Recombinant Yersinia pestis UPF0266 membrane protein YPDSF_1369 (YPDSF_1369)

What is Recombinant Yersinia pestis UPF0266 membrane protein YPDSF_1369?

Recombinant Yersinia pestis UPF0266 membrane protein YPDSF_1369 is a membrane-associated protein from the bacterial pathogen Yersinia pestis (strain Pestoides F). This protein belongs to the UPF0266 protein family, which includes uncharacterized protein families with limited functional annotation. The recombinant form is laboratory-expressed for research purposes, typically with 153 amino acids covering the expression region 1-153 . The protein is characterized by its hydrophobic transmembrane domains and contains several highly conserved regions that suggest functional importance in bacterial membrane processes.

How should researchers store and handle Recombinant Yersinia pestis UPF0266 membrane protein YPDSF_1369 samples?

For optimal stability and activity, store Recombinant Yersinia pestis UPF0266 membrane protein YPDSF_1369 at -20°C for regular use, or at -80°C for extended storage periods. The protein is typically supplied in a Tris-based buffer containing 50% glycerol optimized for stability . When working with the protein, prepare working aliquots to be stored at 4°C for no more than one week to minimize protein degradation. Avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity and activity . For experiments requiring extended use, prepare multiple single-use aliquots during initial thawing.

What experimental controls should be included when working with YPDSF_1369?

When designing experiments with YPDSF_1369, several controls are essential:

• Negative controls: Include buffer-only samples and non-recombinant expression systems (lacking the YPDSF_1369 gene) to establish baseline measurements.

• Positive controls: When available, use characterized membrane proteins from the same organism with known activities or a well-studied protein from the same UPF0266 family.

• Denatured protein control: Include heat-inactivated YPDSF_1369 to distinguish between specific and non-specific effects, similar to methodologies used with Ail protein studies where heat-treated proteins showed altered binding properties .

• Tag-only control: If the recombinant protein contains affinity tags, test the tag alone to ensure observed effects are not tag-mediated.

These controls help establish experimental validity by controlling for extraneous variables that might confound results, maintaining the principles of sound experimental design .

How can researchers determine the membrane topology of YPDSF_1369?

Determining the membrane topology of YPDSF_1369 requires multiple complementary approaches:

• Computational prediction: Begin with bioinformatic tools like TMHMM, HMMTOP, or PredictProtein to predict transmembrane regions based on the amino acid sequence: MSVTDLVLVVFIALLLIYAIYDEFIMNMMKGKTRLQVHLKRKNKLDCMIFVGLIGILIYN NVMAHGAPLTTYLLVGLALVAVYISYIRWPKLLFKNTGFFYANTFIEYSRIKSMNLSEDG ILVIDLEQRRLLIQVKKLDDLEKIYNFFIENQS .

• Protease protection assays: Express the protein in membrane vesicles and treat with proteases. Protected fragments indicate regions embedded within the membrane or facing the vesicle lumen.

• Cysteine scanning mutagenesis: Systematically replace residues with cysteine and probe accessibility with membrane-impermeable reagents.

• Epitope tagging: Insert epitope tags at various positions and determine their accessibility using antibodies.

• Nanodiscs reconstitution: Similar to the approach used for Ail protein , incorporate YPDSF_1369 into nanodiscs for structural studies.

Data from these approaches should be compiled in a formatted table as shown below:

What techniques are most appropriate for studying protein-protein interactions of YPDSF_1369?

For investigating protein-protein interactions of YPDSF_1369, consider these methodologies:

• Pull-down assays: Use affinity-tagged YPDSF_1369 to identify binding partners from cell lysates, following with mass spectrometry identification.

• Cross-linking coupled with mass spectrometry: Chemical cross-linking can capture transient interactions before protein complex analysis.

• Bacterial two-hybrid systems: Especially useful for membrane proteins, these systems can detect interactions in a cellular context.

• Surface Plasmon Resonance (SPR): Provides quantitative binding kinetics for purified interaction partners.

• ELISA-based binding assays: Similar to those used to demonstrate Ail-vitronectin interactions , ELISA can be adapted to study YPDSF_1369 interactions with potential binding partners.

• Nanodiscs reconstitution: Incorporating YPDSF_1369 into nanodiscs provides a native-like membrane environment for interaction studies.

When designing these experiments, ensure proper controls for non-specific binding and implement a true experimental design with randomization of conditions to establish causality in observed interactions .

How should researchers design experiments to investigate the function of YPDSF_1369 in Yersinia pestis?

To investigate YPDSF_1369 function, implement a multi-faceted experimental design:

• Gene knockout/complementation: Create a YPDSF_1369 deletion mutant and complemented strain to assess phenotypic changes.

• Controlled variable manipulation: Systematically manipulate independent variables (e.g., growth conditions, stress factors) while measuring dependent variables (e.g., bacterial survival, membrane integrity) .

• Comparative analysis with known membrane proteins: Compare phenotypes with those of characterized membrane proteins like Ail, which is known to facilitate survival in host tissues .

• Structure-function analysis: Generate targeted mutations based on the sequence (MSVTDLVLVVFIALLLIYAIYDEFIMNMMKGKTRLQVHLKRKNKLDCMIFVGLIGILIYNN VMAHGAPLTTYLLVGLALVAVYISYIRWPKLLFKNTGFFYANTFIEYSRIKSMNLSEDGILVI DLEQRRLLIQVKKLDDLEKIYNFFIENQS) to identify functional domains.

• Controlled infection models: Compare wild-type and mutant strains in relevant infection models, measuring survival, dissemination, and host response.

Use a randomized block design to control for extraneous variables, clearly defining independent variables (genetic modifications, environmental conditions) and dependent variables (measurable outcomes) .

How can researchers differentiate between the functions of YPDSF_1369 and other membrane proteins like Ail in Yersinia pestis pathogenesis?

Differentiating the functions of YPDSF_1369 from other membrane proteins like Ail requires sophisticated experimental approaches:

• Double knockout and complementation studies: Generate single and double knockout mutants (ΔYPDSF_1369, ΔAil, and ΔYPDSF_1369/ΔAil) with corresponding complemented strains to identify unique and overlapping functions.

• Comparative binding assays: Assess binding to host components (ECM proteins, complement regulators) similar to Ail's known interactions with fibronectin, laminin, and vitronectin . Use purified proteins in ELISA or SPR to quantify differential binding.

• Domain swap experiments: Create chimeric proteins exchanging domains between YPDSF_1369 and Ail to map functional regions.

• Differential transcriptomics: Compare host cell transcriptional responses to wild-type, ΔYPDSF_1369, and ΔAil strains to identify protein-specific host responses.

• Competitive infection assays: Co-infect with differentially tagged wild-type and mutant strains to assess fitness contributions of each protein.

When analyzing results, use multivariate statistical approaches to differentiate direct protein effects from secondary consequences of membrane disruption.

What methodologies are appropriate for resolving data contradictions in YPDSF_1369 functional studies?

When faced with contradictory data in YPDSF_1369 functional studies, employ these methodological approaches:

• Standardize experimental conditions: Ensure consistency in protein preparation, buffer composition, and assay conditions to eliminate technical variability.

• Independent verification: Use multiple techniques to test the same hypothesis, similar to how Ail-vitronectin binding was confirmed by multiple methods including ELISA and direct binding assays .

• Temporal resolution studies: Investigate if contradictory results reflect different time points in a dynamic process rather than true contradictions.

• Context-dependent function analysis: Test if YPDSF_1369 functions differently in various cellular contexts or membrane environments.

• Statistical meta-analysis: When multiple datasets exist, perform formal meta-analysis to identify sources of heterogeneity.

• Collaborative cross-validation: Establish collaborations with independent laboratories to cross-validate findings using standardized protocols.

When reporting contradictory results, present data in tables featuring the following columns: Experimental Condition, Method, Observation, Statistical Significance, and Potential Confounding Factors .

How can researchers design experiments to investigate potential interactions between YPDSF_1369 and host immune system components?

To investigate YPDSF_1369 interactions with host immune components, design experiments following these principles:

• Binding assays with immune factors: Assess direct binding to complement components, antibodies, and pattern recognition receptors using techniques similar to those that demonstrated Ail binding to C4b-binding protein and vitronectin .

• Complement resistance assays: Compare serum resistance of wild-type, ΔYPDSF_1369, and complemented strains to determine if YPDSF_1369 contributes to complement evasion similar to Ail .

• Immune cell interaction studies: Quantify phagocytosis rates, survival in macrophages, and neutrophil activation using wild-type and mutant strains.

• Structural determination in complex with immune components: Use X-ray crystallography or cryo-EM to resolve structures of YPDSF_1369 bound to identified immune factors.

• In vivo immune evasion studies: Compare infection progression in immunocompetent versus immunodeficient animal models.

Implement a true experimental design with appropriate controls and randomization to establish causality . Create a comprehensive data table documenting all experimental variables and outcomes, formatted according to standard scientific guidelines .

What are the best practices for purifying recombinant YPDSF_1369 while maintaining native conformation?

To purify recombinant YPDSF_1369 while preserving native conformation:

• Membrane protein extraction optimization:

  • Use mild detergents (DDM, LDAO, or OG) at concentrations just above CMC

  • Extract at 4°C with gentle agitation

  • Include protease inhibitors to prevent degradation

• Purification strategy:

  • Implement affinity chromatography using carefully positioned tags that don't interfere with protein folding

  • Follow with size exclusion chromatography to isolate properly folded monomers

  • Consider using nanodiscs or liposomes for reconstitution similar to methods used with Ail protein

• Conformation verification:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Limited proteolysis to assess proper folding

  • Functional binding assays to verify activity

• Storage conditions:

  • Store in Tris-based buffer with 50% glycerol at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles as noted in product guidelines

  • Monitor protein stability over time using activity assays

Document purification yields and specific activity at each step to identify critical points where native conformation might be compromised.

How should researchers design data tables for YPDSF_1369 binding studies?

When designing data tables for YPDSF_1369 binding studies, follow these guidelines:

• Table organization:

  • List the independent variable (e.g., concentrations, conditions) in the leftmost column

  • Place dependent variables (e.g., binding percentages, affinity constants) in subsequent columns

  • Include columns for each experimental trial and statistical parameters

• Essential information:

  • Include clear headers with units of measurement

  • Document experimental conditions affecting binding

  • Report both raw data and calculated parameters (Kd, Bmax)

• Statistical representation:

  • Include standard deviation or standard error

  • Add columns for p-values when comparing conditions

  • Note sample size for each measurement

Example binding study data table:

What considerations should researchers make when designing comparative studies between YPDSF_1369 and other bacterial membrane proteins?

When designing comparative studies between YPDSF_1369 and other bacterial membrane proteins:

• Protein selection criteria:

  • Include proteins with similar structural features for valid comparisons

  • Select proteins from the same organism (other Y. pestis membrane proteins like Ail )

  • Include proteins from the same family from different organisms

  • Choose proteins with well-characterized functions as benchmarks

• Standardization of experimental conditions:

  • Use identical expression systems and purification methods

  • Ensure equivalent protein concentrations and buffer conditions

  • Standardize membrane mimetic environments (nanodiscs, liposomes)

• Multifaceted functional comparison:

  • Compare binding profiles to the same panel of host factors

  • Assess membrane integrity contributions

  • Evaluate functional complementation in knockout models

• Structural comparison considerations:

  • Use consistent structural determination methods

  • Compare topology models generated by identical methods

  • Analyze sequence conservation in functional domains

• Experimental design principles:

  • Implement a true experimental design with appropriate controls

  • Use randomized block design to control for extraneous variables

  • Document all experimental variables systematically

When reporting results, include a comparison table with standardized metrics across all proteins studied to facilitate direct comparison of functional properties.

What are the key considerations for integrating YPDSF_1369 research findings into broader studies of Yersinia pestis pathogenesis?

Integrating YPDSF_1369 research findings into broader Yersinia pestis pathogenesis studies requires careful consideration of several factors:

• Contextual interpretation: Position YPDSF_1369 findings within the established framework of Y. pestis virulence mechanisms, particularly in relation to other membrane proteins like Ail that facilitate survival in host tissues .

• Systems biology approach: Incorporate YPDSF_1369 data into network analyses of Y. pestis pathogenesis to identify functional relationships with other virulence factors.

• Temporal expression patterns: Correlate YPDSF_1369 expression with disease progression stages to understand its role in the infection cycle.

• Host-pathogen interface: Analyze how YPDSF_1369 functions complement or synergize with other bacterial factors at the host-pathogen interface, similar to how Ail contributes to attachment and complement resistance .

• Translational relevance: Evaluate findings for potential applications in diagnostic development, vaccine design, or novel therapeutic approaches.

• Methodological standardization: Ensure experimental approaches align with established protocols in the field to facilitate data integration and meta-analysis.

By contextualizing YPDSF_1369 research within the broader understanding of Y. pestis pathogenesis, researchers can contribute to a more comprehensive model of plague pathophysiology and potentially identify new targets for intervention strategies.

What future research directions should be prioritized for understanding YPDSF_1369 function?

Priority research directions for advancing understanding of YPDSF_1369 function include:

• High-resolution structural studies: Determine the three-dimensional structure of YPDSF_1369 using X-ray crystallography or cryo-EM to inform structure-function relationships.

• Comprehensive interactome mapping: Identify the complete set of host and bacterial proteins that interact with YPDSF_1369 using proximity labeling techniques coupled with mass spectrometry.

• In vivo significance assessment: Develop animal models to evaluate the contribution of YPDSF_1369 to Y. pestis virulence and tissue tropism.

• Evolutionary analysis: Compare YPDSF_1369 across Yersinia species and strains to understand selective pressures and functional conservation.

• Host response characterization: Determine how YPDSF_1369 modulates host immune responses, particularly in comparison to well-characterized membrane proteins like Ail .

• Therapeutic targeting evaluation: Assess YPDSF_1369 as a potential target for novel anti-virulence therapeutics or vaccine development.

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics approaches to position YPDSF_1369 within regulatory networks controlling Y. pestis adaptation to host environments.