Recombinant Receptor polysaccharide phosphotransferase wefF (wefF)

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Product Specs

Form
Lyophilized powder
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Lead Time
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Notes
Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.
Reconstitution
Centrifuge the vial briefly before opening to collect the contents. Reconstitute the protein in sterile, deionized water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard glycerol concentration is 50% and serves as a guideline.
Shelf Life
Shelf life depends on various factors including storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is essential for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
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Synonyms
wefF; Receptor polysaccharide phosphotransferase WefF; EC 2.7.-.-; Stealth protein WefF
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-333
Protein Length
full length protein
Purity
>85% (SDS-PAGE)
Species
Streptococcus mitis
Target Names
wefF
Target Protein Sequence
MVEKIDFVVA WVDGNDPVWR KKKAQYDGTI NTSKEGMNSD KAYREWGTFK YWFRGVEKFA PWVNKVYLVT DNQKPSWLEL NSDKLVLVDH TEIICNDYLP VFSANPIESN IHRIPGLSEC FVFFNDDVYL TAPVEPTDFF SDDGLPKYVT ALAPITTERY GTGHFQMNDM GIITTHFTRE EILKNGKFFS IKQGVKGIAK SLLYRHSKFI CGFWENHLTH PLLKSTMELV WEKEKDVLEK TSASRFRSPS DTNVWLFKYW QIASGQYAIG NPKLGGLFSL DNAGPDFWKL LNSRKYKVMC INDGFNVQNE NQVMDDFIKA MNQLFPDKSS FEI
Uniprot No.

Target Background

Function
This protein is part of the type 2G receptor polysaccharide (RPS) biosynthesis locus. It is essential for cell surface RPS production and the host-like Gal β1-3GalNAc (G) motif of the RPS. It likely encodes a 1-3GalNAc α-transferase.
Protein Families
Stealth family

Q&A

What is the bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system and how does WefF fit into this system?

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) is a complex bacterial mechanism that performs dual roles in cellular metabolism. Primarily, it catalyzes the transport and phosphorylation of various sugars and sugar derivatives across the cell membrane. Additionally, the PTS carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, chemotaxis, potassium transport, and even virulence in certain pathogens .

The PTS functions as a phosphorylation cascade, where phosphoryl groups from PEP are transferred through a series of proteins including Enzyme I (EI), HPr, and EIIA/EIIB pairs before phosphorylating the target carbohydrate bound to the membrane-spanning EIIC component . WefF (Recombinant Receptor polysaccharide phosphotransferase) operates within this broader PTS framework, likely involved in the phosphorylation of specific polysaccharide substrates in the bacterial cell envelope.

What are the structural domains of WefF and their functional significance?

WefF, like other PTS components, likely contains specific domains that participate in the phosphorylation cascade. Based on known PTS structures, WefF would be expected to contain phosphorylation domains that either acquired a PTS domain by fusion to their N or C termini, integrated a specific PTS regulation domain (PRD), or developed unique sites for PTS-mediated phosphorylation .

The functional significance of these domains relates to protein-protein interactions that occur with either phosphorylated or unphosphorylated PTS components. These interactions can either stimulate or inhibit the function of target proteins, allowing the PTS to regulate numerous proteins and form an extensive regulatory network responsive to the phosphorylation state of various PTS components .

How does the phosphorylation state of WefF influence its regulatory functions?

The phosphorylation state of PTS components, including WefF, serves as a signal that varies according to substrate availability and the metabolic state of the cell. This phosphorylation state is critical for the regulatory functions of the system . For WefF specifically, its phosphorylation state would determine its ability to interact with and regulate other proteins in the bacterial cell.

The regulation mechanisms are based on either direct phosphorylation of target proteins or phosphorylation-dependent interactions . When the cell has abundant PTS substrates, the phosphorylation level of PTS components decreases as phosphoryl groups are transferred to incoming sugars. Conversely, when PTS substrates are scarce, the phosphorylation levels of PTS components increase. These changes in phosphorylation state trigger appropriate regulatory responses, helping bacteria adapt to changing nutrient conditions .

What are the optimal experimental approaches to study WefF phosphorylation dynamics in vitro?

To effectively study WefF phosphorylation dynamics in vitro, researchers should implement a multivariate experimental design rather than the traditional one-variable-at-a-time (OVAT) approach. The experimental design should account for interactions among variables, as these interactions are central to understanding the complex regulatory functions of phosphotransferase systems .

A factorial design approach would be most appropriate, where multiple factors (such as substrate concentration, temperature, pH, and presence of potential regulatory molecules) are varied simultaneously according to a predetermined pattern . This approach not only provides global knowledge across the entire experimental domain but also yields higher quality information with fewer experiments than traditional approaches.

For example, if studying the effects of four variables on WefF phosphorylation kinetics, a 2^4 factorial design would require only 16 experiments to map the entire response surface, including all interaction effects. Researchers should consider the following methodological steps:

  • Define the experimental domain (ranges for each variable)

  • Select the appropriate model (typically starting with a first-order model with interactions)

  • Plan the experiments according to the chosen experimental design

  • Perform the experiments in random order to avoid systematic errors

  • Analyze the data to build a mathematical model of WefF phosphorylation dynamics

How can researchers accurately quantify the effects of amino acid substitutions on WefF substrate specificity?

Quantifying the effects of amino acid substitutions on WefF substrate specificity requires a systematic approach combining structural analysis, site-directed mutagenesis, and kinetic measurements. Researchers should focus on amino acid residues likely involved in substrate recognition and binding based on structural homology with other phosphotransferase systems.

The experimental approach should include:

  • Identification of candidate residues for mutagenesis based on:

    • Sequence alignment with other phosphotransferases

    • Structural modeling to identify residues in proximity to predicted binding sites

    • Evolutionary conservation analysis

  • Generation of a panel of WefF variants with single or multiple amino acid substitutions

  • Expression and purification of recombinant wild-type and mutant WefF proteins

  • Kinetic characterization using a diverse panel of potential substrates, measuring:

    • Substrate binding affinity (Km)

    • Catalytic efficiency (kcat/Km)

    • Phosphoryl transfer rates

    • Competitive inhibition profiles

  • Analysis of results using multivariate statistical methods to develop structure-function relationships

This systematic approach will enable researchers to construct a detailed map of how specific amino acid positions contribute to substrate recognition and catalytic function in WefF.

What are the key considerations when designing experiments to study WefF-mediated regulatory networks in bacteria?

When designing experiments to study WefF-mediated regulatory networks, researchers must account for the complex interconnections between phosphotransferase systems and other cellular processes. The PTS regulation network not only controls carbohydrate uptake and metabolism but also interfaces with nitrogen and phosphorus utilization and can affect virulence in certain pathogens .

Key experimental design considerations include:

  • System-wide perspective: Rather than studying isolated components, researchers should adopt approaches that capture the entire regulatory network, such as:

    • Transcriptomics to identify genes whose expression is affected by WefF

    • Proteomics to identify proteins whose abundance or modification state changes

    • Metabolomics to track changes in relevant metabolite pools

    • Phosphoproteomics to identify proteins phosphorylated by or dependent on WefF

  • Temporal dynamics: Regulatory networks operate on different time scales, requiring time-course experiments that capture both rapid phosphorylation events and slower transcriptional responses.

  • Environmental conditions: Test multiple environmental conditions that affect PTS activity, including:

    • Varying carbon sources (PTS and non-PTS sugars)

    • Nitrogen limitation

    • Phosphate limitation

    • Various stress conditions

  • Genetic approaches: Utilize:

    • WefF deletion mutants

    • Phosphorylation site mutants (e.g., histidine to alanine substitutions)

    • Reporter gene fusions to monitor activity of regulated genes

    • Suppressor screens to identify genetic interactions

  • Data integration: Develop computational models that integrate experimental data to predict system behavior under untested conditions

What qualitative research methods are most effective for investigating WefF function in complex bacterial communities?

Investigating WefF function in complex bacterial communities requires qualitative research methods that can reveal underlying patterns and mechanisms. The most effective approaches include:

  • Ethnographic research in microbial ecology: Observing bacterial communities in their natural environments to understand WefF's role in inter-species interactions. This involves techniques such as:

    • Fluorescence in situ hybridization (FISH) to identify specific bacterial populations

    • Confocal microscopy to visualize spatial organization

    • Time-lapse imaging to track community dynamics

  • Case study research: Detailed examination of specific bacterial communities where WefF plays a crucial role, such as:

    • Biofilm communities

    • Host-associated microbiomes

    • Environmental microbial consortia

  • Qualitative observations: Systematic documentation of phenotypic changes in bacterial communities when WefF function is altered, focusing on:

    • Colony morphology

    • Biofilm architecture

    • Interspecies spatial relationships

    • Succession patterns

These qualitative methods should complement quantitative approaches to provide a more comprehensive understanding of WefF function in complex microbial systems.

How can researchers accurately measure the phosphorylation state of WefF in vivo?

Accurately measuring the phosphorylation state of WefF in vivo presents significant challenges due to the labile nature of histidine and cysteine phosphorylations typical in PTS proteins and the rapid turnover of these phosphoryl groups. Researchers should consider the following methodological approaches:

  • Phosphoproteomic analysis with special preservation techniques:

    • Rapid quenching of cellular metabolism using trichloroacetic acid precipitation

    • Use of phosphatase inhibitors specifically designed to preserve histidine phosphorylation

    • Enrichment of phosphopeptides using titanium dioxide or immobilized metal affinity chromatography

    • Mass spectrometry analysis with fragmentation methods optimized for phosphohistidine detection

  • In vivo genetic reporters:

    • Construction of split fluorescent protein systems where reconstitution depends on the phosphorylation state of WefF

    • FRET-based sensors that change conformation upon phosphorylation

    • Phosphorylation-dependent protein-protein interaction reporters

  • Radioactive labeling approaches:

    • Pulse-chase experiments with 32P-labeled phosphoenolpyruvate

    • Selective capture of labeled phosphoproteins

    • Quantification of label transfer kinetics

  • Conformation-specific antibodies:

    • Development of antibodies that specifically recognize the phosphorylated or unphosphorylated forms of WefF

    • Immunoprecipitation followed by western blotting to quantify relative amounts

These methods should be calibrated using in vitro phosphorylated WefF as a standard, and researchers should be aware of the potential for rapid changes in phosphorylation state during sample processing .

What statistical approaches are most appropriate for analyzing WefF experimental data with weighted samples?

When analyzing experimental data involving WefF with weighted samples, researchers must carefully consider how weighting affects the precision of their estimates. The Weighting Effect Factor (WEFF) is a critical statistical measure for determining the loss of precision after sample weighting .

The WEFF is defined as:

WEFF=1+σw2μw2\text{WEFF} = 1 + \frac{\sigma^2_w}{\mu_w^2}

where σw2\sigma^2_w is the population variance of the weights and μw\mu_w is the mean of the weights. Alternatively, it can be calculated as:

WEFF=ni=1nxi2(i=1nxi)2\text{WEFF} = \frac{n\sum_{i=1}^{n}x_i^2}{(\sum_{i=1}^{n}x_i)^2}

where n is the number of weights and x is the weight variable .

To determine the effective sample size after weighting, researchers should divide the actual sample size by the WEFF. A smaller WEFF value is desirable as it indicates a smaller loss of precision.

Appropriate statistical approaches for weighted WefF data include:

  • Weighted regression analysis: To account for different levels of reliability in different observations

  • Hierarchical modeling: When data comes from different experimental conditions or bacterial strains

  • Bootstrap resampling with weights: To generate confidence intervals that properly account for the weighting scheme

  • Propensity score methods: When attempting to control for confounding variables in observational studies

It's crucial to note that a low WEFF does not necessarily mean a representative sample. The variables chosen for weighting should be carefully selected based on their biological relevance to WefF function .

How should researchers interpret contradictory results about WefF function from different experimental systems?

When faced with contradictory results about WefF function from different experimental systems, researchers should apply a systematic approach to reconcile these discrepancies:

  • Evaluate experimental contexts thoroughly:

    • Compare the bacterial strains used (laboratory vs. clinical isolates)

    • Assess growth conditions (rich vs. minimal media, aerobic vs. anaerobic)

    • Examine the genetic backgrounds (presence of suppressor mutations)

    • Consider environmental signals (carbon source availability, stress conditions)

  • Analyze methodological differences:

    • In vitro vs. in vivo studies may yield different results due to the complex regulatory networks in living cells

    • Recombinant protein studies may not capture native protein modifications

    • Different assay sensitivities and dynamic ranges may lead to apparently contradictory results

  • Consider strain-specific variations:

    • WefF function may genuinely differ between bacterial species or strains

    • Genetic context may alter WefF regulatory networks

    • Create a comparison table documenting key differences between experimental systems:

Experimental SystemOrganismGrowth ConditionsWefF Expression LevelObserved FunctionKey Regulatory PartnersStudy Limitations
System AE. coli K12Minimal media, glucoseEndogenousSugar transport regulationHPr, EILimited to in vitro assays
System BP. aeruginosaLB media, aerobicOverexpressedVirulence factor regulationUnknownArtificial expression level
System CB. subtilisBiofilm conditionsDeletion mutantSporulation effectsSpoA, CcpAPolar effects on downstream genes
  • Develop integrative models:

    • Create testable hypotheses that could explain apparent contradictions

    • Design experiments specifically to test these hypotheses

    • Consider that WefF may have multiple functions depending on cellular context

What are the most common pitfalls in interpreting WefF phosphorylation cascade data?

Interpreting data from WefF phosphorylation cascade studies presents several common pitfalls that researchers should be aware of:

  • Overlooking the rapid dynamics of phosphoryl transfer:

    • PTS phosphorylation states can change in milliseconds

    • Sample preparation time may alter the observed phosphorylation state

    • Static measurements may miss important kinetic aspects of the cascade

  • Misinterpreting in vitro results:

    • Isolated components may behave differently than in the cellular context

    • Buffer conditions strongly influence phosphoryl transfer rates

    • Recombinant protein modifications may differ from native proteins

  • Failing to account for cross-talk between different PTS systems:

    • Phosphoryl groups can be transferred between different PTS pathways

    • Regulatory effects may be indirect through other PTS components

    • Non-PTS proteins may intercept phosphoryl groups

  • Misattributing regulatory effects:

    • Changes in gene expression may result from indirect effects

    • Phenotypic changes may have multiple causes beyond WefF

    • Compensatory mutations may mask true WefF functions

  • Confusing correlation with causation:

    • Coincidental changes in phosphorylation and cellular processes

    • Failure to perform appropriate control experiments

    • Not testing direct causality through site-directed mutagenesis

To avoid these pitfalls, researchers should:

  • Use multiple complementary approaches to confirm findings

  • Include appropriate time-resolved measurements

  • Perform careful genetic controls, including point mutations of phosphorylation sites

  • Consider the entire PTS system rather than isolated components

How can researchers differentiate between direct and indirect regulatory effects of WefF phosphorylation?

Differentiating between direct and indirect regulatory effects of WefF phosphorylation is essential for accurately mapping its regulatory network. Researchers should employ the following strategies:

  • Phosphorylation site mapping and mutation:

    • Identify specific phosphorylation sites in WefF using mass spectrometry

    • Create phosphomimetic (e.g., His→Asp) and phosphoablative (e.g., His→Ala) mutations

    • Compare phenotypes of these mutants to distinguish direct effects

    • Analyze how these mutations affect downstream phosphorylation events

  • Temporal resolution studies:

    • Use time-course experiments with high temporal resolution

    • Direct effects typically occur rapidly after WefF phosphorylation

    • Indirect effects often appear with a time delay

    • Apply mathematical modeling to distinguish immediate vs. delayed responses

  • Protein-protein interaction studies:

    • Use techniques like bacterial two-hybrid assays or co-immunoprecipitation

    • Compare interactions of wild-type WefF vs. phosphorylation site mutants

    • Apply crosslinking approaches to capture transient interactions

    • Determine if interactions depend on phosphorylation state

  • In vitro reconstitution experiments:

    • Reconstitute minimal systems with purified components

    • Add components systematically to identify which ones are necessary for observed effects

    • Directly test phosphoryl transfer using radiolabeled phosphoenolpyruvate

    • Compare rates and specificities to in vivo observations

  • Genetic approaches to establish causality:

    • Create a heterologous expression system where only the components of interest are present

    • Use genetic suppressor screens to identify genes that can bypass WefF function

    • Apply synthetic genetic array analysis to map genetic interactions

    • Use CRISPRi to create partial loss-of-function phenotypes for more nuanced analysis

How can structural biology approaches enhance our understanding of WefF function?

Structural biology approaches provide crucial insights into WefF function by revealing the molecular architecture that underlies its catalytic and regulatory activities. Researchers should consider the following approaches:

  • X-ray crystallography studies:

    • Crystallize WefF in different phosphorylation states

    • Co-crystallize with substrate analogs and regulatory partners

    • Analyze conformational changes associated with phosphorylation

    • Create a structural basis for understanding substrate specificity

  • Nuclear Magnetic Resonance (NMR) spectroscopy:

    • Determine solution structures of WefF domains

    • Study dynamics of phosphorylation-induced conformational changes

    • Analyze protein-protein interactions in solution

    • Investigate transient interactions with regulatory partners

  • Cryo-electron microscopy (cryo-EM):

    • Visualize larger complexes involving WefF and its partners

    • Study membrane-associated complexes in near-native environments

    • Capture different conformational states during the catalytic cycle

    • Reconstruct the complete phosphorylation cascade machinery

  • Molecular dynamics simulations:

    • Model conformational changes upon phosphorylation

    • Predict effects of amino acid substitutions on protein dynamics

    • Simulate substrate binding and phosphoryl transfer

    • Integrate experimental structural data with computational modeling

These structural approaches should be combined with functional studies to correlate structural features with specific aspects of WefF activity and regulation .

What are the best approaches for studying WefF evolution and its implications for bacterial adaptation?

Studying WefF evolution provides insights into bacterial adaptation mechanisms. Researchers should employ these approaches:

  • Comparative genomics:

    • Analyze WefF sequences across diverse bacterial lineages

    • Identify conserved domains and variable regions

    • Map evolutionary relationships using phylogenetic analysis

    • Correlate sequence variations with ecological niches

  • Ancestral sequence reconstruction:

    • Infer ancestral WefF sequences using maximum likelihood methods

    • Synthesize and characterize reconstructed ancestors

    • Compare biochemical properties of ancestral and modern WefF proteins

    • Track the evolution of substrate specificity and regulatory functions

  • Horizontal gene transfer analysis:

    • Identify evidence of horizontal transfer events involving WefF

    • Analyze codon usage patterns and GC content

    • Investigate genomic context across species

    • Assess the contribution of horizontal transfer to functional diversity

  • Experimental evolution:

    • Subject bacterial populations to selective pressures relevant to WefF function

    • Sequence evolved strains to identify adaptive mutations in WefF

    • Characterize the phenotypic effects of these mutations

    • Reconstruct the fitness landscape of WefF variants

  • Structure-function correlation across evolutionary history:

    • Map sequence variations onto structural models

    • Identify co-evolving residues that maintain function

    • Analyze the evolution of protein-protein interaction interfaces

    • Study how WefF has co-evolved with its interaction partners

How can systems biology approaches be used to model the complex regulatory networks involving WefF?

Systems biology approaches are essential for understanding the complex regulatory networks involving WefF. Researchers should implement:

  • Network reconstruction and analysis:

    • Integrate data from transcriptomics, proteomics, and metabolomics

    • Construct directed graphs representing regulatory interactions

    • Identify network motifs such as feedback loops and feed-forward loops

    • Analyze network properties like robustness and modularity

  • Ordinary differential equation (ODE) models:

    • Develop mathematical models of phosphoryl transfer kinetics

    • Incorporate enzyme kinetics parameters for all components

    • Simulate dynamic responses to environmental changes

    • Validate models using time-course experimental data

  • Stochastic modeling approaches:

    • Account for intrinsic noise in biochemical reactions

    • Model cell-to-cell variability in WefF phosphorylation

    • Simulate rare events that may have regulatory significance

    • Investigate how noise affects regulatory network function

  • Constraint-based modeling:

    • Integrate WefF regulation into genome-scale metabolic models

    • Apply flux balance analysis to predict metabolic consequences

    • Use regulatory constraints to refine metabolic predictions

    • Model growth phenotypes under various conditions

  • Multi-scale modeling:

    • Connect molecular events to cellular phenotypes

    • Bridge timescales from rapid phosphorylation to slower growth effects

    • Model population-level behaviors emerging from single-cell processes

    • Incorporate spatial aspects of cellular organization

These approaches should be iteratively refined through experimental validation, creating a cycle where models generate predictions that guide new experiments whose results improve the models .

What are the most promising future research directions for WefF phosphotransferase studies?

The most promising future research directions for WefF phosphotransferase studies include:

  • Integration with bacterial physiological responses:

    • Investigating how WefF connects carbon metabolism to other cellular processes

    • Exploring the role of WefF in bacterial stress responses

    • Understanding how WefF contributes to metabolic flexibility

    • Mapping the complete interactome of WefF under various conditions

  • Applications in synthetic biology:

    • Engineering WefF variants with novel substrate specificities

    • Creating synthetic regulatory circuits incorporating WefF

    • Developing biosensors based on WefF phosphorylation

    • Utilizing WefF in metabolic engineering applications

  • Therapeutic targeting opportunities:

    • Exploring WefF as a potential antibiotic target

    • Developing inhibitors specific to pathogen-associated WefF variants

    • Investigating how WefF modulates virulence in pathogens

    • Understanding how host-microbe interactions affect WefF function

  • Advanced technological approaches:

    • Applying CRISPR interference for precise temporal control of WefF expression

    • Utilizing optogenetic tools to manipulate WefF activity with spatial and temporal precision

    • Developing improved phosphoproteomic methods for studying histidine phosphorylation

    • Creating high-throughput screening platforms for WefF function

These directions will likely yield significant insights into bacterial physiology and potentially lead to novel biotechnological applications and therapeutic strategies .

How can researchers effectively collaborate across disciplines to advance WefF research?

Effective interdisciplinary collaboration is essential for advancing WefF research. Researchers should consider these approaches:

  • Establish clear communication frameworks:

    • Develop a shared vocabulary across disciplines

    • Create visual models that can be understood by all team members

    • Hold regular interdisciplinary meetings with structured knowledge exchange

    • Document and share discipline-specific methodologies and assumptions

  • Integrate diverse experimental approaches:

    • Combine structural biology with functional genomics

    • Integrate computational modeling with wet-lab validation

    • Connect molecular mechanisms to ecological significance

    • Link fundamental research with applied biotechnology

  • Develop collaborative research platforms:

    • Establish standardized protocols for WefF characterization

    • Create shared databases of WefF variants and their properties

    • Develop open-source computational tools for data analysis

    • Implement collaborative electronic lab notebooks

  • Design truly interdisciplinary experiments:

    • Formulate research questions that require multiple disciplines

    • Create experimental designs with input from all disciplines

    • Analyze data using complementary approaches from different fields

    • Interpret results through multiple disciplinary lenses

  • Train researchers in interdisciplinary thinking:

    • Organize workshops on methods from complementary disciplines

    • Establish co-mentoring of students across disciplines

    • Create joint publications with balanced contributions

    • Develop funding proposals that genuinely integrate disciplines

These collaborative approaches will accelerate progress in understanding the complex roles of WefF in bacterial physiology and potential applications .

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