Recombinant Salmonella choleraesuis UPF0266 membrane protein yobD (yobD)

How does yobD compare structurally and functionally to other membrane proteins in Salmonella species?

The yobD protein represents one of many membrane proteins in Salmonella enterica serotype Choleraesuis, which is notable for its high predilection to cause systemic infections in humans rather than gastroenteritis . Unlike well-characterized membrane proteins involved in virulence and antibiotic resistance, yobD's function remains largely unknown.

Comparative sequence analysis with other membrane proteins in Salmonella reveals that yobD lacks the characteristic domains associated with known virulence factors, secretion systems, or antibiotic efflux pumps. This suggests yobD may serve a more fundamental housekeeping role in membrane organization or cellular homeostasis. Further structural and functional studies are needed to fully characterize its role in Salmonella biology and potential contributions to pathogenesis .

What expression systems are most effective for producing recombinant yobD protein?

Based on current research protocols, E. coli expression systems have proven effective for producing recombinant yobD protein with N-terminal His-tags . For optimal expression, consider the following methodological approaches:

• Expression vector selection: pET-based expression systems with T7 promoters typically yield high expression levels for bacterial membrane proteins.

• E. coli strain optimization: BL21(DE3) or C41(DE3)/C43(DE3) strains are recommended, as they are engineered for membrane protein expression.

• Induction conditions: IPTG concentration (0.1-1.0 mM), temperature (16-30°C), and duration (4-24 hours) should be optimized.

• Extraction methods: Given its membrane nature, detergent solubilization is critical for isolation. Consider trying multiple detergents including DDM, LMNG, or SMA polymers for native nanodiscs extraction .

The expression region typically encompasses residues 1-152, capturing the full-length protein . Experimental validation through SDS-PAGE and Western blotting should be performed to confirm successful expression.

What are the optimal storage conditions for recombinant yobD protein to maintain structural integrity?

To maintain the structural and functional integrity of recombinant yobD protein, adhere to the following evidence-based storage protocols:

Importantly, repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and aggregation. For practical use, it is recommended to:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% for freeze storage

• Prepare multiple small working aliquots to minimize freeze-thaw cycles

This systematic approach to storage will help maintain protein stability and activity for experimental applications.

What methodologies are most effective for studying protein-lipid interactions of yobD?

For investigating yobD protein-lipid interactions, contemporary methodological approaches include:

• Native nanodiscs extraction: This approach allows for isolation of membrane proteins while preserving their native lipid environment. Styrene maleic acid (SMA) copolymers can extract membrane proteins directly into native nanodiscs without requiring detergents, maintaining the native membrane context .

• Tunable proteoliposomes: Reconstituting yobD into liposomes with defined lipid compositions enables systematic investigation of how specific lipids influence protein structure and function. Mass spectrometric analysis can then identify specific lipid binding preferences .

• Lipid specificity assays: Native mass spectrometry now allows direct analysis of membrane protein-lipid complexes, helping identify specific lipid interactions that may be crucial for yobD function .

• Nanoscale spatial organization studies: Single-molecule resolution imaging techniques can reveal how yobD organizes within membrane environments and whether it forms oligomeric structures or interacts with other membrane components .

When designing these experiments, it's critical to consider the native lipid environment of Salmonella membranes to most accurately represent physiological conditions.

How can researchers effectively design ELISA-based assays for detecting and quantifying yobD protein?

To design effective ELISA-based assays for yobD protein detection and quantification, researchers should implement the following methodological framework:

• Antibody selection/development:

  • Primary antibody: Generate polyclonal antibodies against full-length yobD or peptide antibodies targeting unique extramembrane regions

  • Detection antibody: Use anti-His tag antibodies for recombinant His-tagged yobD

• Assay format optimization:

  • Direct ELISA: Immobilize yobD on plates for antibody binding studies

  • Sandwich ELISA: Use capture and detection antibodies for higher specificity

  • Competitive ELISA: For samples with potential interfering substances

• Protocol refinement:

  • Coating buffer: Carbonate buffer (pH 9.6) for efficient protein immobilization

  • Blocking agent: 1-5% BSA or milk powder to minimize background

  • Sample preparation: Detergent solubilization (0.1-0.5% DDM or Triton X-100) for membrane protein extraction

• Standardization:

  • Generate a standard curve using purified recombinant yobD (50 μg is available commercially)

  • Include positive and negative controls to validate assay performance

• Data analysis:

  • Apply four-parameter logistic regression for standard curve fitting

  • Calculate detection limits and quantitative range

This methodological approach ensures robust, reproducible detection and quantification of yobD protein in research contexts.

How can nanoscale spatial resolution techniques be applied to study yobD organization in bacterial membranes?

Advanced nanoscale spatial resolution techniques offer powerful approaches to investigate yobD organization within native bacterial membranes. The following methodological framework can be implemented:

• Direct membrane extraction using SMA lipid particles (SMALPs): This method enables extraction of membrane proteins while preserving their native lipid environment and oligomeric state. The styrene maleic acid copolymer forms nanodiscs (10-30 nm diameter) around membrane proteins, maintaining their native context .

• Single-molecule imaging protocols: After extraction, single-molecule fluorescence microscopy can resolve individual yobD proteins and their oligomeric organization. This approach can detect:

  • Monomeric vs. oligomeric distribution

  • Spatial clustering patterns

  • Changes in organization upon environmental stimuli

• Correlative electron microscopy: Combining fluorescence localization with electron microscopy provides structural context to functional observations.

• Quantitative analysis workflow:

  • Spatial point pattern analysis to detect non-random distributions

  • Nearest neighbor measurements to characterize clustering

  • Pair correlation functions to determine organization patterns

Recent studies applying these techniques to diverse membrane proteins have revealed previously undetectable oligomerization states and spatial organization patterns that correlate with function . Applied to yobD, these approaches could reveal critical insights into its membrane arrangement and potential interaction partners.

What role might yobD play in Salmonella choleraesuis pathogenicity and virulence mechanisms?

While the specific function of yobD remains to be fully characterized, methodological approaches to investigate its potential role in pathogenicity include:

• Comparative genomics analysis: Examining the conservation and expression patterns of yobD across Salmonella serotypes with different virulence profiles. Serotype Choleraesuis shows the highest predilection to cause systemic infections in humans, suggesting its membrane proteins may contribute to this phenotype .

• Gene knockout studies: Systematically deleting the yobD gene and assessing impacts on:

  • Bacterial growth and survival under stress conditions

  • Invasion efficiency in cell culture models

  • Virulence in animal infection models

  • Antimicrobial resistance profiles

• Interactome mapping: Identifying protein-protein interactions involving yobD using:

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation coupled with mass spectrometry

  • Proximity-based labeling in living bacteria

• Transcriptional regulation analysis: Determining if yobD expression changes in response to:

  • Host cell contact

  • Intracellular environment

  • Antimicrobial exposure

The emergence of multidrug-resistant Salmonella Choleraesuis strains highlights the importance of understanding membrane proteins that may contribute to virulence or antibiotic resistance mechanisms . While direct evidence for yobD's role is still emerging, its membrane localization makes it a candidate for involvement in processes such as adhesion, invasion, or environmental sensing.

How can native mass spectrometry be optimized for studying yobD structure and interactions?

Native mass spectrometry (MS) offers powerful capabilities for studying membrane proteins like yobD in their near-native state. To optimize this approach:

• Sample preparation protocol development:

  • Extract yobD using SMA polymers to maintain native lipid interactions

  • Alternative approach: Solubilize using MS-compatible detergents (DDM, C8E4)

  • Buffer exchange into volatile MS-compatible buffers (ammonium acetate)

• Instrument parameters optimization:

  • Ion source: Nano-electrospray ionization with controlled capillary voltage

  • Pressure gradient: Manipulate collision gas pressure to maintain native structure

  • Quadrupole settings: Wide mass range with optimized transmission for large complexes

• Data acquisition strategy:

  • Monitor multiple charge states to ensure complete mass coverage

  • Implement collision-induced dissociation for subunit composition analysis

  • Apply native top-down MS/MS for structural characterization

• Data analysis workflow:

  • Deconvolution algorithms specific for membrane protein complexes

  • Mass matching to identify bound lipids, ligands, or interacting proteins

  • Quantitative assessment of binding stoichiometries

Recent methodologies for direct native MS analysis from membrane environments have significantly improved the study of membrane protein-lipid specificity . Applied to yobD, these approaches could reveal critical structural features, oligomerization states, and specific lipid interactions that influence its function.

What are common challenges in purifying recombinant yobD protein and how can they be addressed?

Purification of membrane proteins like yobD presents several challenges. Here's a systematic approach to address common issues:

When working with the commercial His-tagged recombinant yobD protein, reconstitution should follow manufacturer recommendations: reconstitute in deionized sterile water to 0.1-1.0 mg/mL and add glycerol to a final concentration of 50% for storage . Following purification, SDS-PAGE analysis should confirm purity greater than 90% , with additional validation by mass spectrometry to verify the intact mass and sequence coverage.

How should researchers interpret and reconcile contradictory experimental data regarding yobD function?

When facing contradictory experimental data about yobD function, implement this methodological framework for robust data analysis and reconciliation:

• Systematic experimental variable assessment:

  • Protein preparation: Compare detergent-solubilized vs. nanodisc-embedded protein

  • Expression system differences: E. coli vs. native Salmonella expression

  • Buffer composition effects: Test stability and activity across buffer conditions

  • Tag interference: Evaluate whether purification tags affect observed functions

• Multi-technique validation approach:

  • Employ orthogonal techniques to probe the same functional question

  • Quantitatively compare results across different methodologies

  • Develop ranking system for reliability of different approaches

• Biological context consideration:

  • Native membrane environment vs. in vitro systems

  • Growth conditions and expression timing

  • Interaction with other bacterial components

• Statistical robustness analysis:

  • Apply appropriate statistical tests for each data type

  • Identify outliers through standardized statistical approaches

  • Conduct power analysis to ensure sufficient sample sizes

• Computational modeling integration:

  • Use structural models to predict functional capabilities

  • Simulate protein behavior under different conditions

  • Generate testable hypotheses to resolve contradictions

When facing contradictory data, it's essential to recognize that UPF0266 family proteins like yobD have uncharacterized functions, making functional assignments preliminary. A comprehensive understanding will require integration of genomic, proteomic, and phenotypic approaches, with careful attention to experimental conditions that may reveal condition-specific functions.

What quality control metrics should be applied when working with recombinant yobD preparations?

Implementing rigorous quality control metrics is essential for reproducible research with recombinant yobD protein. The following comprehensive QC framework is recommended:

• Purity assessment protocol:

  • SDS-PAGE with Coomassie/silver staining (target: >90% purity)

  • Densitometry analysis for quantitative purity determination

  • Western blot with anti-His or specific anti-yobD antibodies

• Structural integrity verification:

  • Circular dichroism to confirm secondary structure elements

  • Fluorescence spectroscopy to assess tertiary structure

  • Mass spectrometry to verify intact mass and detect modifications

• Functional validation assays:

  • Lipid binding assays using native MS or fluorescence approaches

  • Thermal stability studies under varying conditions

  • Activity assays once specific function is identified

• Batch consistency metrics:

  • Lot-to-lot comparison of critical parameters

  • Retention of reference samples for comparative analysis

  • Standardized acceptance criteria for each parameter

• Storage stability monitoring:

  • Time-course analysis of protein under storage conditions

  • Freeze-thaw stability testing

  • Accelerated degradation studies to predict shelf-life

Quality control documentation should include detailed methodologies and acceptance criteria for each parameter. Commercial recombinant yobD preparations typically provide certificates with purity >90% as determined by SDS-PAGE , but independent verification is recommended for critical research applications.

What genomic and proteomic approaches can advance our understanding of yobD's evolutionary significance?

Advancing our understanding of yobD's evolutionary significance requires integrating multiple genomic and proteomic methodological approaches:

• Comparative genomics framework:

  • Phylogenetic analysis across Salmonella serotypes and related enterobacteria

  • Synteny analysis to examine conservation of genomic context

  • Selection pressure analysis (dN/dS ratios) to identify evolutionarily constrained regions

  • Identification of paralogs and potential functional redundancy

• Structural genomics integration:

  • Homology modeling based on related UPF0266 family proteins

  • Prediction of functional sites through evolutionary conservation mapping

  • Structure-based functional annotation using computational approaches

• Transcriptomic profiling strategy:

  • RNA-Seq analysis under various environmental conditions

  • Co-expression network analysis to identify functional associations

  • Transcriptional response to host-relevant stresses

• Proteome-wide interaction mapping:

  • Bacterial two-hybrid screening for protein-protein interactions

  • Co-purification mass spectrometry to identify stable interactors

  • Protein-lipid interaction profiling using lipidomic approaches

This integrated approach could provide crucial insights into why yobD has been conserved in Salmonella choleraesuis, which causes distinctly invasive infections compared to other Salmonella serotypes . The findings may reveal unexpected roles in virulence, stress response, or metabolic adaptation that contribute to Salmonella choleraesuis' unique pathogenicity profile.

How might nanoscale extraction methods improve structural studies of yobD protein?

Nanoscale extraction methodologies represent a paradigm shift in structural studies of membrane proteins like yobD. A systematic approach includes:

• SMA polymer-based extraction protocol:

  • Direct extraction from native membranes into nanodiscs (10-30 nm diameter)

  • Preservation of native lipid environment and oligomeric state

  • Elimination of detergent-induced artifacts in structural studies

• Implementation of native nanodiscs for structural analysis:

  • Cryo-electron microscopy of nanodisc-embedded yobD

  • Single-particle analysis for high-resolution structural determination

  • Subtomogram averaging to observe structural heterogeneity

• Functional characterization in nanodiscs:

  • Lipid composition manipulation to assess structural dependencies

  • Single-molecule studies of conformational dynamics

  • Native mass spectrometry for stoichiometry and ligand binding analysis

• Comparative methodological assessment:

  • Parallel structural studies using conventional detergent solubilization

  • Quantitative comparison of structural features and functional properties

  • Identification of context-dependent structural elements

Recent developments in proteome-wide quantitative platforms for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs enable unprecedented structural studies with preserved native environments . Applied to yobD, these approaches could reveal structural features lost in conventional detergent-based studies, potentially uncovering functional mechanisms that have remained elusive.

What implications does antibiotic resistance in Salmonella Choleraesuis have for research on membrane proteins like yobD?

The emergence of multidrug-resistant Salmonella Choleraesuis strains has significant implications for membrane protein research:

• Mechanistic investigation framework:

  • Screen for yobD expression changes in response to antibiotic exposure

  • Assess co-localization with known resistance determinants

  • Evaluate potential roles in membrane permeability or efflux systems

• Protein-antibiotic interaction studies:

  • Direct binding assays between yobD and antibiotics

  • Structural analysis of potential interaction sites

  • Functional impact of antibiotics on yobD activity or oligomerization

• Genetic manipulation approach:

  • Generate yobD knockout and overexpression strains

  • Determine minimum inhibitory concentrations across antibiotic classes

  • Assess contribution to fitness during antibiotic stress

• Translational research considerations:

  • Evaluate yobD as a potential target for novel antimicrobials

  • Develop inhibitors specific to bacterial UPF0266 family proteins

  • Assess conservation across resistant clinical isolates

Salmonella Choleraesuis has been shown to acquire drug resistance genes through recombination of virulence and resistance plasmids , highlighting the close relationship between virulence and resistance mechanisms. While yobD's specific contribution to resistance remains to be determined, its membrane localization places it at the critical interface where many antibiotics act, making it relevant to understanding the membrane biology of resistant strains.