Recombinant Ubiquinol oxidase subunit 2 (cyaB)
Description
Introduction to Ubiquinol Oxidase Subunit 2 (cyaB)
Ubiquinol oxidase subunit 2, encoded by the cyaB gene, is a membrane protein that belongs to the cytochrome c oxidase subunit 2 family . This protein constitutes a vital component of the respiratory chain in various prokaryotic organisms, particularly in bacteria such as Acetobacter aceti . Within the context of cellular function, cyaB participates in electron transport chains and contributes to the generation of proton motive force, a fundamental process in energy metabolism.
The recombinant form of cyaB represents a bioengineered version of this protein, produced through molecular cloning and heterologous expression systems. This approach allows for enhanced production, purification, and study of the protein outside its native cellular environment, providing valuable insights into its structural and functional characteristics.
Monomeric and Dimeric Forms
One of the significant findings from recent structural studies is that ubiquinol oxidases can exist in both monomeric and dimeric forms. The cryo-EM structure of E. coli cytochrome bo3 ubiquinol oxidase revealed that this protein can form dimers with C2 symmetry . The dimerization interface is primarily formed between subunit II of one monomer and subunit IV of another monomer, with a large channel between the two monomers .
Table 1: Comparison of Monomeric and Dimeric Forms of Ubiquinol Oxidase
| Feature | Monomeric Form | Dimeric Form |
|---|---|---|
| Symmetry | N/A | C2 symmetry |
| Buried Interface Area | N/A | ~556 Ų (~0.6% of total surface area) |
| Interface Nature | N/A | Predominantly hydrophobic |
| Key Structural Changes | Reference state | Movement of loop in subunit IV (residues 67-74) by ~12 Å |
| Resolution of Structural Data | 3.15 Å | 3.46 Å |
The dimerization appears to induce minimal structural changes in each monomer, with the most significant difference being the movement of a loop in subunit IV (residues 67-74) . This loop shifts by approximately 12 Å to accommodate the dimer formation, suggesting a potential functional relevance for the dimeric state.
Role in Respiratory Chain
Ubiquinol oxidase subunit 2 plays a crucial role in the respiratory chain by facilitating the oxidation of ubiquinol and the reduction of oxygen to water . This process is coupled to proton translocation across the membrane, contributing to the formation of a proton gradient that drives ATP synthesis.
In the E. coli system, cytochrome bo3 ubiquinol oxidase functions as a proton pump in the aerobic respiratory chain . The enzyme contains a binding site for ubiquinone-8 (UQ8), which serves as the substrate for the oxidation reaction. The isoprene side chain of UQ8 is positioned within a hydrophobic groove in subunit I, stabilized by a transmembrane helix (TM0) that is specific to quinol oxidases and absent in cytochrome c oxidases .
Enzymatic Activities
In some bacterial systems, proteins encoded by cyaB genes function as adenylyl cyclases that synthesize cyclic adenosine 3′,5′-monophosphate (cAMP) . In Pseudomonas aeruginosa, CyaB belongs to the Class III adenylyl cyclase family and serves as a key regulator of virulence . The activity of this enzyme is stimulated by inorganic carbon and regulated by its N-terminal MASE2 domain, which acts as a membrane anchor .
Studies have identified activating mutations in CyaB that enhance cAMP reporter activity, providing insights into the regulatory mechanisms of this enzyme . These findings have potential implications for understanding bacterial pathogenesis and developing therapeutic strategies.
Expression Systems
Recombinant Ubiquinol oxidase subunit 2 can be produced using various expression systems, including:
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Escherichia coli
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Yeast
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Baculovirus-infected insect cells
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Mammalian cell lines
The choice of expression system depends on the specific requirements for protein folding, post-translational modifications, and downstream applications. The bacterial expression system (E. coli) is commonly used due to its simplicity, cost-effectiveness, and high yield, particularly for functional and structural studies .
Purification Methods
The purification of recombinant cyaB typically involves a multi-step process to achieve high purity. The protein can be purified using:
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Affinity chromatography (utilizing tags incorporated into the recombinant protein)
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Ion exchange chromatography
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Size exclusion chromatography
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Hydrophobic interaction chromatography
For membrane proteins like cyaB, detergents or amphipols are often used to maintain solubility during purification. Recent structural studies have utilized styrene-maleic acid copolymer (SMA) nanodiscs and membrane scaffold protein (MSP) nanodiscs to stabilize the protein in a native-like membrane environment .
Quality Control
The purity of recombinant cyaB is typically assessed by SDS-PAGE, with commercial preparations achieving greater than or equal to 85% purity . Additional quality control measures may include:
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Western blotting to confirm protein identity
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Mass spectrometry for precise molecular weight determination
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Circular dichroism spectroscopy to assess secondary structure
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Functional assays to verify enzymatic activity
Functional Investigations
Functional studies utilizing recombinant cyaB have explored various aspects of its activity, including:
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Catalytic mechanisms of ubiquinol oxidation
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Proton translocation pathways across the membrane
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Regulation of enzyme activity by environmental factors
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Identification of activating mutations that enhance enzyme function
In P. aeruginosa, studies on CyaB have revealed that it belongs to the Class IIIb subfamily of adenylyl cyclases and is stimulated by inorganic carbon . The enzyme is regulated by its N-terminal MASE2 domain, which serves as a membrane anchor and influences catalytic activity .
Inhibition Studies
Recombinant cyaB has been used to study the inhibition of ubiquinol oxidases by various compounds, which may have potential therapeutic applications:
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Aurachin D, a specific inhibitor of quinol oxidation by bd oxidases
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Identification of selective inhibitors for CyaB, which exhibit a unique inhibition profile compared to other adenylyl cyclases
These inhibition studies provide insights into the design of selective inhibitors that could target specific bacterial enzymes without affecting mammalian counterparts.
Table 2: Key Structural and Functional Studies on cyaB and Related Ubiquinol Oxidases
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during the production process. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Q&A
What is the structural composition of cytochrome bo3 ubiquinol oxidase?
Cytochrome bo3 ubiquinol oxidase in Escherichia coli is a four-subunit heme-copper oxidase that serves as a terminal oxidase in the bacterial aerobic respiratory chain. The enzyme contains two heme groups - heme b and heme o3 - and functions as a proton pump. Recent cryo-EM studies have revealed that this enzyme can exist in both monomeric and dimeric forms, with structures determined to resolutions of 3.15 Å and 3.46 Å, respectively . The dimer exhibits C2 symmetry with the dimerization interface maintained through interactions between subunit II of one monomer and subunit IV of the other monomer .
How do the monomeric and dimeric structures of ubiquinol oxidase differ?
The monomeric subunit in the ubiquinol oxidase dimer structure is remarkably similar to the isolated monomer structure, with a main chain RMSD of 0.624 Å based on alignment of 1204 residues . The most significant structural difference is located in a loop in subunit IV (residues 67–74) . In the monomeric state, this loop swings outward to the protein surface, while in the dimeric state, subunit II of the second monomer forces this loop to move away from the dimer interface to avoid steric clash. This movement results in a maximum shift of approximately 12 Å between the monomer and dimer conformations .
What experimental techniques are most effective for structural characterization of ubiquinol oxidase?
The most effective techniques for structural characterization of ubiquinol oxidase include:
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Cryogenic electron microscopy (cryo-EM): Recent studies have successfully employed cryo-EM single particle reconstruction to determine the structures of both monomeric and dimeric forms of E. coli cytochrome bo3 ubiquinol oxidase to high resolution .
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X-ray crystallography: Earlier structures of the enzyme were determined by this method, though some regions (residues 552-656 in subunit I) were missing in the first crystallographic structure but were visible in cryo-EM maps .
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Molecular dynamics (MD) simulations: These computational methods have been valuable for modeling the binding of ubiquinones to the enzyme and investigating structural dynamics during substrate interactions .
What methodological approaches can separate monomeric and dimeric forms of ubiquinol oxidase?
Separating monomeric and dimeric forms of ubiquinol oxidase requires sophisticated techniques:
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Computational particle enrichment: When both forms exist in the same sample, researchers have successfully used CNN-based particle pickers like Topaz to enrich particles for each oligomeric state . This method involves several rounds of training and picking, resulting in progressively purer sets of monomeric or dimeric particles.
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Blue Native PAGE (BN-PAGE): This technique has been employed to assess the oligomeric state of purified ubiquinol oxidase, revealing a monomer to dimer ratio of approximately 50:1 in some preparations .
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2D classification in cryo-EM: Initial 2D classification of particles can identify both monomers and dimers, though standard particle picking methods often favor the more abundant monomeric form .
| Technique | Application | Advantages | Challenges |
|---|---|---|---|
| CNN-based particle picking | Enrichment of specific oligomeric states | Progressive purification of particles | Requires specialized software and expertise |
| BN-PAGE | Assessment of oligomeric distribution | Simple technique, semi-quantitative | Limited resolution of structural details |
| 2D classification | Identification of different oligomeric states | Direct visualization of different forms | Bias toward abundant species |
How does ubiquinone binding affect the conformational dynamics of respiratory chain complexes?
Ubiquinone binding plays a crucial role in the conformational dynamics of respiratory chain complexes:
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Induced structural perturbations: When ubiquinones bind to the reaction site near the Fe-S cluster N2, they induce structural perturbations in the protein backbone . Areas of higher fluctuations include the loop connecting TMHs 5-6 of the ND1 subunit, the central loop of the PSST subunit, and a segment of 49-kDa subunit carrying Asp 160, all critical for catalytic function .
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Access pathway dynamics: The binding of oversized ubiquinones (OS-UQs) suggests that the access path for ubiquinone may be more open than previously thought in the channel model . Molecular dynamics simulations show that during transition through the channel, rodlike substituents of OS-UQs undergo marked deformation (bending) due to significant steric hindrance .
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Structural changes and proton pumping: The reaction characteristics of different ubiquinones affect the structural changes of the quinone reaction site required for triggering proton translocation, as evidenced by the varying proton-translocating efficiencies of OS-UQs with different side-chain structures .
What factors influence dimerization of ubiquinol oxidase and its functional implications?
Several factors may influence the dimerization of ubiquinol oxidase:
How can researchers optimize expression and purification of recombinant ubiquinol oxidase?
Optimizing expression and purification of recombinant ubiquinol oxidase requires careful consideration of several factors:
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Membrane mimetic selection: Different solubilization methods have been used successfully, including detergents like DDM, amphipol, styrene maleic acid co-polymer (SMA), and nanodiscs . The choice of membrane mimetic should balance protein stability, functionality, and compatibility with downstream applications.
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Preserving cofactor binding: Some detergents, such as Triton X-100, can strip bound ubiquinone from the protein . Researchers should select purification conditions that maintain all cofactors if functional studies are planned.
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Oligomeric state assessment: Methods like BN-PAGE should be employed to assess the oligomeric distribution of the purified protein . If a specific oligomeric form is desired, additional purification steps may be necessary.
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Quality control: Cryo-EM analysis with 2D classification can be used to verify the structural integrity and homogeneity of the purified enzyme .
What approaches can effectively measure electron transfer activity in ubiquinol oxidase?
Electron transfer activity in ubiquinol oxidase can be measured using several approaches:
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Substrate analog studies: Researchers have used various ubiquinone analogs, including oversized ubiquinones (OS-UQs), to study electron transfer activity . By varying the chemical structure of these analogs, researchers can probe structural requirements for substrate binding and catalysis.
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Inhibitor sensitivity profiling: The electron transfer activities can be assessed in the presence of different quinone-site inhibitors (piericidin A, rotenone, fenpyroximate, or bullatacin) to confirm that reduction takes place at the physiological reaction site .
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Dose-response analysis: Dose-response curves for inhibition can characterize the sensitivity of the enzyme to specific inhibitors and provide insights into the binding site properties .
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Coupled proton translocation measurements: Since ubiquinol oxidase functions as a proton pump, measuring proton translocation coupled with electron transfer can provide insights into the mechanistic coupling between these processes .
How do experimental contradictions between isolated systems and native environments inform research design?
Experimental contradictions between isolated systems and native environments provide important considerations for research design:
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System-dependent behavior: The observation that OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes, contrary to observations in submitochondrial particles (SMPs), highlights how enzyme behavior can differ between contexts .
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Multiple validation approaches: Researchers should design experiments using complementary approaches that bridge between isolated systems and more native-like environments. This might include:
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Progressive simplification from native membranes to reconstituted systems
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Comparison of different membrane mimetics with varying lipid compositions
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Inclusion of additional components from the native system
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Mechanistic hypotheses testing: Contradictory results can be leveraged to test specific mechanistic hypotheses. For example, differences in behavior between isolated and native systems might suggest the involvement of additional factors or specific lipid requirements.
How can structural data from cryo-EM be integrated with functional studies of ubiquinol oxidase?
Integrating structural and functional data provides a more comprehensive understanding of ubiquinol oxidase:
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Structure-guided mutagenesis: High-resolution structural data from cryo-EM can guide the design of site-directed mutations to test the functional importance of specific residues, particularly those at the dimerization interface or in regions that show conformational changes .
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Computational modeling: Molecular dynamics simulations based on cryo-EM structures can model substrate binding and predict conformational changes, generating hypotheses that can be tested experimentally .
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Correlation analysis: Researchers can correlate structural features (e.g., dimensions of access channels, distances between redox centers) with functional parameters (e.g., enzyme activity, substrate specificity) to identify structure-function relationships.
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Oligomeric state comparison: Functional studies can be performed with preparations enriched for either monomeric or dimeric forms to determine if oligomeric state affects catalytic properties or stability .
What analytical framework should be used to interpret ubiquinone binding site accessibility?
Interpreting ubiquinone binding site accessibility requires a sophisticated analytical framework:
What are common pitfalls in structural studies of membrane proteins like ubiquinol oxidase?
Structural studies of membrane proteins face several common pitfalls:
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Loss of cofactors: Certain detergents can strip bound cofactors from the protein, such as ubiquinone being lost from ubiquinol oxidase during purification . This may affect both structural integrity and function.
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Sample heterogeneity: The presence of multiple oligomeric states (monomers and dimers) can complicate structural determination . Special computational approaches may be needed to separate different particle populations.
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Conformational variability: Membrane proteins often exhibit conformational flexibility that can limit resolution in structural studies . Classification approaches may be needed to identify distinct conformational states.
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Non-native environment effects: The choice of membrane mimetic may affect protein behavior. For example, the properties of amphipol, detergent, SMA, or nanodiscs may not perfectly replicate the native membrane environment .
How can researchers troubleshoot contradictory results in electron transfer activity assays?
When faced with contradictory results in electron transfer activity assays, researchers should consider:
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Substrate accessibility considerations: The ability of substrates to access the reaction site may differ between experimental systems. For example, OS-UQs with shorter spacers (OS-UQ1) showed lower electron transfer activity than those with longer spacers (OS-UQ2) despite better water solubility, suggesting steric hindrance issues .
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Inhibitor specificity validation: Different inhibitors may have varying mechanisms and binding sites. Complete inhibition by diverse quinone-site inhibitors (piericidin A, rotenone, fenpyroximate, or bullatacin) can confirm that reduction occurs at the physiological reaction site .
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System-dependent effects: Reconcile differences between isolated complexes and membrane-embedded systems by considering the lipid environment, protein-protein interactions, and potential structural constraints .
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Concentration-dependent effects: For inhibitors like S1QEL2.3 that show incomplete inhibition (~60% residual activity), consider the mechanism of inhibition (direct vs. indirect modulation) and compare dose-response curves across different substrates .
This FAQ collection provides a comprehensive resource for researchers working with recombinant ubiquinol oxidase, addressing both fundamental concepts and advanced methodological considerations based on recent structural and functional studies.
