Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B UPF0060 membrane protein YE2027 (YE2027)

What is the basic structure and function of the YE2027 membrane protein?

YE2027 is a UPF0060 family membrane protein from Yersinia enterocolitica serotype O:8 / biotype 1B. It consists of 108 amino acids with the sequence: MLKASLLFFVTALAEIIGCFLPYLWLRKGASMWLLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVATALIWLRVVDDVKLSLFDWVGAAVALVGMLIIVAGWRVN . As a membrane protein, it weaves through the cellular membrane, though its specific functional role is still being characterized. The UPF0060 designation indicates it belongs to a family of proteins with uncharacterized function, presenting opportunities for novel research into its biological significance. Membrane proteins like YE2027 typically function in various cellular processes including signal transduction, transport, or structural roles in the membrane architecture.

How is recombinant YE2027 protein typically expressed and purified?

The recombinant YE2027 protein is typically expressed in E. coli expression systems. According to available data, the full-length protein (amino acids 1-108) is often fused with an N-terminal His-tag to facilitate purification . For expression, researchers commonly use E. coli strains optimized for membrane protein production, such as Lemo21(DE3), which allows for tunable T7 expression to achieve optimal assembly of transmembrane proteins .

For purification, immobilized metal affinity chromatography (IMAC) is the primary method, taking advantage of the His-tag's affinity for nickel ions. The purified protein is typically available as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage stability at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week.

What is the significance of Yersinia enterocolitica serotype O:8 in research?

Yersinia enterocolitica serotype O:8 has special significance in research due to its clinical relevance and unique properties. It has been associated with septicemia cases, with the first blood culture-positive case in Japan documented in research literature . While this serotype is considered rare in some regions, chromosomal DNA analysis suggests it may persist latently in healthy carriers throughout certain geographic areas .

From a research perspective, proteins from this serotype, including YE2027, provide valuable insights into bacterial pathogenesis, membrane structure, and potential diagnostic applications. The study of recombinant proteins from Y. enterocolitica has shown promise in improving the specificity of serological assays, particularly in patients with chronic, non-specific infections compared to using native antigens .

What challenges are associated with expressing multipass membrane proteins like YE2027, and how can these be overcome?

Expressing multipass membrane proteins like YE2027 presents several significant challenges. The hydrophobic nature of transmembrane segments often leads to protein aggregation and misfolding during recombinant expression . Additionally, overexpression can saturate the membrane protein biogenesis pathway, resulting in cell death and/or inclusion body formation .

To overcome these challenges, researchers should consider the following strategies:

• Expression moderation: Use expression systems like Lemo21(DE3) that allow precise regulation of target gene transcription through a T7 RNA polymerase inhibitor protein (LysY) . This strain enables sampling of a wide range of expression levels to find optimal conditions for each unique target protein.

• Expression temperature optimization: Lower temperatures (16-25°C) often improve proper folding of membrane proteins by slowing down the production rate.

• Membrane-mimetic environments: Include appropriate detergents or lipids during purification to maintain the native-like environment required for proper folding.

• Consider co-expression with chaperones: Recent research has revealed the importance of chaperone complexes, such as the PAT complex, in facilitating the biogenesis of multipass membrane proteins like rhodopsin . Similar approaches may be beneficial for YE2027 expression.

• Optimization of induction conditions: Adjust inducer concentration and induction time to minimize toxicity while maintaining adequate expression levels.

For YE2027 specifically, the principle that "less expression often results in more functional protein" should be considered when designing expression protocols .

How does the structural characterization of YE2027 compare with other UPF0060 family proteins, and what computational tools are most effective for its analysis?

Structural characterization of YE2027 can be compared with other UPF0060 family proteins using computational models and structural predictions. While specific experimental structural data for YE2027 itself may be limited, computational approaches have been used for related proteins like UPF0060 membrane protein DSY4157 from Desulfitobacterium hafniense Y51 .

For effective analysis, researchers should consider:

• AlphaFold or similar AI-based structure prediction tools: These have demonstrated remarkable accuracy in predicting membrane protein structures. For instance, the UPF0060 membrane protein DSY4157 has been modeled with a global pLDDT (predicted local distance difference test) score of 83.43, indicating confident structure prediction .

• Confidence metrics: When using computational models, assess confidence scores like pLDDT, which provides per-residue confidence between 0 and 100. For membrane proteins, some regions below 50 pLDDT may be unstructured in isolation .

• Comparative analysis: Using sequence similarity searches (with cutoffs at 30%, 50%, 70%, 90%, 95%, and 100% identity) to identify structural homologs can provide insights into conserved structural features across the UPF0060 family .

• Transmembrane topology prediction: Tools like TMHMM, Phobius, or TOPCONS can predict the membrane-spanning regions and their orientation.

Researchers should note that computational predictions should ideally be validated with experimental approaches when possible, as "there are no experimental data to verify the accuracy of computed structure models" for many of these proteins .

What role might YE2027 play in Yersinia enterocolitica pathogenesis, and how can this be experimentally investigated?

While the specific role of YE2027 in Y. enterocolitica pathogenesis remains largely uncharacterized, its nature as a membrane protein suggests potential involvement in host-pathogen interactions, bacterial survival in host environments, or virulence mechanisms. To experimentally investigate these possibilities, researchers could employ several approaches:

• Gene knockout/knockdown studies: Generate YE2027-deficient mutants and evaluate changes in virulence, colonization ability, or stress resistance in both in vitro and in vivo models.

• Protein-protein interaction studies: Use techniques like bacterial two-hybrid systems, co-immunoprecipitation, or crosslinking followed by mass spectrometry to identify interaction partners that might suggest functional roles.

• Host response analysis: Examine host immune responses to recombinant YE2027 protein, including cytokine production, immune cell activation, or antibody responses, which could indicate its role during infection.

• Comparative genomics: Analyze the conservation and variation of YE2027 across different Yersinia strains and serotypes, particularly those with varying virulence profiles.

• Subcellular localization: Determine the precise localization of YE2027 within the bacterial membrane using fluorescent protein fusions or immunogold electron microscopy.

• Structural-functional analysis: Perform systematic mutagenesis of conserved residues to identify functionally important regions of the protein.

These investigations could provide valuable insights into whether YE2027 contributes to pathogenesis processes like adhesion, invasion, immune evasion, or survival in host environments, which are critical aspects of Y. enterocolitica infections like the rare but serious septicemia documented in the literature .

What are the optimal conditions for solubilizing and reconstituting YE2027 for functional studies?

Optimal solubilization and reconstitution of YE2027 for functional studies require careful consideration of various factors to maintain the protein's native structure and function. Based on established membrane protein methodologies:

• Initial solubilization: For YE2027, which is provided as a lyophilized powder, reconstitution should begin with deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

• Buffer selection: Tris/PBS-based buffers at pH 8.0 with 6% trehalose have been documented as appropriate storage buffers for the solubilized protein . This provides a starting point for functional studies.

• Detergent selection: For membrane proteins like YE2027, mild detergents such as n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltoside (DM), or digitonin are often effective for initial solubilization while preserving protein structure.

• Reconstitution into proteoliposomes: For functional studies, reconstitution into lipid bilayers may be necessary. This typically involves:

  • Mixing the detergent-solubilized protein with appropriate lipids (e.g., E. coli total lipid extract or defined mixtures of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol)

  • Removing detergent gradually using dialysis, Bio-Beads, or cyclodextrin

  • Verifying successful reconstitution using techniques like freeze-fracture electron microscopy or dynamic light scattering

• Stability considerations: Addition of glycerol (5-50% final concentration) enhances protein stability for long-term storage at -20°C/-80°C . The recommendation of avoiding repeated freeze-thaw cycles suggests that aliquoting the reconstituted protein is advisable.

• Functional validation: Following reconstitution, functional validation using appropriate assays should be performed to ensure the protein has maintained its native activity.

For specific functional studies of YE2027, researchers may need to optimize these conditions empirically, as the optimal environment may depend on the particular function being investigated.

How can researchers effectively use YE2027 in immunological studies or for developing diagnostic assays?

Using YE2027 in immunological studies or diagnostic assay development requires strategic approaches to leverage this recombinant protein effectively:

• Antibody development: Purified recombinant YE2027 can serve as an antigen for generating specific antibodies. These antibodies can be used for:

  • Immunolocalization studies to determine the spatial distribution of the protein in bacterial cells

  • Western blotting to detect expression levels under different conditions

  • Immunoprecipitation to identify interaction partners

• Diagnostic assay development: Drawing from methodologies used with other Yersinia recombinant proteins, YE2027 could be incorporated into various diagnostic platforms:

  • ELISA: Similar to approaches with Yop proteins, YE2027 could be used as a highly specific antigen in ELISA tests for detecting antibodies in patient sera . This may increase specificity compared to native antigen preparations, particularly for chronic, non-specific infections.

  • Recom-dot assays: YE2027 could be immobilized on nitrocellulose strips for dot-blot based detection, which provides a simple and potentially field-deployable diagnostic option .

  • Multiplex assays: Combining YE2027 with other Yersinia antigens in multiplex platforms could improve diagnostic sensitivity and specificity.

• Study population selection: When validating immunological assays, include appropriate control groups. Previous studies with Yersinia recombinant proteins used both patients suspected of infection (n=74) and healthy blood donors (n=41) to characterize immune responses .

• Comparative analysis: Researchers should compare results from YE2027-based assays with established methods (e.g., commercial western-blot assays) to validate performance .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with antibodies against related pathogens to ensure diagnostic specificity.

• Epitope mapping: Identify immunodominant regions of YE2027 that might be particularly useful for diagnostic purposes or as vaccine candidates.

These approaches could potentially contribute to improved serodiagnosis of yersiniosis, especially in cases where traditional methods may lack specificity or sensitivity.

What experimental design considerations are crucial when studying membrane protein biogenesis pathways using YE2027 as a model?

When using YE2027 as a model to study membrane protein biogenesis pathways, several crucial experimental design considerations should be addressed:

• Expression system selection: Choose expression systems carefully, considering that the standard iterative model of membrane protein insertion through Sec61's lateral gate may not apply universally. Recent research has challenged this conventional model, showing that specific chaperones like the PAT complex play critical roles in multipass membrane protein biogenesis . For YE2027, researchers should consider:

  • Using tunable expression systems like Lemo21(DE3) that allow precise control of expression levels

  • Evaluating alternative systems beyond traditional E. coli if toxicity or misfolding issues arise

• Temporal analysis: Design experiments to capture the dynamics of membrane insertion:

  • Use pulse-chase experiments with radioisotope labeling or inducible systems

  • Employ time-resolved crosslinking to identify transient interactions during biogenesis

  • Consider synchronization methods to capture specific stages of membrane insertion

• Interaction partner identification: Recent research highlights the importance of chaperone complexes in membrane protein biogenesis :

  • Co-immunoprecipitation under native conditions to identify interacting partners

  • Proximity labeling approaches (BioID, APEX) to identify spatially adjacent proteins during biogenesis

  • Genetic screens to identify factors affecting YE2027 membrane insertion efficiency

• Structural intermediates: Capture folding intermediates during the biogenesis process:

  • Design truncated constructs representing potential folding intermediates

  • Use limited proteolysis to identify protected domains during folding

  • Employ single-molecule techniques to observe folding trajectories

• Comparative analysis: Include control membrane proteins with well-characterized biogenesis pathways in parallel experiments:

  • Single-pass membrane proteins that use the established Sec61 pathway

  • Other multipass proteins with varying numbers of transmembrane domains

  • Rhodopsin or similar proteins known to utilize the PAT complex

• Validation of models: Test hypotheses about insertion mechanisms using targeted approaches:

  • Site-directed mutagenesis of key residues in transmembrane domains

  • Domain swapping experiments to identify regions critical for proper insertion

  • In vitro reconstitution experiments with purified components of the insertion machinery

• Consider lipid environment: The composition of the membrane can significantly affect insertion efficiency and folding:

  • Manipulate membrane lipid composition through genetic or chemical means

  • Evaluate insertion into different membrane systems (bacterial vs. mammalian)

These considerations align with recent scientific advances showing that the biogenesis of multipass membrane proteins is more complex than previously thought, involving specialized chaperone complexes and potentially multiple insertion pathways .

What are the potential applications of YE2027 in synthetic biology and membrane protein engineering?

YE2027 presents several promising applications in synthetic biology and membrane protein engineering due to its specific properties as a bacterial membrane protein:

• Scaffold for designer membrane proteins: YE2027's relatively small size (108 amino acids) makes it an attractive scaffold for engineering membrane-embedded biosensors or signaling components. Researchers could:

  • Introduce binding domains for specific analytes

  • Modify transmembrane regions to alter membrane permeability or signaling properties

  • Create chimeric proteins by fusing functional domains from other proteins

• Minimal membrane protein model: As a compact membrane protein, YE2027 could serve as a simplified model system for studying fundamental aspects of membrane protein folding and stability:

  • Systematic mutation studies to establish sequence-structure-function relationships

  • Template for computational design of novel membrane proteins with desired properties

  • Platform for testing membrane protein design algorithms and folding predictions

• Membrane-anchored display systems: YE2027 could be engineered as an anchoring component for displaying peptides or proteins at the cell surface:

  • Development of bacterial display libraries for antibody discovery

  • Creation of engineered bacteria with surface-displayed antigens for vaccine development

  • Design of cellular biosensors with membrane-displayed recognition elements

• Study of minimal requirements for membrane insertion: The compact nature of YE2027 makes it valuable for determining the minimal sequence requirements for successful membrane integration:

  • Systematic truncation or substitution studies

  • Investigation of topology determinants

  • Evaluation of the relative contributions of different insertion machinery components

• Platform for studying membrane protein evolution: YE2027 could serve as a starting point for directed evolution experiments:

  • Evolution of new functions or substrate specificities

  • Development of membrane proteins with enhanced stability in non-native environments

  • Investigation of evolutionary pathways for membrane protein complexity

These applications would benefit from the established expression and purification protocols for YE2027, including its successful expression in E. coli systems with His-tag purification strategies . The growing understanding of membrane protein biogenesis pathways, including the roles of specialized chaperones , would further inform these engineering efforts.

How might comparative genomics approaches enhance our understanding of YE2027 function across different Yersinia species?

Comparative genomics approaches offer powerful tools for elucidating the function of YE2027 by examining its conservation, variation, and evolutionary context across Yersinia species and beyond:

• Sequence conservation analysis: Examining the conservation patterns of YE2027 across different Yersinia species and strains can reveal:

  • Functionally critical residues that remain conserved despite evolutionary divergence

  • Variable regions that might reflect adaptation to different niches or hosts

  • Potential horizontal gene transfer events that might indicate functional importance

• Synteny analysis: Examining the genomic context of YE2027 across species can provide functional insights:

  • Conservation of gene neighborhoods often indicates functional relationships

  • Co-evolution with specific gene clusters may suggest involvement in particular pathways

  • Changes in genomic context can reflect acquisition of new functions

• Phylogenetic profiling: Correlating the presence/absence of YE2027 with specific phenotypic traits:

  • Association with virulence characteristics across Yersinia species

  • Correlation with adaptation to specific environmental conditions

  • Relationship to host range or tissue tropism

• Structural homology detection: Identifying distant structural homologs even when sequence similarity is low:

  • Using tools like AlphaFold to generate structural models for YE2027 orthologs

  • Comparing predicted structures to identify conserved structural features

  • Relating structural conservation to potential functional sites

• Natural variation analysis: Examining polymorphisms in YE2027 across clinical isolates:

  • Analysis of single nucleotide polymorphisms (SNPs) in relation to disease severity

  • Identification of variants associated with specific clinical presentations

  • Assessment of selection pressures acting on different protein regions

• Integration with experimental data: Combining comparative genomics with experimental approaches:

  • Targeting experimental studies based on predictions from comparative analyses

  • Validating predicted functional sites through site-directed mutagenesis

  • Testing hypothesized functions in diverse Yersinia strains

This approach is particularly relevant for Yersinia enterocolitica serotype O:8, which has been shown to have unique clinical significance, such as in the reported case of septicemia in Japan . Comparative analysis could help explain why certain serotypes like O:8 might persist latently in healthy carriers while causing significant disease in others.

What new insights could be gained by applying emerging membrane protein structural determination techniques to YE2027?

Applying cutting-edge structural determination techniques to YE2027 could provide unprecedented insights into its structure-function relationships and membrane integration:

• Cryo-electron microscopy (cryo-EM): Recent advances in cryo-EM have revolutionized membrane protein structural biology:

  • Single-particle analysis could reveal YE2027's structure at near-atomic resolution

  • Visualization of YE2027 in different functional states if it undergoes conformational changes

  • Structural studies of YE2027 in complex with potential interaction partners or in native-like lipid environments

• Integrative structural biology approaches: Combining multiple experimental techniques:

  • Cross-linking mass spectrometry (XL-MS) to identify spatial relationships between protein regions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map solvent-exposed regions and conformational dynamics

  • Solid-state NMR to study YE2027 in membrane environments, providing atomic-level insights into protein-lipid interactions

• Advanced computational methods: Beyond traditional homology modeling:

  • AlphaFold2 and RoseTTAFold have demonstrated remarkable accuracy for membrane proteins

  • Molecular dynamics simulations in explicit membrane environments to study dynamic behavior

  • Enhanced sampling techniques to explore conformational landscapes

• Microcrystal electron diffraction (MicroED): Emerging technique for small crystalline samples:

  • Could overcome challenges associated with traditional X-ray crystallography for membrane proteins

  • Requires minimal amount of sample, potentially addressing purification yield challenges

  • Can provide high-resolution structures from very small crystals

• Serial femtosecond crystallography (SFX): X-ray free-electron laser-based approach:

  • Room-temperature structures that avoid potential artifacts from cryogenic conditions

  • Time-resolved studies to capture dynamic structural changes if YE2027 has any enzymatic activity

  • Radiation damage-free structures due to the "diffraction-before-destruction" principle

• In-cell structural studies: Examining YE2027 in its native environment:

  • Cellular cryo-electron tomography to visualize YE2027 in its native membrane context

  • In-cell NMR to study dynamics and interactions in the cellular environment

  • Correlative light and electron microscopy to connect structural data with cellular localization

These advanced techniques could provide insights into several key questions:

• How YE2027 is integrated into the membrane and what factors influence this process

• Whether YE2027 functions independently or as part of a larger protein complex

• How membrane composition affects YE2027 structure and function

• The structural basis for any potential role in Yersinia pathogenesis

The emerging understanding of membrane protein biogenesis pathways, including the specialized chaperones and insertion mechanisms revealed in recent research , provides important context for interpreting these structural studies and relating them to YE2027's cellular role.