Recombinant Yersinia pseudotuberculosis serotype O:3 UPF0266 membrane protein YPK_2467 (YPK_2467)

How should YPK_2467 be properly stored and handled in laboratory settings?

For optimal stability and functionality of recombinant YPK_2467 protein:

• Store the stock protein solution at -20°C for regular use

• For extended storage periods, maintain at -20°C or preferably -80°C

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

• Avoid repeated freeze-thaw cycles as this significantly compromises protein integrity

• Typical storage buffers include Tris-based buffers with 50% glycerol, specifically optimized for membrane proteins

The protein is typically supplied in solutions containing stabilizing agents and cryoprotectants such as glycerol that maintain structural integrity during freeze-thaw cycles .

What expression systems are recommended for producing recombinant YPK_2467?

E. coli expression systems are commonly used for the production of recombinant YPK_2467, as evidenced by commercial sources of the protein . For membrane proteins like YPK_2467, the following methodological considerations are important:

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

• Use of specialized vectors containing strong but controllable promoters

• Growth at lower temperatures (16-25°C) after induction to allow proper folding

• Addition of specific detergents during cell lysis and purification to maintain protein solubility

For higher yields of functional protein, mammalian expression systems might be considered, though with greater complexity and cost. HEK293T cells have shown superior performance for the production of complex recombinant proteins compared to suspension cells cultivated in serum-free medium .

What is the biological context of Yersinia pseudotuberculosis proteins?

Yersinia pseudotuberculosis (Yptb) is a gram-negative bacterium that causes intestinal infection with the potential to spread to the liver, where it can induce hemosiderosis, abscesses, and hepatitis. The bacterium employs various mechanisms to evade host immune responses, including:

• Expression of at least six plasmid-encoded Yersinia outer proteins belonging to the Type III secretion system

• Production of chromosome-encoded protein toxins involved in anti-phagocytic defense

• Modulation of immune cell functions to facilitate bacterial colonization of lymphoid organs

Understanding the specific role of membrane proteins like YPK_2467 within this pathogenic context provides important insights into bacterial virulence mechanisms and potential therapeutic targets .

What experimental approaches are most appropriate for studying the membrane interactions of YPK_2467?

For investigating YPK_2467's membrane interactions, several sophisticated techniques can be employed:

• Fluorescence Correlation Spectroscopy (FCS): This technique can quantitatively characterize reversible protein-membrane interactions. Recent advances in FCS methodology allow for accurate determination of partition coefficients (Kx) for membrane proteins, accounting for spontaneous protein-membrane dissociation and reassociation to the same or different lipid vesicles .

• Surface Plasmon Resonance (SPR): For studying real-time binding kinetics between YPK_2467 and various membrane components.

• Microscale Thermophoresis (MST): Useful for measuring binding affinities in near-native conditions.

A comprehensive experimental design should consider:

• Preparation of appropriate model membrane systems (liposomes with defined lipid compositions)

• Protein labeling strategies that minimally affect function

• Controls for non-specific binding

• Validation using multiple complementary techniques

When designing FCS experiments specifically, researchers should account for the statistical equilibrium distribution of proteins on lipid vesicles and utilize appropriate mathematical models to extract accurate partition coefficients .

How can researchers design experiments to investigate potential functions of YPK_2467 in bacterial pathogenesis?

Given the limited characterized functions of YPK_2467, a systematic experimental approach is essential:

• Generate knockout mutants of YPK_2467 in Yersinia pseudotuberculosis following the basic experimental design principles:

  • Define clear variables (independent: presence/absence of YPK_2467; dependent: various virulence phenotypes)

  • Formulate specific, testable hypotheses

  • Design appropriate controls including complementation strains

  • Assign experimental groups using appropriate statistical approaches

  • Measure multiple dependent variables to capture potential phenotypes

• Perform infection studies comparing wild-type and knockout strains:

  • In vitro infection of relevant cell types (especially phagocytes)

  • Ex vivo tissue models

  • In vivo animal models when appropriate and ethically approved

• Investigate potential interactions with host immune components:

  • Assess impacts on phagocytosis

  • Examine effects on inflammatory responses

  • Evaluate possible role in M2 macrophage polarization, which has been observed with other Yersinia proteins

What techniques are recommended for studying potential protein-protein interactions involving YPK_2467?

To characterize the interactome of YPK_2467, multiple complementary approaches should be considered:

• Pull-down assays using His-tagged YPK_2467 followed by mass spectrometry

  • Use crosslinking strategies to capture transient interactions

  • Include appropriate negative controls (e.g., non-specific His-tagged proteins)

  • Validate findings with reciprocal pull-downs

• Bacterial two-hybrid or yeast two-hybrid systems adapted for membrane proteins

  • Consider split-ubiquitin yeast two-hybrid system specifically designed for membrane proteins

  • Screen against genomic libraries of both Yersinia and potential host organisms

• Proximity-labeling approaches (BioID or APEX2)

  • Fusion of YPK_2467 with a promiscuous biotin ligase

  • In situ labeling of proximal proteins

  • Mass spectrometry identification of biotinylated proteins

• Förster Resonance Energy Transfer (FRET) for confirming direct interactions

  • Design appropriate fluorescent protein fusions

  • Control for proper membrane localization of fusion proteins

  • Quantify FRET efficiency using appropriate controls

Each identified interaction should undergo rigorous validation using multiple independent techniques to minimize false positives .

How can researchers effectively analyze the structure-function relationship of YPK_2467?

A comprehensive structure-function analysis would involve:

• Computational structural prediction:

  • Use homology modeling based on related UPF0266 family proteins

  • Apply membrane protein-specific prediction algorithms

  • Identify conserved domains and potential functional motifs

  • Use molecular dynamics simulations to predict membrane interactions

• Site-directed mutagenesis:

  • Target conserved residues identified from sequence alignments

  • Create systematic alanine scanning mutants

  • Generate domain deletion constructs

  • Design chimeric proteins with related membrane proteins

• Functional characterization of mutants:

  • Membrane localization assays

  • Interaction studies with identified partners

  • Assessment of effects on bacterial phenotypes

  • In vitro biochemical assays for specific activities

• Structural biology approaches:

  • X-ray crystallography (challenging for membrane proteins)

  • Cryo-electron microscopy

  • NMR spectroscopy for smaller domains

  • Hydrogen-deuterium exchange mass spectrometry

This multifaceted approach can provide insights into structure-function relationships even for poorly characterized membrane proteins like YPK_2467 .

What are the methodological considerations for studying the biophysical properties of YPK_2467 in membrane environments?

Investigating the biophysical properties of YPK_2467 requires specialized approaches for membrane proteins:

• Reconstitution strategies:

  • Selection of appropriate detergents for solubilization

  • Choice of lipid compositions mimicking bacterial membranes

  • Methods for protein incorporation (direct incorporation versus detergent removal)

  • Verification of proper orientation in the membrane

• Biophysical characterization techniques:

  • Circular dichroism (CD) spectroscopy for secondary structure determination

  • Differential scanning calorimetry (DSC) for thermal stability

  • Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) for orientation determination

  • Solid-state NMR for detailed structural information

• Dynamic measurements:

  • Fluorescence recovery after photobleaching (FRAP) for lateral mobility

  • Single-particle tracking for diffusion behavior

  • Atomic force microscopy (AFM) for topography and mechanical properties

• Partition coefficient determination:

  • Fluorescence correlation spectroscopy (FCS) with appropriate mathematical models

  • Account for statistical and phase equilibria in reversible binding scenarios

  • Consider the Cramer-Rao bound to establish limits of Kx determination

  • Validate findings with computational simulations

How can researchers troubleshoot common issues in recombinant YPK_2467 expression and purification?

When working with YPK_2467, researchers may encounter several challenges:

• Low expression yields:

  • Optimize codon usage for the expression host

  • Test multiple promoter strengths and induction conditions

  • Consider fusion partners that enhance solubility (e.g., MBP, SUMO)

  • Evaluate different E. coli strains specialized for membrane proteins

• Protein aggregation:

  • Screen various detergents and detergent concentrations

  • Include stabilizing agents such as glycerol in buffers

  • Reduce expression temperature to allow proper folding

  • Test extraction directly into amphipols or nanodiscs

• Low purity after affinity chromatography:

  • Increase washing stringency (higher imidazole for His-tagged proteins)

  • Add secondary purification steps (ion exchange, size exclusion)

  • Consider on-column refolding protocols

  • Optimize detergent concentration in purification buffers

• Loss of function during purification:

  • Minimize time between cell lysis and final purification

  • Include protease inhibitors throughout purification

  • Maintain consistent temperature during handling

  • Consider stabilizing ligands if known

Creating a structured troubleshooting decision tree can help systematically address expression and purification challenges for this membrane protein .

What specialized techniques are recommended for analyzing YPK_2467 interactions with host cell membranes?

For studying YPK_2467 interactions with host cell membranes, consider:

• Giant unilamellar vesicles (GUVs) as model systems:

  • Prepare GUVs with compositions mimicking host cell membranes

  • Include fluorescently labeled lipids for visualization

  • Use micromanipulation techniques for controlled experiments

  • Apply quantitative image analysis for interaction measurements

• Supported lipid bilayers (SLBs) for surface-sensitive techniques:

  • Prepare SLBs on appropriate substrates (mica, glass, silicon)

  • Use quartz crystal microbalance with dissipation (QCM-D) to measure binding kinetics

  • Apply total internal reflection fluorescence (TIRF) microscopy for spatial distribution

  • Consider atomic force microscopy (AFM) for topographical changes

• Live cell imaging approaches:

  • Express fluorescently tagged YPK_2467 for localization studies

  • Apply super-resolution techniques (STED, PALM, STORM) for detailed distribution

  • Use FRAP to measure diffusion behavior in cellular membranes

  • Consider correlative light and electron microscopy for ultrastructural context

• Biochemical fractionation:

  • Isolate different membrane compartments after controlled exposure

  • Use gradient centrifugation for separation of membrane fractions

  • Apply proteomics to identify enriched cellular components

  • Validate findings with immunofluorescence microscopy

The combination of biophysical and cell biological approaches provides complementary insights into membrane interaction mechanisms .

What are the recommended controls and validation steps for YPK_2467 functional studies?

Rigorous controls and validation are critical for functional studies:

• Essential controls for protein characterization:

  • Inactive mutants (site-directed mutagenesis of predicted functional residues)

  • Heat-denatured protein samples

  • Related but functionally distinct membrane proteins

  • Empty vector or irrelevant protein controls

• Validation of localization:

  • Multiple detection methods (different antibodies or tags)

  • Subcellular fractionation followed by Western blotting

  • Correlation with known membrane markers

  • Super-resolution microscopy to confirm specific localization

• Functional validation approaches:

  • Complementation of knockout phenotypes

  • Dose-dependent effects when applicable

  • Time-course experiments to establish causality

  • Epistasis analysis with related pathways

• Reproducibility considerations:

  • Technical replicates to assess method variability

  • Biological replicates (different protein preparations, bacterial cultures)

  • Testing in multiple experimental systems

  • Validation by independent research techniques

How might YPK_2467 research contribute to understanding bacterial pathogenesis mechanisms?

Research on YPK_2467 has significant potential to advance our understanding of bacterial pathogenesis:

• Novel virulence mechanisms:

  • Identification of previously uncharacterized membrane-associated virulence factors

  • Elucidation of unique mechanisms for evading host immunity

  • Understanding bacterial adaptation to different host environments

  • Discovery of new protein-protein interaction networks involved in pathogenesis

• Bacterial membrane biology:

  • Insights into membrane organization in Gram-negative bacteria

  • Understanding of protein trafficking and localization mechanisms

  • Characterization of membrane microdomains and their functional significance

  • Elucidation of bacterial outer membrane biogenesis

• Host-pathogen interactions:

  • Mechanisms of bacterial attachment to host tissues

  • Understanding of how bacteria modulate host cell signaling

  • Insights into immune evasion strategies

  • Characterization of phagocyte responses to bacterial membrane components

As Yersinia pseudotuberculosis is known to modulate immune responses through various mechanisms, membrane proteins like YPK_2467 might play roles in bacterial survival within macrophages and potentially influence macrophage polarization toward an M2 phenotype, which could contribute to bacterial persistence .

What approaches are recommended for identifying potential inhibitors of YPK_2467 function?

A systematic approach to inhibitor discovery would include:

• High-throughput screening strategies:

  • Development of function-based assays amenable to screening

  • Adaptation to microplate format for automation

  • Establishment of clear readouts correlating with protein function

  • Creation of robust statistical parameters for hit identification

• Structure-based drug design:

  • In silico docking studies based on predicted protein structure

  • Fragment-based screening approaches

  • Virtual screening of compound libraries

  • Molecular dynamics simulations to predict binding stability

• Peptide-based inhibitor development:

  • Design of peptides mimicking interaction interfaces

  • Phage display screening against purified protein

  • Stapled peptides for enhanced stability and membrane permeability

  • Cyclization strategies to improve protease resistance

• Validation of inhibitor specificity:

  • Counter-screening against related bacterial proteins

  • Evaluation of effects on host proteins

  • Assessment of activity in bacterial cultures

  • Testing in infection models

This multifaceted approach combines empirical screening with rational design to maximize the chances of identifying effective inhibitors .

How can researchers integrate YPK_2467 studies with broader systems biology approaches?

Integrating YPK_2467 research into systems biology frameworks requires:

• Multi-omics integration:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to map protein interaction networks

  • Metabolomics to identify affected metabolic pathways

  • Lipidomics to characterize membrane composition changes

• Network analysis approaches:

  • Construction of protein-protein interaction networks

  • Pathway enrichment analysis for functional context

  • Identification of regulatory nodes and feedback mechanisms

  • Cross-species network comparison for evolutionary insights

• Mathematical modeling:

  • Ordinary differential equation models of relevant pathways

  • Agent-based models of host-pathogen interactions

  • Flux balance analysis for metabolic impacts

  • Machine learning approaches for pattern identification

• Integration with structural biology:

  • Incorporation of structural constraints into network models

  • Molecular dynamics simulations at multiple scales

  • Prediction of allosteric regulation mechanisms

  • Modeling of membrane protein complexes

This integrated approach places YPK_2467 in its broader biological context, enhancing understanding of its role in bacterial physiology and pathogenesis .

How does YPK_2467 compare to similar proteins in other bacterial species?

A comprehensive comparative analysis reveals:

• Evolutionary relationships:

  • YPK_2467 belongs to the UPF0266 membrane protein family

  • Homologs are present across multiple bacterial phyla

  • Sequence conservation patterns reveal potentially functional domains

  • Phylogenetic analysis can indicate horizontal gene transfer events

• Structural comparisons:

  • Conservation of transmembrane domains across species

  • Variability in loop regions may indicate host-specific adaptations

  • Prediction of common structural motifs despite sequence divergence

  • Identification of conserved residues that may be functionally critical

• Functional diversity:

  • Related proteins may have characterized functions in other species

  • Genomic context analysis can reveal associated pathways

  • Co-expression patterns might indicate functional associations

  • Presence/absence patterns correlating with virulence phenotypes

This evolutionary perspective can provide valuable insights into the function and importance of YPK_2467 by leveraging knowledge from better-characterized homologs in other bacterial species .

What databases and bioinformatic tools are most useful for analyzing YPK_2467?

For comprehensive bioinformatic analysis:

• Primary databases:

  • UniProt (Q66BY5 for the related YPTB1631 protein)

  • Protein Data Bank (PDB) for structural information of homologs

  • NCBI Protein and Genome databases

  • Pathogen-specific databases like PATRIC

• Specialized tools for membrane proteins:

  • TMHMM or HMMTOP for transmembrane domain prediction

  • PSIPRED for secondary structure prediction

  • MPEx for membrane protein topology analysis

  • PPM server for positioning proteins in membranes

• Functional prediction tools:

  • InterProScan for domain and motif identification

  • BLAST and PSI-BLAST for homology detection

  • STRING for protein-protein interaction network prediction

  • Phyre2 for structural modeling

• Evolutionary analysis tools:

  • MUSCLE or CLUSTAL for multiple sequence alignment

  • PhyML or RAxML for phylogenetic tree construction

  • PAML for selection pressure analysis

  • ConSurf for conservation mapping onto structures

These resources can be integrated into a comprehensive workflow for thorough characterization of YPK_2467's potential functions based on sequence, structure, and evolutionary information .