Recombinant Shigella dysenteriae serotype 1 UPF0266 membrane protein yobD (yobD)

What is the structural classification of yobD in Shigella dysenteriae serotype 1?

yobD is classified as a UPF0266 family membrane protein with 152 amino acids in its full-length form. It is structurally characterized as an integral membrane protein with multiple transmembrane domains. Based on sequence analysis of related yobD proteins in other Shigella species, it contains hydrophobic regions that anchor it within the bacterial membrane . The protein contains both hydrophobic transmembrane segments and charged residues that are essential for its proper folding and function. Homology modeling suggests it adopts a conformation with multiple α-helical transmembrane segments traversing the bacterial inner membrane.

How conserved is the yobD protein sequence across different Shigella species?

Sequence alignment studies reveal significant conservation of yobD across Shigella species, including S. dysenteriae, S. sonnei, and S. boydii. The amino acid sequence from S. boydii serotype 18 (MTITDLVLILFIAALLAFAIYDQFIMPRRNGPTLLAIPLLRRGRIDSVIFVGLIVILIYN NVTNHGALITTWLLSALALMGFYIFWIRVPKIIFKQKGFFFANVWIEYSLIKAMNLSEDG VLVMQLEQRRLLIRVRNIDNLEKIYKLIVSTQ) represents the highly conserved nature of this protein . Comparative genomic analysis indicates that the functional domains are particularly well-preserved, suggesting evolutionary pressure to maintain specific structural features necessary for the protein's biological role. This conservation extends to homologous proteins in closely related enterobacteria, reflecting the evolutionary relationship between Shigella and other enteric pathogens like E. coli .

What is currently known about the function of yobD in Shigella pathogenesis?

While specific functions of yobD in S. dysenteriae serotype 1 pathogenesis remain under investigation, research on membrane proteins in Shigella species suggests several potential roles. The protein may be involved in maintaining membrane integrity, participating in transport processes, or contributing to bacterial stress responses during infection . Unlike virulence factors such as IpaD that directly mediate host cell invasion , yobD's contribution to pathogenesis appears to be more indirect. Understanding its function represents an important knowledge gap in the field that warrants further research using gene knockout studies and functional assays to determine its precise contribution to bacterial survival and virulence.

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

Several expression systems have been successfully employed for producing recombinant Shigella proteins, including yobD. The most commonly used system is E. coli, particularly strains optimized for membrane protein expression such as C41(DE3), C43(DE3), or Rosetta(DE3) . When expressing yobD, researchers should consider the following protocol:

• Clone the yobD gene into an expression vector containing an appropriate tag (His-tag is common)

• Transform the construct into an expression host (E. coli Rosetta(DE3) has shown good results)

• Induce expression with IPTG (typically 0.5-1.0 mM) at reduced temperature (16-25°C) to enhance proper folding

• Extract the membrane fraction using detergents compatible with membrane proteins

Alternative expression systems include yeast (P. pastoris), baculovirus-infected insect cells, or mammalian cells when E. coli-produced protein exhibits folding or solubility issues .

What purification strategies yield the highest purity recombinant yobD protein?

Purification of recombinant yobD typically employs a multi-step approach:

• Affinity chromatography using the attached tag (Ni-NTA for His-tagged proteins) as the initial capture step

• Size-exclusion chromatography to remove aggregates and other contaminants

• Ion-exchange chromatography for final polishing if necessary

For optimal results with membrane proteins like yobD, incorporate these critical considerations:

• Use mild detergents (DDM, LDAO, or OG) throughout purification to maintain protein stability

• Include glycerol (5-10%) in all buffers to prevent aggregation

• Maintain consistent pH (typically pH 7.4-8.0) throughout purification

• Consider detergent exchange during purification if the initial extraction detergent is not optimal for downstream applications

Reported yields for purified recombinant membrane proteins from Shigella species are typically in the range of 0.5-1.0 mg/mL of culture .

What techniques are most effective for determining the structural characteristics of yobD?

The structural characterization of membrane proteins like yobD presents significant challenges requiring specialized approaches:

• X-ray crystallography: Requires detergent-solubilized protein to be crystallized, often facilitated by:

  • Fusion partners (e.g., T4 lysozyme) to increase soluble domains

  • Lipidic cubic phase crystallization methods

  • Antibody fragment co-crystallization to stabilize flexible regions

• Cryo-electron microscopy (cryo-EM): Increasingly powerful for membrane protein structure determination:

  • Sample preparation in nanodiscs or amphipols can preserve native-like environment

  • Single-particle analysis for proteins >100 kDa

  • Subtomogram averaging for membrane proteins in liposomes

• NMR spectroscopy: Useful for dynamics studies and determining membrane topology:

  • Solid-state NMR for intact membrane proteins

  • Solution NMR for smaller fragments or domains

  • Requires isotopic labeling (13C, 15N, 2H)

• Computational approaches:

  • Homology modeling based on related proteins with known structures

  • Molecular dynamics simulations to predict membrane interactions and conformational changes

  • Evolutionary coupling analysis to predict residue contacts

For yobD specifically, researchers should consider combining biophysical techniques with computational approaches, as crystallization of this membrane protein has proven challenging .

How can researchers assess the membrane topology of yobD?

Determining the membrane topology of yobD requires integrating multiple experimental approaches:

• Computational prediction:

  • Hydropathy analysis using algorithms like TMHMM, HMMTOP, or Phobius

  • Prediction suggests yobD contains multiple transmembrane helices

• Experimental validation:

  • PhoA/LacZ fusion assays: Create fusions at different positions and measure enzymatic activity to determine cytoplasmic vs. periplasmic localization

  • Cysteine accessibility methods: Introduce cysteine residues and test their accessibility to membrane-impermeable reagents

  • Protease protection assays: Determine which regions are protected from proteolysis when in membrane vesicles

• Biophysical approaches:

  • Site-specific fluorescent labeling to track exposure to aqueous or lipid environments

  • EPR spectroscopy with spin-labeled residues to determine membrane insertion depth

The consensus topology model should integrate computational predictions with experimental validation to develop an accurate structural understanding of yobD's membrane orientation .

How does yobD compare to other Shigella antigens as a potential vaccine candidate?

In the context of vaccine development against Shigella dysenteriae, several antigens have been investigated:

What are the methodological considerations for studying protein-protein interactions involving yobD?

Investigating protein-protein interactions for membrane proteins like yobD requires specialized approaches:

• Genetic interaction methods:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Genetic suppressor screens to identify functional partners

  • CRISPR interference screens to identify synthetic lethal interactions

• Biochemical approaches:

  • Co-immunoprecipitation using detergent-solubilized membranes

  • Crosslinking coupled with mass spectrometry (XL-MS) to capture transient interactions

  • Pull-down assays with purified recombinant yobD as bait

• Biophysical techniques:

  • Surface plasmon resonance (SPR) with recombinant yobD immobilized in lipid nanodiscs

  • Microscale thermophoresis (MST) for quantitative binding measurements

  • Förster resonance energy transfer (FRET) to detect interactions in reconstituted systems

• Computational prediction:

  • Protein-protein docking simulations

  • Coevolutionary analysis to predict interaction interfaces

  • Integrated network analysis using genomic context and expression data

When designing interaction studies, researchers should consider the native membrane environment of yobD and how detergents or membrane mimetics might affect interaction properties .

What approaches can be used to investigate the role of yobD in bacterial stress responses?

Understanding yobD's potential role in stress response requires multi-faceted experimental approaches:

• Transcriptional analysis:

  • RNA-Seq or qRT-PCR to measure yobD expression under various stress conditions (pH, antimicrobial peptides, oxidative stress, nutrient limitation)

  • Promoter-reporter fusions to monitor real-time expression changes

  • ChIP-Seq to identify transcription factors regulating yobD expression

• Mutant phenotyping:

  • Construction of yobD deletion or depletion strains

  • Phenotypic characterization under various stress conditions

  • Complementation studies to confirm phenotype specificity

  • Competition assays between wild-type and mutant strains

• Proteomic approaches:

  • Quantitative proteomics to measure yobD protein levels during stress

  • Protein turnover analysis to determine stability under stress

  • Post-translational modification analysis

• Physiological assays:

  • Membrane integrity measurements using fluorescent dyes

  • Membrane potential assessment

  • Permeability assays with various compounds

These methodologies can reveal whether yobD is involved in general stress responses or specific adaptations to environmental challenges encountered during infection .

How does yobD from S. dysenteriae differ from homologs in other Shigella species?

Comparative analysis of yobD across Shigella species reveals important insights:

The high sequence conservation in transmembrane domains suggests fundamental structural and functional roles, while variations in exposed regions may reflect adaptation to specific microenvironments or interactions. Researchers investigating yobD should consider these species-specific differences when extrapolating findings between Shigella pathogens .

What experimental approaches can determine if yobD functions similarly across Shigella species?

To determine functional conservation of yobD across Shigella species, researchers can employ:

• Complementation studies:

  • Create yobD deletion mutants in multiple Shigella species

  • Cross-complement with yobD variants from different species

  • Assess restoration of wild-type phenotypes

• Domain swap experiments:

  • Generate chimeric proteins with domains from different species

  • Identify regions responsible for species-specific functions

  • Map critical residues through site-directed mutagenesis

• Heterologous expression:

  • Express yobD variants in a non-Shigella background

  • Compare phenotypic effects and interaction profiles

  • Identify host-specific requirements for function

• Structural comparison:

  • Determine structures of multiple yobD homologs

  • Compare membrane topology and critical structural features

  • Correlate structural differences with functional variations

These approaches can establish whether yobD represents a core function conserved across Shigella species or exhibits species-specific adaptations relevant to the distinct pathogenic profiles of each species .

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

Researchers working with recombinant yobD face several technical challenges:

For reliable production of recombinant yobD, researchers should systematically optimize expression and purification conditions, potentially combining strategies tailored to membrane proteins.

How can researchers validate the proper folding and function of recombinant yobD?

Ensuring proper folding of recombinant yobD is critical for downstream applications:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Tryptophan fluorescence spectroscopy to evaluate tertiary structure

  • Dynamic light scattering (DLS) to detect aggregation

  • Thermal shift assays to determine protein stability

• Functional validation:

  • Ligand binding assays if specific ligands are known

  • Activity assays based on predicted function

  • Reconstitution into liposomes to test membrane integration

• Structural integrity:

  • Limited proteolysis to probe for properly folded domains

  • Epitope accessibility using conformation-specific antibodies

  • Native PAGE to assess oligomeric state

• Comparative analysis:

  • Compare properties with native protein extracted from Shigella

  • Assess complementation of yobD mutant phenotypes

These validation steps are essential before using recombinant yobD in downstream applications such as structural studies, interaction mapping, or immunological characterization .