editing to create isogenic cell lines with NDUFB11 variants

  • CRISPRi/CRISPRa for controlled modulation of NDUFB11 expression

  • Base editing for precise introduction of disease-associated mutations

• Organoid and tissue-specific models:

  • Brain and cardiac organoids to study tissue-specific effects

  • Patient-derived iPSCs differentiated into relevant cell types

  • Tissue-specific knockout models in appropriate organisms

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell-specific responses

  • Spatial transcriptomics to map expression in complex tissues

  • Single-cell proteomics to measure protein-level changes

These technologies will enable more precise characterization of NDUFB11's role in mitochondrial function and disease pathogenesis, potentially leading to therapeutic strategies for NDUFB11-related disorders .

How can findings from NDUFB11 research be translated to therapeutic applications?

Translational potential of NDUFB11 research includes:

• Diagnostic applications:

  • Development of functional assays to assess variants of uncertain significance

  • Biomarker identification for early detection of NDUFB11-related disorders

  • Prenatal and preimplantation genetic testing protocols

• Therapeutic strategies:

  • Gene therapy approaches for X-linked NDUFB11 deficiency

  • Antisense oligonucleotides to correct splicing defects

  • Small molecule screens to identify compounds that stabilize complex I or enhance residual activity

• Personalized medicine approaches:

  • Variant-specific therapeutic strategies

  • Tissue-targeted interventions based on expression patterns

  • Metabolic modifications to bypass complex I deficiency

Researchers should consider potential therapeutic applications early in study design, including the collection of relevant data on tissue specificity, biochemical defects, and mechanistic insights that could inform intervention strategies .