Recombinant Macaca fascicularis NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial (NDUFB5)

What is NDUFB5 and what is its role in cellular metabolism?

NDUFB5 (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial) is a protein subunit of Complex I of the mitochondrial electron transport chain. As part of NADH dehydrogenase, it contributes to the largest of the five complexes in the respiratory chain. The protein is primarily located in the mitochondrial inner membrane and functions within the enzymatic framework that transfers electrons from NADH to ubiquinone, an essential step in cellular respiration and ATP production. NDUFB5 is specifically categorized as a non-catalytic, accessory subunit of Complex I, suggesting its role is more structural or regulatory rather than directly involved in the electron transfer reactions . Unlike the core subunits that contain the redox centers, NDUFB5 is part of the approximately 31 hydrophobic subunits that form the transmembrane region of Complex I, providing structural integrity to the complex within the mitochondrial membrane .

How does recombinant NDUFB5 differ structurally and functionally from the native protein?

Recombinant Macaca fascicularis NDUFB5 is produced using heterologous expression systems, primarily E. coli, and includes modifications that distinguish it from the native protein. These differences must be considered when designing experiments:

• The recombinant protein includes an N-terminal His-tag for purification purposes, which is not present in the native protein .

• The recombinant version typically represents only the mature protein sequence (amino acids 47-189), lacking the mitochondrial targeting sequence present in the nascent protein expressed in vivo .

• Post-translational modifications that may occur in the native mitochondrial environment are likely absent in the E. coli-expressed recombinant protein.

• The recombinant protein is isolated from its native complex partners, whereas in vivo it functions as an integrated component of the multiprotein Complex I assembly.

Despite these differences, the core structural elements of the protein remain preserved, making the recombinant form a valuable tool for studying protein-protein interactions, antibody generation, and structural studies. Researchers should acknowledge these distinctions when extrapolating in vitro findings to physiological contexts, particularly for functional studies where protein-complex assembly and integration into the mitochondrial membrane are critical considerations.

What are the optimal storage and reconstitution protocols for recombinant NDUFB5?

Proper storage and reconstitution of recombinant NDUFB5 are critical for maintaining protein integrity and experimental reproducibility. Based on manufacturer recommendations and standard protein handling practices, the following protocols should be implemented:

Storage Protocol:

• Upon receipt, store lyophilized NDUFB5 at -20°C or -80°C for long-term storage .

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles.

• Working aliquots may be stored at 4°C for up to one week .

• For extended storage of reconstituted protein, add glycerol to a final concentration of 50% and store at -20°C or -80°C .

Reconstitution Protocol:

• Briefly centrifuge the vial containing lyophilized protein prior to opening to ensure the material is at the bottom of the tube .

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Allow the protein to dissolve completely by gentle mixing; avoid vigorous shaking or vortexing.

• For downstream applications requiring buffer exchange, consider using a Tris-based buffer with pH 8.0 .

Following these protocols will help maintain protein stability and activity. It is important to note that repeated freeze-thaw cycles significantly decrease protein integrity, so single-use aliquots are strongly recommended for research applications requiring consistent protein quality .

How can researchers validate the purity and activity of recombinant NDUFB5?

Validating both the purity and functional activity of recombinant NDUFB5 requires a multi-faceted approach:

Purity Assessment Methods:

• SDS-PAGE analysis - Commercial recombinant NDUFB5 typically exhibits purity greater than 90% as determined by SDS-PAGE . Researchers should perform their own verification upon receipt.

• Western blotting - Using specific antibodies against NDUFB5 or the His-tag to confirm protein identity.

• Mass spectrometry - For precise molecular weight determination and sequence verification.

Functional Validation Approaches:

• Protein-protein interaction assays - Since NDUFB5 is part of Complex I, its ability to interact with other complex components can be assessed using pull-down assays.

• Secondary structure analysis - Circular dichroism spectroscopy to confirm proper protein folding compared to predicted secondary structure elements.

• Binding studies - If antibodies or ligands with known affinity for NDUFB5 are available, binding kinetics can be measured.

For researchers studying the protein in the context of complex I function, it's important to note that as a non-catalytic subunit, NDUFB5 itself doesn't possess enzymatic activity to measure directly . Instead, functional validation often focuses on its ability to incorporate into complex I assemblies or interact with known binding partners. When designing validation experiments, researchers should consider the recombinant protein's structural differences from the native form, particularly the presence of the His-tag and the absence of the native mitochondrial environment.

What experimental systems are most appropriate for studying NDUFB5 function?

Selecting the appropriate experimental system for studying NDUFB5 function depends on research objectives and specific questions being addressed. Several platforms offer distinct advantages:

Cell-Based Systems:

• Primate cell lines - Particularly those derived from Macaca fascicularis provide the most physiologically relevant context for studying the native functions of NDUFB5.

• Human cell lines with NDUFB5 knockdown/knockout - Can reveal the functional importance through loss-of-function phenotypes.

• Cell models of mitochondrial dysfunction - Allow investigation of NDUFB5's role in pathological contexts.

Reconstitution Systems:

• Liposome incorporation - Recombinant NDUFB5 can be incorporated into artificial membrane systems to study its membrane interactions.

• Complex I assembly assays - In vitro assembly systems to investigate NDUFB5's role in complex formation.

• Nanodiscs - Allow study of membrane proteins in a controlled, native-like lipid environment.

Structural Biology Approaches:

• Cryo-electron microscopy - Particularly useful for studying NDUFB5 in the context of the entire Complex I structure.

• X-ray crystallography - While challenging for membrane proteins, can provide high-resolution structural information.

• Nuclear magnetic resonance (NMR) - Suitable for studying dynamic interactions and conformational changes.

When selecting an experimental system, researchers should consider that NDUFB5 is a non-catalytic subunit of Complex I with primarily structural roles . Therefore, functional studies often benefit from approaches that examine protein-protein interactions, complex assembly, or membrane integration rather than direct enzymatic activity. Additionally, since the protein contains both hydrophobic transmembrane and hydrophilic domains, experimental systems that accommodate membrane proteins will provide more physiologically relevant insights.

How does NDUFB5 contribute to Complex I assembly and stability?

NDUFB5's contribution to Complex I assembly and stability represents a sophisticated area of investigation that extends beyond basic structural characterization. Current research suggests several key mechanisms:

NDUFB5 contains a highly conserved two-domain structure with an N-terminal hydrophobic domain that forms an alpha helix spanning the inner mitochondrial membrane and a C-terminal hydrophilic domain that interacts with globular subunits of Complex I . This architecture suggests NDUFB5 functions as an anchor point that helps stabilize the massive L-shaped structure of Complex I within the membrane.

The protein's strategic positioning within the transmembrane region allows it to form multiple contact points with neighboring subunits. The hydrophobic domain likely mediates interactions with other membrane-embedded subunits, while the hydrophilic C-terminal domain extends into the mitochondrial matrix where it can interact with the peripheral arm subunits .

Assembly studies indicate that NDUFB5, as part of the membrane-embedded portion of Complex I, is incorporated during the later stages of complex assembly. This suggests its role may be particularly important in the final stabilization and maturation of the holocomplex rather than in early assembly intermediates.

Researchers investigating NDUFB5's assembly contributions should consider using techniques such as:

• Blue native PAGE to track assembly intermediates

• Proximity labeling approaches to identify direct interaction partners

• Site-directed mutagenesis of conserved residues to identify regions critical for assembly

• Pulse-chase experiments to determine the temporal sequence of NDUFB5 incorporation into Complex I

Understanding these mechanisms provides insights into both normal respiratory chain function and the molecular basis of mitochondrial disorders associated with Complex I deficiency.

What are the implications of NDUFB5 mutations or dysfunction in mitochondrial diseases?

While specific mutations in NDUFB5 have not been extensively documented in the literature compared to other Complex I subunits, theoretical and comparative analyses suggest several potential implications for mitochondrial pathophysiology:

• Reduced NADH oxidation capacity

• Decreased electron transfer efficiency

• Impaired proton pumping across the inner mitochondrial membrane

• Increased reactive oxygen species (ROS) production

• Compromised ATP synthesis

The highly conserved nature of the NDUFB5 protein sequence across species suggests functional importance, with evolutionary pressure maintaining its structure . Mutations affecting the hydrophobic N-terminal domain could disrupt membrane anchoring, while alterations in the C-terminal hydrophilic domain might interfere with interactions with other Complex I subunits.

Research approaches to investigate NDUFB5-related dysfunction might include:

• Genetic screening of patients with unexplained Complex I deficiency

• CRISPR/Cas9-mediated gene editing to create cellular models with specific NDUFB5 mutations

• Functional complementation assays using recombinant NDUFB5 in deficient cell lines

• Proteomic analysis of altered protein-protein interactions in the presence of mutant NDUFB5

Researchers should note that since NDUFB5 is a non-catalytic accessory subunit, its dysfunction may produce more subtle phenotypes compared to mutations in core catalytic subunits, potentially contributing to the heterogeneity of mitochondrial disease presentations.

How can recombinant NDUFB5 be used to study protein-protein interactions within Complex I?

Recombinant NDUFB5 provides a valuable tool for dissecting the complex network of protein interactions within the mitochondrial respiratory Complex I. Several methodological approaches leverage this recombinant protein for interaction studies:

Affinity Purification Techniques:
The His-tagged recombinant NDUFB5 can be immobilized on nickel or cobalt affinity matrices to capture and identify interaction partners from mitochondrial lysates. This approach allows for:

• Identification of direct binding partners through pull-down assays

• Mapping of interaction domains using truncated versions of NDUFB5

• Quantitative assessment of binding affinities for different complex subunits

Cross-linking Mass Spectrometry (XL-MS):
Chemical cross-linking of recombinant NDUFB5 with mitochondrial fractions, followed by mass spectrometry analysis, can reveal:

• Spatial relationships between NDUFB5 and other Complex I components

• Identification of transient interactions not captured by standard pull-down approaches

• Structural constraints for molecular modeling of Complex I assembly

Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC):
These biophysical techniques using purified recombinant NDUFB5 enable:

• Determination of binding kinetics (kon and koff rates)

• Measurement of thermodynamic parameters (ΔH, ΔS, and ΔG)

• Quantitative comparison of wild-type vs. mutant interaction properties

Proximity Labeling Approaches:
Modified recombinant NDUFB5 (e.g., with BioID or APEX2 tags) can be expressed in cellular systems to:

• Map the protein's interactome in the native mitochondrial environment

• Identify dynamic changes in interaction partners under different cellular conditions

• Discover previously unknown associations with non-Complex I proteins

When designing these experiments, researchers should account for the non-catalytic nature of NDUFB5 and its dual-domain architecture. The hydrophobic transmembrane domain may require detergent solubilization or membrane mimetics to maintain proper folding and interaction potential. Additionally, the recombinant protein lacks the normal mitochondrial import sequence and may not reflect all post-translational modifications present in the native protein , potentially affecting certain interaction properties.

How does Macaca fascicularis NDUFB5 compare with human and other mammalian orthologs?

Comparative analysis of NDUFB5 across mammalian species reveals important insights into its evolutionary conservation and functional significance:

The Macaca fascicularis NDUFB5 protein shares high sequence homology with human NDUFB5, reflecting their close evolutionary relationship. This conservation extends across the protein's functional domains, particularly in regions critical for Complex I integration and function . The mature protein sequence (amino acids 47-189) used in recombinant protein production represents the most highly conserved region across primates.

Table 1: Comparison of NDUFB5 Across Selected Mammalian Species

*Note: The sequence for Sorex araneus NDUFB5 from search result appears to be a partial sequence of 130 amino acids (based on the ORF nucleotide sequence length of 390bp).

The high conservation of NDUFB5 across mammals suggests strong evolutionary pressure to maintain its structure and function, consistent with its important role in cellular energy metabolism. Particularly conserved are:

This conservation provides researchers with confidence that findings from Macaca fascicularis NDUFB5 studies can often be extrapolated to human systems with reasonable accuracy, making it a valuable model for investigating Complex I biology relevant to human health and disease .

What unique structural or functional features distinguish NDUFB5 from other Complex I subunits?

NDUFB5 possesses several distinguishing characteristics that set it apart from other subunits within Complex I of the respiratory chain:

Structural Distinctiveness:
Unlike the core subunits that contain redox centers (e.g., iron-sulfur clusters or FMN), NDUFB5 is a non-catalytic accessory subunit . Its structure features a distinctive two-domain architecture with:

• A hydrophobic N-terminal domain that forms a membrane-spanning alpha helix

• A hydrophilic C-terminal domain that extends from the membrane to interact with peripheral subunits

This hybrid architecture allows NDUFB5 to serve as a bridge between the membrane domain and peripheral arm of Complex I, potentially facilitating communication between these functionally distinct regions.

Positioning Within Complex I:
NDUFB5 is positioned within the transmembrane region of Complex I, which distinguishes it from:

• The peripheral arm subunits that contain the electron transfer components

• The matrix-facing subunits involved in NADH binding

• The intermembrane space-facing subunits

This strategic positioning suggests a role in maintaining the structural integrity of the membrane domain and potentially participating in the conformational changes that couple electron transfer to proton pumping.

Evolutionary Context:
While many core subunits of Complex I show homology to bacterial counterparts, NDUFB5 belongs to the set of accessory subunits that appeared later in evolution and are absent in prokaryotic systems. This evolutionary pattern suggests that NDUFB5 may have evolved to meet the specialized requirements of the more complex eukaryotic oxidative phosphorylation system, potentially providing regulatory functions not required in simpler organisms.

Understanding these unique features provides context for experimental design when targeting NDUFB5 specifically versus addressing Complex I function more broadly. Researchers should consider these distinctions when interpreting results from structural studies, functional assays, or comparative analyses involving NDUFB5 .

How can evolutionary analysis of NDUFB5 inform research approaches?

Evolutionary analysis of NDUFB5 offers valuable insights that can guide research strategies and experimental design:

Identification of Critical Functional Domains:
Comparative sequence analysis across species reveals regions under strong evolutionary constraint, indicating functional importance. For NDUFB5, the highly conserved two-domain structure suggests that both the membrane-spanning and peripheral interaction domains are critical for function . Researchers can use this information to:

• Target site-directed mutagenesis to highly conserved residues

• Design truncation experiments that preserve intact functional domains

• Focus structural studies on regions showing the highest conservation

Cross-Species Extrapolation Potential:
The high homology between Macaca fascicularis NDUFB5 and human NDUFB5 suggests that experimental findings using the recombinant macaque protein can likely be extrapolated to human systems with reasonable confidence . This enables:

• Validation of research findings across primate models

• Development of therapeutic approaches with higher translational potential

• Identification of species-specific adaptations by focusing on regions of sequence divergence

Table 2: Evolutionary Analysis Applications for NDUFB5 Research

By integrating evolutionary insights into experimental design, researchers can develop more targeted approaches to understanding NDUFB5 function. For example, focusing recombinant protein studies on the most highly conserved regions may yield insights with broader applicability across species, while examining species-specific variations may reveal adaptations to particular metabolic demands or environmental conditions .

What are the primary challenges in working with recombinant NDUFB5 and how can they be addressed?

Working with recombinant NDUFB5 presents several technical challenges common to membrane proteins, as well as unique issues specific to this protein:

Challenge 1: Maintaining Proper Protein Folding
NDUFB5 contains a hydrophobic transmembrane domain that can promote aggregation when expressed recombinantly . To address this:

Solutions:

• Use specialized E. coli strains designed for membrane protein expression

• Express the protein at lower temperatures (16-20°C) to slow folding and reduce aggregation

• Include mild detergents or lipid mimetics in purification buffers

• Consider chaperone co-expression systems to aid proper folding

Challenge 2: Preserving Stability During Storage and Handling
The protein may lose structural integrity during freeze-thaw cycles or extended storage .

Solutions:

• Store the lyophilized protein at -20°C or -80°C until needed

• Add 50% glycerol to reconstituted protein for extended storage

• Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week only

Challenge 3: Mimicking the Native Membrane Environment
As a membrane protein, NDUFB5 requires a lipid environment for proper structure and function.

Solutions:

• Reconstitute into liposomes or nanodiscs for functional studies

• Use non-denaturing detergents that maintain membrane protein structure

• Consider amphipols or other membrane-mimetic systems for structural studies

• Employ lipid-detergent mixed micelles for crystallization attempts

Challenge 4: Assessing Functional Activity
As a non-catalytic subunit, traditional enzymatic assays cannot directly measure NDUFB5 activity .

Solutions:

• Focus on protein-protein interaction assays to assess binding function

• Measure structural integrity through circular dichroism or thermal shift assays

• Use complementation assays in NDUFB5-deficient cell lines

• Assess integration into Complex I using blue native PAGE techniques

By acknowledging these challenges and implementing appropriate technical solutions, researchers can maximize the utility of recombinant NDUFB5 for investigating complex I biology and mitochondrial function.

How can researchers optimize experimental conditions for recombinant NDUFB5 studies?

Optimizing experimental conditions for recombinant NDUFB5 studies requires careful consideration of several factors that can significantly impact results:

Buffer Composition Optimization:
The choice of buffer can dramatically affect protein stability and functionality. For NDUFB5:

• Tris-based buffers at pH 8.0 have been demonstrated to maintain protein stability

• The addition of 6% trehalose helps preserve protein structure during storage

• For functional studies, buffers mimicking the mitochondrial environment (pH 7.2-7.4) may be more physiologically relevant

Protein Concentration Considerations:
The working concentration of recombinant NDUFB5 requires optimization based on the specific application:

• For reconstitution, 0.1-1.0 mg/mL is recommended as an initial concentration

• Higher concentrations may promote aggregation due to the hydrophobic domains

• For interaction studies, titration experiments should determine optimal protein ratios

Temperature and Time Parameters:
Experimental temperature can significantly impact NDUFB5 stability and interaction properties:

• Short-term studies can be conducted at room temperature or 37°C (physiological)

• For extended experiments, 4°C may better preserve protein integrity

• Thermal stability assays can help determine the optimal temperature range for specific applications

Detergent Selection for Membrane Protein Studies:
As a protein with a hydrophobic transmembrane domain, NDUFB5 may require detergents for solubilization:

• Mild non-ionic detergents (e.g., DDM, LMNG) are often suitable for initial trials

• Detergent concentration should be maintained above critical micelle concentration

• Detergent screening may be necessary to identify optimal conditions

Table 3: Optimization Parameters for Recombinant NDUFB5 Experiments

By systematically optimizing these parameters, researchers can develop robust experimental protocols that maximize the utility of recombinant NDUFB5 for investigating Complex I biology and mitochondrial function .

What advanced analytical techniques are most suitable for NDUFB5 characterization?

Advanced analytical techniques offer powerful approaches for comprehensive characterization of recombinant NDUFB5, providing insights into structure, interactions, and functional properties:

High-Resolution Structural Analysis:

• Cryo-Electron Microscopy (Cryo-EM): Particularly valuable for visualizing NDUFB5 within the context of the entire Complex I structure, revealing its spatial relationships with neighboring subunits without requiring crystallization.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Useful for analyzing specific domains of NDUFB5, particularly the more soluble C-terminal hydrophilic region, and for studying dynamic interactions with binding partners.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Provides information about protein folding, dynamics, and solvent accessibility of different regions, helping map interaction interfaces.

Interaction and Assembly Analysis:

• Surface Plasmon Resonance (SPR): Enables real-time, label-free measurement of binding kinetics between NDUFB5 and potential interaction partners from Complex I.

• Microscale Thermophoresis (MST): Allows determination of binding affinities in solution with minimal sample consumption, suitable for membrane protein studies.

• Blue Native PAGE with Western Blotting: Valuable for studying the incorporation of NDUFB5 into Complex I assemblies and identifying assembly intermediates.

Functional Characterization:

• Reconstitution into Proteoliposomes: Enables study of NDUFB5's role in maintaining Complex I structure and function within a membrane environment.

• Electron Paramagnetic Resonance (EPR) Spectroscopy: With site-directed spin labeling, can provide information about structural changes and conformational dynamics.

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET): Allows investigation of dynamic conformational changes that may occur during Complex I assembly or function.

Advanced Mass Spectrometry Approaches:

• Cross-linking Mass Spectrometry (XL-MS): Provides spatial constraints for modeling protein-protein interactions by identifying residues in close proximity.

• Native Mass Spectrometry: Can analyze intact protein complexes, providing insights into stoichiometry and stability of NDUFB5-containing assemblies.

• Targeted Proteomics (MRM/PRM): Enables precise quantification of NDUFB5 and associated proteins in complex biological samples.

When selecting analytical techniques, researchers should consider the specific research questions being addressed and the technical limitations of each method, particularly regarding membrane proteins like NDUFB5. Integration of multiple complementary techniques often provides the most comprehensive characterization by overcoming the limitations of individual methods.