Recombinant Inner membrane ABC transporter permease protein ycjO (ycjO)
Description
Fundamental Characteristics of YcjO
YcjO is classified as a putative transport protein that functions as a membrane component of an ATP-dependent sugar transporter system in Escherichia coli K-12 and related bacterial species . The protein belongs to the ATP-binding cassette (ABC) superfamily, a diverse group of membrane transport proteins that utilize ATP hydrolysis to translocate various substrates across cellular membranes . In its native context, YcjO is specifically located in the bacterial inner membrane, where it forms part of a multi-component transport complex .
The gene encoding YcjO (ycjO) is located at map position [1,371,246 -> 1,372,127] in the E. coli genome, corresponding to approximately 29.55 centisomes or 106° on the circular chromosome . This gene produces a protein with a length of 293 amino acids, translated from an 882-base pair coding sequence . YcjO has been assigned various database identifiers, including UniProt accession number P0AFR7, which facilitates cross-referencing across different biological databases and research platforms .
Functional Role in Transport Systems
YcjO serves as a critical component of a putative ATP-dependent sugar transport system in bacteria . In this capacity, the protein functions as a permease, creating a channel or pathway through which specific substrates can cross the inner membrane. Based on sequence similarity and genomic context, researchers predict that YcjO works in concert with other proteins to facilitate the import of carbohydrate substrates into the bacterial cell .
The transport complex of which YcjO is a part consists of multiple components, each with a specialized role in the transport process. Specifically, YcjO operates alongside YcjP (another membrane component), YcjN (a periplasmic binding component), and YcjV (an ATP-binding protein) . Together, these proteins form a functional transport system that is believed to be involved in the uptake of specific sugars or sugar derivatives.
The substrate specificity of the YcjO-containing transport system has not been definitively established, but genomic context provides valuable clues. The ycjO gene is located within an operon that includes genes involved in carbohydrate import and metabolism . This genomic arrangement suggests that YcjO likely participates in the transport of specific carbohydrates that serve as substrates for the enzymatic activities encoded by neighboring genes.
YcjO in the Multicomponent Transport Complex
YcjO functions as part of a multicomponent ABC transport system with the following composition:
This transport system follows the typical operational mechanism of ABC importers in bacteria. The periplasmic binding protein (YcjN) captures the substrate from the extracellular environment and delivers it to the membrane components (YcjO and YcjP), which form a pathway for the substrate to cross the membrane . The ATP-binding component (YcjV) provides energy for this process through ATP hydrolysis .
Recent research on YcjN has provided further insights into the transport complex's function. Crystal structure analysis of YcjN has revealed that it belongs to subcluster D-I of substrate binding proteins, which includes the well-characterized maltose binding protein (MBP) . This structural similarity suggests that the YcjO-containing transport system may be involved in the import of maltose-like carbohydrates or related sugar compounds.
Genomic Context and Metabolic Significance
The ycjO gene exists within a larger gene cluster that encodes multiple proteins involved in carbohydrate metabolism . This genomic context provides valuable clues about the physiological role of YcjO and its associated transport complex. The ycj operon includes genes for several enzymes that process specific carbohydrates, suggesting that YcjO participates in the import of substrates for these enzymes.
Notable enzymes encoded in the same operon include:
The presence of these enzymes suggests that the YcjO transport system may import specific sugars that are subsequently metabolized through pathways involving these enzymes. Particularly significant is the finding that YcjQ, YcjR, and YcjS constitute a previously unrecognized metabolic pathway for the transformation of D-gulosides to D-glucosides via 3-keto-D-guloside and 3-keto-D-glucoside intermediates . This suggests that YcjO might be involved in the import of D-gulosides, D-glucosides, or related compounds that serve as substrates for this pathway.
Essentiality and Growth Characteristics
Experimental studies have investigated the essentiality of YcjO for bacterial growth under various conditions. Knockout experiments, in which the ycjO gene was inactivated, have provided valuable insights into the protein's physiological importance. Available data indicate that YcjO is not essential for bacterial growth under standard laboratory conditions .
The table below summarizes growth characteristics of ycjO knockout strains under various conditions:
These results suggest that while YcjO may contribute to optimal nutrient acquisition under specific conditions, it is not absolutely required for bacterial viability in standard growth media. This non-essentiality is consistent with YcjO's putative role in the transport of specialized carbohydrates that may be dispensable when other carbon sources are available.
Related Research on the YcjN Component
Recent research on YcjN, the periplasmic binding component that works with YcjO, provides additional context for understanding the transport complex. A crystal structure of YcjN has been determined to a resolution of 1.95 Å, revealing structural similarities to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP) .
Interestingly, studies have shown that recombinant overexpression of YcjN results in the formation of a lipidated form that is posttranslationally diacylated at cysteine 21 . This lipidated YcjN (Lipo-YcjN) exhibits different physicochemical properties compared to the non-lipidated form (ΔYcjN), particularly in terms of aggregation behavior in solution. Specifically, size-exclusion chromatography and dynamic light scattering measurements have demonstrated that Lipo-YcjN tends to aggregate in solution via its lipid moiety, while ΔYcjN remains monomeric .
These findings have implications for understanding how the YcjO-containing transport complex may be organized and regulated in the bacterial cell. The lipidation of YcjN suggests a potential mechanism for anchoring the periplasmic component to the membrane, which could facilitate interaction with the membrane components YcjO and YcjP.
Future Research Directions and Applications
Despite significant progress in characterizing YcjO and its associated transport system, several questions remain unanswered. Future research directions may include:
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Determination of the precise substrate specificity of the YcjO-containing transport system
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Elucidation of the three-dimensional structure of YcjO, alone and in complex with other transport components
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Investigation of the regulation of ycjO expression and its integration into cellular metabolic networks
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Exploration of potential applications in biotechnology, such as engineered transport systems for specific molecules
Recombinant YcjO has valuable applications in basic research, providing insights into the mechanisms of ABC transporters and bacterial nutrient acquisition. Additionally, understanding YcjO's structure and function may contribute to the development of new antimicrobial strategies targeting bacterial transport systems.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: sfl:SF1317
Q&A
What is Inner membrane ABC transporter permease protein ycjO and what is its biological significance?
The Inner membrane ABC transporter permease protein ycjO is a critical component of ATP-binding cassette (ABC) transport systems in bacterial cells. It functions as a transmembrane permease that facilitates substrate transport across cellular membranes. According to molecular characterization, ycjO is found in organisms such as Shigella flexneri with Uniprot identification number P0AFR8 . The protein is part of a larger complex that typically consists of substrate-binding proteins, permease domains, and nucleotide-binding domains that collectively enable active transport of various substrates against concentration gradients. ABC transporters are involved in numerous biological processes including nutrient uptake, drug resistance, and cell signaling, making ycjO an important target for understanding bacterial physiology and potentially developing antimicrobial strategies.
How does ycjO relate to other components in its transport system?
The ycjO protein functions in coordination with other components of its ABC transporter system, particularly with YcjN, which is a substrate-binding protein expressed from the same gene cluster involved in carbohydrate import and metabolism . While ycjO serves as the transmembrane component (permease), YcjN acts as the substrate-binding protein that captures target molecules and delivers them to the transporter complex. Recent structural studies have determined the crystal structure of YcjN to a resolution of 1.95 Å, revealing structural similarity to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein . This relationship suggests that the ycjO-containing transporter system may be involved in carbohydrate transport, though specific substrates remain to be fully characterized.
What are the recommended methods for recombinant expression of ycjO?
For successful recombinant expression of ycjO, researchers should consider the following methodological approach:
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Vector Selection: Choose expression vectors with inducible promoters (such as T7 or tac) that allow tight regulation of membrane protein expression to prevent toxicity.
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Host Selection: Use specialized E. coli strains designed for membrane protein expression, such as C41(DE3), C43(DE3), or Lemo21(DE3), which can better tolerate membrane protein overexpression.
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Expression Conditions: Optimize temperature (typically lower temperatures of 16-25°C), inducer concentration, and expression duration to maximize properly folded protein yield.
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Membrane Fraction Isolation: Employ differential centrifugation techniques to isolate membrane fractions containing the expressed ycjO protein.
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Solubilization: Select appropriate detergents (such as DDM, LDAO, or DMNG) for efficient solubilization of the membrane-embedded ycjO while maintaining its native structure.
For better yield control, consider adding fusion tags (such as His-tag, MBP, or SUMO) to facilitate purification and potentially enhance solubility. Expression should be verified using Western blotting with antibodies against the fusion tag or the protein itself.
What purification strategies are most effective for recombinant ycjO?
Purifying membrane proteins like ycjO requires specialized approaches:
| Purification Step | Methodology | Key Considerations |
|---|---|---|
| Solubilization | Detergent extraction (DDM, LDAO, DMNG) | Maintaining protein stability and native conformation |
| Affinity Chromatography | IMAC (for His-tagged protein) | Buffer composition with detergent above CMC |
| Size Exclusion Chromatography | Superdex 200 or similar | Assessing oligomeric state and protein homogeneity |
| Ion Exchange Chromatography | Optional polishing step | Based on theoretical pI of the protein |
When working with recombinant ycjO, it's crucial to maintain an appropriate detergent concentration throughout the purification process to prevent protein aggregation. Additionally, including stabilizing agents such as glycerol (20-50%) can help preserve protein activity during storage . Quality control should include SDS-PAGE analysis, Western blotting, and functional assays to confirm the identity and activity of the purified protein.
How can researchers evaluate the proper folding and functional state of purified ycjO?
Assessing the proper folding and functional integrity of purified ycjO involves multiple complementary approaches:
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Circular Dichroism (CD) Spectroscopy: Analyze secondary structure content to verify that the protein has the expected high α-helical content typical of membrane transporters.
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Thermal Stability Assays: Use differential scanning fluorimetry (DSF) with appropriate membrane protein dyes to assess protein stability.
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Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): Determine the oligomeric state and homogeneity of the protein-detergent complex.
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Substrate Binding Assays: Develop fluorescence-based or isothermal titration calorimetry (ITC) assays to measure binding of potential substrates.
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Reconstitution into Liposomes or Nanodiscs: Incorporate purified ycjO into artificial membrane systems to assess transport activity using fluorescent substrates or radiolabeled compounds.
Researchers should also consider lipidation state analysis, as related proteins like YcjN have been shown to undergo post-translational diacylation at specific cysteine residues, which can affect their function and membrane association .
What experimental designs are recommended for studying substrate specificity of ycjO?
Investigating the substrate specificity of ycjO requires a systematic approach using design of experiments (DOE) principles. A comprehensive experimental design should include:
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Candidate Substrate Screening: Based on the relationship to YcjN and its similarity to carbohydrate transporters, screen various sugar molecules and derivatives as potential substrates.
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Transport Assays in Reconstituted Systems: Reconstitute purified ycjO along with its ABC transporter partners into proteoliposomes or nanodiscs. Measure transport activity using:
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Fluorescently labeled substrates with fluorescence quenching assays
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Radiolabeled substrates with scintillation counting
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FRET-based assays to detect conformational changes during transport
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Factorial Design Approach: Implement full factorial designs to simultaneously test multiple factors affecting transport activity, such as substrate concentration, pH, temperature, and ATP concentration .
This systematic approach allows researchers to model the behavior of ycjO's transport activity as a function of multiple factors and determine whether these factors interact in their effect on transport efficiency . Statistical analysis of the results enables building a mathematical model to predict optimal conditions for specific substrate transport.
How can researchers investigate the structure-function relationship of ycjO?
To elucidate structure-function relationships in ycjO, researchers should consider the following methodological framework:
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Site-Directed Mutagenesis: Systematically mutate key residues in the transmembrane helices and substrate-binding pocket based on sequence alignments with related transporters and computational predictions.
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Cysteine Scanning Mutagenesis: Introduce cysteine residues at specific positions and use thiol-reactive probes to monitor conformational changes during the transport cycle.
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Cryo-EM Analysis: Given the challenges in crystallizing membrane proteins, cryo-electron microscopy offers a powerful alternative for structural determination of ycjO in different conformational states.
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Molecular Dynamics Simulations: Perform simulations of ycjO in a lipid bilayer environment to predict substrate pathways and conformational changes during transport.
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Cross-linking Studies: Investigate protein-protein interactions within the ABC transporter complex using chemical cross-linking coupled with mass spectrometry.
The integration of these approaches allows for a comprehensive understanding of how specific structural features of ycjO contribute to its transport mechanism and substrate specificity.
What are the considerations for studying ycjO interactions with other ABC transporter components?
Investigating the interactions between ycjO and other components of its ABC transporter system requires careful experimental design:
| Interaction Component | Methodology | Expected Outcome |
|---|---|---|
| Substrate-Binding Protein (YcjN) | Pull-down assays, Surface Plasmon Resonance | Binding kinetics, interaction interfaces |
| Nucleotide-Binding Domain | ATPase activity assays, co-purification | Coupling efficiency between ATP hydrolysis and transport |
| Lipid Environment | Lipid composition variation in reconstituted systems | Effect of membrane environment on transport activity |
| Associated Proteins | Co-immunoprecipitation, proteomics | Identification of interaction partners |
When designing these experiments, researchers should consider the dynamic nature of ABC transporter complexes. The transporter undergoes conformational changes during the transport cycle, so capturing different states may require specific conditions or the use of ATP analogs that trap the complex in specific conformations. Additionally, the lipidation state of associated proteins like YcjN may affect complex formation and activity .
How does ycjO compare with other bacterial ABC transporter permease proteins?
Comparative analysis of ycjO with other bacterial ABC transporter permease proteins reveals important evolutionary and functional relationships:
| ABC Transporter Permease | Organism | Substrate Specificity | Sequence Similarity to ycjO |
|---|---|---|---|
| MalG | E. coli | Maltose/maltodextrins | Moderate (based on YcjN's similarity to maltose binding protein) |
| BtuC | E. coli | Vitamin B12 | Low |
| HisM | S. typhimurium | Histidine | Low to moderate |
| ModB | E. coli | Molybdate | Low |
What systems biology approaches can be used to understand the role of ycjO in bacterial metabolism?
Understanding ycjO's role in the broader context of bacterial metabolism requires integrated systems biology approaches:
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Transcriptomic Analysis: RNA-seq under various growth conditions to identify co-regulated genes and metabolic pathways associated with ycjO expression.
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Metabolomic Profiling: Compare metabolite profiles between wild-type and ycjO knockout strains to identify affected metabolic pathways.
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Flux Balance Analysis: Construct computational models incorporating ycjO transport activities to predict metabolic flux distributions.
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Protein-Protein Interaction Networks: Use interactomics approaches to map ycjO's interactions within the larger cellular network.
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Phenotypic Microarrays: Screen growth phenotypes on different carbon sources and stress conditions to identify functional roles.
These approaches should be designed following DOE principles to efficiently explore the multidimensional space of experimental conditions and identify significant factors affecting ycjO's role in bacterial metabolism .
