Recombinant Salmonella paratyphi C UPF0266 membrane protein yobD (yobD)

What is Salmonella paratyphi C and how does it differ from other Salmonella Paratyphi serovars?

Salmonella paratyphi C is one of the serovars of Salmonella enterica that causes paratyphoid fever in humans. Salmonella enterica serovars Paratyphi A, B (tartrate negative), and C are identified as etiologic agents of Salmonella Paratyphi infections . While sharing some characteristics with other Paratyphi serovars, S. paratyphi C has distinct genomic features and epidemiological patterns.

The infection process typically involves ingestion of food or water contaminated with the stool or urine of a carrier. Most cases of paratyphoid fever are acquired during international travel to endemic regions including parts of Asia, Africa, and Latin America . The incubation period typically ranges from 1 to 10 days, and the disease remains communicable as long as paratyphoid bacilli are present in excreta .

Clinical presentations include sustained fever, headache, malaise, anorexia, relative bradycardia, and varying gastrointestinal symptoms, though mild and atypical infections may also occur . Unlike some other Salmonella species, S. paratyphi has humans as its only known reservoir.

What is the current understanding of YobD protein structure and why is it significant for research?

The UPF0266 membrane protein YobD in Salmonella paratyphi C belongs to a family of uncharacterized proteins found in bacterial membranes. As a membrane protein, structural analysis requires specialized techniques such as those outlined in the five-step approach for membrane protein structure determination:

• Preparation of uniformly 13C/15N labeled protein in proteoliposomes

• Resolution of individual signals using Magic Angle Spinning (MAS) solid-state NMR

• Assignment of signals to specific residues

• Measurement of orientation-dependent frequencies and distance restraints

• Calculation of the three-dimensional structure

The significance of YobD lies in its potential role in bacterial membrane function, which could impact pathogenesis, survival mechanisms, or antimicrobial resistance. Understanding its structure-function relationship provides insights into basic bacterial physiology and potential therapeutic targets.

What expression systems are most effective for producing functional recombinant YobD protein?

Multiple expression systems should be evaluated when working with membrane proteins like YobD:

When selecting an expression system, consider:

For structural studies specifically, the solid-state NMR approach requires uniformly 13C/15N labeled protein, necessitating expression in minimal media with labeled precursors .

What analytical techniques are essential for confirming the identity and purity of recombinant YobD?

A multi-technique approach is essential for validating recombinant YobD protein:

• Biochemical identification:

  • SDS-PAGE for size verification and initial purity assessment

  • Western blotting with tag-specific or YobD-specific antibodies

  • Mass spectrometry for precise mass determination and sequence verification

  • N-terminal sequencing to confirm proper processing

• Structural integrity assessment:

  • Circular dichroism to evaluate secondary structure elements

  • Fluorescence spectroscopy to assess tertiary structure

  • Thermal stability assays to determine protein folding quality

  • Limited proteolysis to identify properly folded domains

• Homogeneity analysis:

  • Size exclusion chromatography to assess oligomeric state

  • Dynamic light scattering for polydispersity measurement

  • Analytical ultracentrifugation for precise molecular weight determination

  • Native gel electrophoresis for non-denatured size analysis

• Functional validation:

  • Lipid binding assays to verify membrane interaction capabilities

  • Reconstitution into liposomes to assess membrane insertion

  • Activity assays based on predicted functions (if known)

For membrane proteins specifically, additional considerations include detergent screening to maintain native-like structure and assessing protein-lipid interactions that may be essential for function.

How can researchers optimize membrane protein isolation protocols specifically for YobD?

Optimizing YobD isolation requires systematic evaluation of each purification step:

• Membrane fraction isolation:

  • Gentle cell lysis methods to preserve membrane integrity (e.g., enzymatic lysis, French press at moderate pressure)

  • Differential centrifugation to separate membrane fractions (e.g., 5,000×g to remove debris, followed by 100,000×g to collect membranes)

  • Multiple washing steps with high salt buffers (300-500 mM NaCl) to remove peripheral proteins

• Solubilization optimization:

  • Systematic detergent screening using the matrix approach:

• Chromatographic purification:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • On-column detergent exchange during washing steps

  • Size exclusion chromatography as final polishing step

  • Monitoring protein quality at each step via activity or structural assays

• Sample concentration considerations:

  • Careful concentration using centrifugal devices with appropriate molecular weight cutoffs

  • Addition of glycerol (10-15%) to prevent aggregation

  • Adjustment of critical micelle concentration (CMC) of detergents during concentration

The structural analysis approach described in search results emphasizes the importance of reconstituting purified YobD into proteoliposomes for solid-state NMR studies, requiring careful optimization of lipid composition and protein-to-lipid ratios .

What are the current technical challenges in determining the high-resolution structure of YobD using solid-state NMR?

Solid-state NMR structure determination of membrane proteins like YobD faces several technical challenges:

• Sample preparation complexities:

  • Achieving homogeneous reconstitution in proteoliposomes

  • Optimizing protein-to-lipid ratios for spectral quality

  • Ensuring complete isotopic labeling (13C/15N) for multidimensional experiments

  • Maintaining protein stability during extended data acquisition periods

• Spectroscopic challenges:

  • Signal overlap in larger membrane proteins

  • Limited sensitivity requiring extended acquisition times

  • Hardware requirements for specialized solid-state NMR experiments

  • Need for both Magic Angle Spinning (MAS) and Oriented Sample (OS) approaches

• Data analysis and interpretation:

  • Complex assignment strategies for membrane protein spectra

  • Extraction of structural restraints from orientation-dependent frequencies

  • Integration of distance and angular constraints into structure calculations

  • Computational demands of structure refinement

As outlined in the research literature, a comprehensive approach requires:

• High-field NMR spectrometers (700-750 MHz or higher)

• Specialized probes for solid-state experiments (e.g., 3.2 mm low-E triple resonance probes)

• Two- and three-dimensional separated local field (SLF) experiments for angular constraints

• Integration of multiple NMR parameters into structure calculations

The optimization process typically requires iterative refinement of sample conditions, pulse sequences, and data analysis methods to achieve sufficient resolution for structure determination.

How can metabolomic approaches enhance our understanding of YobD function during infection?

Metabolomic analysis provides valuable insights into YobD function through the detection of altered metabolite profiles:

• Experimental design considerations:

  • Comparison between wild-type and YobD knockout strains

  • Analysis of host cell responses to infection with each strain

  • Time-course studies to capture dynamic metabolic changes

  • Integration with transcriptomic data for pathway analysis

• Analytical techniques:

  • Two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) has proven effective for detecting metabolite signals in Salmonella infections

  • The approach can identify hundreds of individual metabolite peaks (695 in one study) with high sensitivity

  • Supervised pattern recognition methods can identify significant metabolite profiles that distinguish between different infection states

• Data analysis strategies:

  • Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) for metabolite profile differentiation

  • Receiver-operating characteristic (ROC) curves to assess diagnostic potential of metabolite combinations

  • Identification of key metabolites with highest discriminatory power

Research on Salmonella Typhi and Paratyphi A has demonstrated that a combination of just six metabolites (ethanolamine, gluconic acid, monosaccharide, phenylalanine, pipecolic acid, and saccharide) can provide high discriminatory power between infection states, suggesting similar approaches could reveal YobD's metabolic impact .

What methodological approaches can resolve contradictory findings regarding YobD's role in pathogenesis?

Resolving contradictory findings requires systematic methodological approaches:

• Standardization of experimental systems:

  • Establish reference strains and constructs

  • Define precise genetic modifications (clean deletions vs. insertional inactivation)

  • Standardize culture conditions and growth phases

  • Use multiple infection models with defined parameters

• Multi-level phenotypic analysis:

  • Conduct comprehensive virulence testing in multiple models

  • Perform detailed in vitro phenotypic characterization

  • Analyze subcellular localization under identical conditions

  • Examine protein-protein interactions systematically

• Molecular genetic approaches:

  • Create allelic series with point mutations targeting specific domains

  • Perform complementation with homologs from related species

  • Use controlled expression systems to determine dose-dependency

  • Construct reporter fusions to monitor expression in different conditions

• Data integration framework:

  • Implement meta-analysis of published and unpublished data

  • Establish a standardized database of YobD experimental results

  • Develop mathematical models to reconcile apparently contradictory observations

  • Adopt Bayesian approaches to weigh evidence from different methodologies

• Collaborative resolution strategies:

  • Organize multi-laboratory studies with standardized protocols

  • Implement round-robin testing of key findings

  • Establish a consortium approach for comprehensive phenotyping

  • Develop consensus reporting standards for YobD research

This systematic approach can reconcile contradictory findings by identifying condition-dependent effects and methodological variables that influence experimental outcomes.

What techniques can effectively characterize the membrane topology and orientation of YobD?

Multiple complementary techniques should be employed to determine YobD membrane topology:

• Biochemical mapping approaches:

  • Cysteine scanning mutagenesis with selective labeling of accessible residues

  • Protease protection assays with site-specific proteases

  • Chemical modification accessibility studies

  • Glycosylation mapping with engineered sites

• Genetic fusion strategies:

  • Reporter fusions (PhoA, LacZ, GFP) at different positions

  • Dual reporter constructs to distinguish cytoplasmic vs. periplasmic locations

  • Systematic truncation analysis with terminal reporters

  • Split protein complementation to identify membrane-spanning segments

• Spectroscopic methods:

  • Oriented Sample (OS) solid-state NMR to determine angular constraints

  • Site-directed spin labeling with EPR spectroscopy

  • FRET analysis with strategically placed fluorophores

  • Hydrogen-deuterium exchange mass spectrometry

• Computational integration:

  • Topology prediction algorithms (TMHMM, TOPCONS)

  • Integration of experimental data with computational models

  • Molecular dynamics simulations in membrane environments

  • Evolutionary analysis of conserved topology features

• Structural validation:

  • The five-step NMR approach described in research provides angular constraints that directly define orientation

  • Two- and three-dimensional separated local field (SLF) spectra resolve orientation-dependent frequencies

  • These frequencies translate directly into angular constraints for structure calculation

The integration of these approaches provides a robust characterization of YobD's membrane topology and orientation, essential for understanding its functional mechanisms.

How can researchers identify potential interaction partners of YobD in host and bacterial membranes?

A systematic multi-method approach is required to identify YobD interaction partners:

• Proximity-based identification methods:

  • In vivo crosslinking followed by mass spectrometry

  • BioID or APEX2 proximity labeling

  • Split-protein complementation assays

  • Resonance energy transfer approaches (FRET/BRET)

• Affinity-based approaches:

  • Co-immunoprecipitation with quantitative proteomics

  • Tandem affinity purification with gentle solubilization

  • Pull-down assays with recombinant YobD as bait

  • Yeast two-hybrid using membrane yeast two-hybrid variants

• Genetic and functional screening:

  • Suppressor screens to identify functional partners

  • Synthetic genetic array analysis for genetic interactions

  • Transcriptional response profiling after YobD perturbation

  • Chemical genetic profiling to identify functional pathways

• Computational prediction and validation:

  • Machine learning approaches for interaction prediction

  • Co-evolution analysis to identify potential binding interfaces

  • Molecular docking simulations with candidate partners

  • Network analysis of protein-protein interaction databases

• Validation and characterization:

  • Reconstitution of protein complexes in proteoliposomes

  • Structure determination of complexes using the five-step NMR approach

  • Mutagenesis of predicted interaction interfaces

  • Functional assays to assess biological significance of interactions

The combination of these approaches provides a comprehensive strategy for identifying and validating YobD interaction partners in both bacterial and host cellular contexts.

What are the optimal NMR parameters for studying membrane proteins like YobD in phospholipid bilayers?

Optimizing NMR parameters for YobD structural studies requires attention to several technical aspects:

As described in the research literature, optimal experimental conditions include:

• Instrumentation requirements:

  • High-field NMR spectrometer (700-750 MHz or higher)

  • Specialized probes for solid-state experiments (3.2 mm low-E triple resonance probe)

  • Custom-built probes for oriented sample (OS) solid-state NMR with solenoid coils and strip shields to minimize sample heating

• Pulse sequence optimization:

  • Separated local field (SLF) experiments for resolving orientation-dependent frequencies

  • Multiple-quantum coherence experiments for improved resolution

  • Dipolar recoupling sequences for distance measurements

  • Temperature compensation in pulse programs

• Sample optimization:

  • Protein-to-lipid ratios typically 1:50 to 1:100

  • Hydration levels carefully controlled

  • Sample temperature maintained above lipid phase transition

  • Buffer conditions optimized for protein stability

• Data acquisition strategies:

  • Non-uniform sampling for multidimensional experiments

  • Long experimental times (days to weeks) for complete datasets

  • Multiple complementary experiments for cross-validation

These parameters must be empirically optimized for each specific membrane protein system to achieve the resolution required for structure determination .

What strategies can overcome expression challenges in producing recombinant YobD for structural studies?

Overcoming expression challenges for YobD requires a systematic troubleshooting approach:

• Genetic construct optimization:

  • Screen multiple construct boundaries to identify stable domains

  • Test various affinity and solubility tags (His6, MBP, SUMO, Trx)

  • Optimize codon usage for expression host

  • Consider synthetic gene design with optimized mRNA folding

  • Remove potential toxic elements (internal promoters, RBS sites)

• Expression system selection:

  • Test multiple E. coli strains specialized for membrane proteins (C41/C43, Lemo21)

  • Evaluate eukaryotic systems for complex membrane proteins (Pichia, insect cells)

  • Consider cell-free expression systems for direct incorporation into lipid environments

  • Use dual-plasmid systems with chaperones or membrane insertion machinery

• Culture condition optimization matrix:

• Extraction and purification optimization:

  • Implement high-throughput detergent screening

  • Test alternative solubilization methods (SMALPs, nanodiscs)

  • Optimize buffer components for stability

  • Implement on-column detergent exchange

• Quality control integration:

  • Develop rapid folding assays to guide optimization

  • Implement in-process monitoring of protein quality

  • Use GFP fusion reporters to monitor folding in real-time

  • Apply thermal shift assays to assess stability improvements

This methodical approach addresses the multiple factors that influence successful expression of challenging membrane proteins for structural studies.

What protocols produce the most reliable reconstitution of YobD into membrane mimetics for functional studies?

Reliable reconstitution of YobD requires systematic optimization of multiple parameters:

• Selection of appropriate membrane mimetic:

• Lipid composition optimization:

  • Screen natural E. coli lipid extracts vs. synthetic lipid mixtures

  • Test varying ratios of PC, PE, PG, and cardiolipin

  • Evaluate cholesterol or ergosterol incorporation effects

  • Consider native lipids from Salmonella membranes

• Reconstitution methodology:

  • Detergent removal techniques:

    • Dialysis (slow, gentle)

    • Bio-beads adsorption (controlled rate)

    • Cyclodextrin complexation (rapid)

    • Dilution method (simple but lower efficiency)

  • Physical parameter control:

    • Temperature during reconstitution

    • Protein-to-lipid ratios (typically 1:100 to 1:1000)

    • Buffer ionic strength and pH

    • Presence of stabilizing additives

• Quality control methods:

  • Freeze-fracture electron microscopy to assess distribution

  • Dynamic light scattering for size distribution

  • Sucrose density gradients to verify incorporation

  • Protease protection assays to confirm orientation

• Functional validation:

  • Specific activity measurements in the reconstituted system

  • Comparison with native membrane activity

  • Stability assessment over time

  • Structural integrity verification by spectroscopic methods

For NMR studies specifically, the five-step approach described in research emphasizes the importance of uniformly 13C/15N labeled YobD reconstituted into proteoliposomes with optimized conditions for spectral quality .

How can researchers design definitive experiments to determine YobD's role in Salmonella virulence?

Designing definitive experiments for YobD virulence studies requires rigorous methodology:

• Genetic manipulation strategies:

  • Clean deletion mutants (unmarked, in-frame)

  • Complementation controls (chromosomal, single-copy)

  • Point mutations targeting specific functional domains

  • Regulated expression systems to titrate YobD levels

  • Tagged variants for localization studies

• In vitro infection model matrix:

• In vivo infection approaches:

  • Multiple animal models with different susceptibilities

  • Competitive index assays (wild-type vs. mutant)

  • Tissue-specific bacterial burden determination

  • Immune response characterization

  • Long-term colonization assessment

• Molecular mechanism investigations:

  • Transcriptomic analysis of host and bacterial responses

  • Metabolomic profiling using GCxGC/TOFMS as described in research

  • Protein-protein interaction mapping

  • Membrane integrity and function assessment

• Systems biology integration:

  • Multi-omics data integration

  • Network analysis of affected pathways

  • Mathematical modeling of host-pathogen interaction

  • Machine learning approaches to identify key virulence signatures

This comprehensive experimental framework provides multiple lines of evidence to definitively establish YobD's role in Salmonella virulence through complementary approaches and rigorous controls.

What are the most sensitive analytical methods for detecting conformational changes in YobD upon ligand binding?

Multiple biophysical techniques can detect YobD conformational changes with varying sensitivity:

• Spectroscopic approaches:

  • Circular dichroism (CD) spectroscopy for secondary structure changes

  • Fluorescence spectroscopy with intrinsic tryptophan or introduced fluorophores

  • FTIR spectroscopy for hydrogen bonding network alterations

  • EPR spectroscopy with site-directed spin labeling for domain movement

• NMR-based methods:

  • Chemical shift perturbation mapping in solution NMR

  • Solid-state NMR frequency changes in reconstituted systems

  • Hydrogen-deuterium exchange rates for accessibility changes

  • Relaxation dispersion measurements for microsecond-millisecond dynamics

• Thermodynamic and hydrodynamic techniques:

  • Isothermal titration calorimetry (ITC) for binding energetics

  • Differential scanning calorimetry (DSC) for stability changes

  • Analytical ultracentrifugation for shape and oligomerization changes

  • Size exclusion chromatography coupled with multi-angle light scattering

• Single-molecule approaches:

  • FRET with strategically placed fluorophores

  • Atomic force microscopy for topography changes

  • Single-molecule force spectroscopy for unfolding energy landscapes

  • Nanopore analysis for conductance changes

• Structural biology integration:

  • Time-resolved structural methods (TR-NMR, TR-crystallography)

  • Cryo-EM of ligand-bound vs. unbound states

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Cross-linking mass spectrometry (XL-MS) for distance constraint changes

For membrane proteins specifically, solid-state NMR approaches as described in research offer particular advantages in detecting subtle conformational changes in membrane environments, as they can measure orientation-dependent frequencies that directly reflect structural changes .