Recombinant Pseudomonas syringae pv. syringae UPF0250 protein Psyr_4360 (Psyr_4360)

What are the fundamental structural and biochemical properties of Psyr_4360?

Psyr_4360 is a small hypothetical protein from Pseudomonas syringae pv. syringae B728a with a molecular weight of approximately 10.0 kDa and an isoelectric point (pI) of 6.08 . The protein has a negative charge of -2.08 at physiological pH (7.0) and a Kyte-Doolittle Hydrophobicity Value of -0.120, indicating it is slightly hydrophilic . The protein is encoded on the negative strand of the chromosome at position 5213599-5213874 .

For determining these properties experimentally, researchers should consider using:

• Size exclusion chromatography for molecular weight confirmation

• Isoelectric focusing gels for pI verification

• Circular dichroism spectroscopy for secondary structure analysis

• Hydrophobicity plot analysis using multiple algorithms beyond Kyte-Doolittle (e.g., Hopp-Woods, Eisenberg) for comprehensive characterization

How can I optimize expression of recombinant Psyr_4360 protein in E. coli?

Optimizing recombinant Psyr_4360 expression requires systematic evaluation of expression parameters. Drawing from established recombinant protein expression protocols, such as those used for GALE protein expression , consider the following methodology:

• Vector selection: pET-based vectors with T7 promoter systems typically provide high expression levels for small bacterial proteins

• Codon optimization: Analyze and optimize the Psyr_4360 coding sequence for E. coli codon usage

• Expression conditions testing matrix:

• Solubility assessment: Test extraction under native conditions using different buffer systems (phosphate, Tris-HCl) at various pH values (6.0-8.0)

• Affinity tag selection: Compare N-terminal vs. C-terminal His6-tag performance, considering TEV protease cleavage sites for tag removal

Monitor expression levels using SDS-PAGE and Western blotting, targeting >95% purity similar to other successfully expressed recombinant proteins .

What purification strategy should I employ for recombinant Psyr_4360?

For high-purity Psyr_4360 preparation suitable for functional and structural studies, implement a multi-step purification strategy:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin if expressing with a His-tag

• Intermediate purification: Ion exchange chromatography based on Psyr_4360's negative charge at pH 7.0 (-2.08)

• Polishing step: Size exclusion chromatography to achieve >95% purity

Consider the following buffer optimization approach:

Assess purity at each stage using SDS-PAGE, targeting final purity comparable to other research-grade recombinant proteins (>95%) . Confirm identity through mass spectrometry and N-terminal sequencing.

How can I assess the potential membrane-association properties of Psyr_4360?

Given the slight hydrophilicity of Psyr_4360 (Kyte-Doolittle value: -0.120) , but considering that many bacterial proteins with similar profiles interact transiently with membranes, use the following methodological approach:

• In silico analysis:

  • Run transmembrane prediction algorithms (TMHMM, Phobius)

  • Amphipathic helix prediction (HeliQuest)

  • Membrane binding motif identification

• Experimental validation:

  • Liposome binding assays using different lipid compositions, similar to methods used for HrpZ(Psph)

  • Membrane fractionation of bacterial cells expressing Psyr_4360

  • Planar lipid bilayer experiments to detect potential pore-forming activity

• Quantitative assessment:

  • Surface plasmon resonance (SPR) for binding kinetics

  • Fluorescence resonance energy transfer (FRET) assays using labeled protein and liposomes

  • Microscale thermophoresis for interaction studies

Drawing from HrpZ research methodologies, assess whether Psyr_4360 can associate with synthetic bilayer membranes and measure any resulting conductance changes using electrophysiological techniques .

How does Psyr_4360 compare phylogenetically across different Pseudomonas species and what might this reveal about its function?

To conduct a comprehensive phylogenetic analysis of Psyr_4360 orthologs:

• Data extraction and sequence alignment:

  • Retrieve all 535 members of the Pseudomonas Ortholog Group POG001685

  • Include representatives from the 49 genera identified as containing this protein

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

• Phylogenetic tree construction:

  • Apply maximum likelihood methods (RAxML or IQ-TREE)

  • Validate with Bayesian approaches (MrBayes)

  • Assess node support through bootstrapping (1000 replicates)

• Functional inference methodology:

  • Map presence/absence patterns against known pathogenicity traits

  • Identify conserved motifs using MEME Suite

  • Apply context-based gene neighborhood analysis

  • Correlate with bacterial adaptation to different hosts or environments

The fact that Psyr_4360 is found in both pathogenic and non-pathogenic strains suggests a core cellular function rather than a direct virulence role. Detailed comparative genomics could reveal co-evolution patterns with other bacterial systems, potentially illuminating its biological significance.

What experimental approaches would best determine whether Psyr_4360 interacts with plant immune systems?

Given that some Pseudomonas syringae proteins interact with plant defense mechanisms, including via the type III secretion system , investigating Psyr_4360's potential role in plant-pathogen interactions requires a multi-faceted approach:

• Secretion system dependency testing:

  • Generate deletion mutants of key type III secretion system components

  • Assess Psyr_4360 secretion in wild-type vs. mutant backgrounds using immunoblotting

  • Conduct heterologous expression in Yersinia enterocolitica to test type III secretion capability as done for HrpZ

• Plant response assays:

  • Infiltrate purified recombinant Psyr_4360 into plant leaves

  • Monitor reactive oxygen species (ROS) production

  • Measure defense gene expression via qRT-PCR

  • Assess hypersensitive response development

• Protein-protein interaction studies:

  • Yeast two-hybrid screening against plant immunity components

  • Co-immunoprecipitation assays from infected plant tissue

  • Bimolecular fluorescence complementation (BiFC) for in planta interaction validation

• Functional genomics validation:

  • Generate Psyr_4360 knockout strains

  • Conduct plant infection assays comparing wild-type and mutant strains

  • Perform complementation studies to confirm phenotypes

Though Psyr_4360 is found in both pathogenic and non-pathogenic strains , which might suggest a non-virulence role, systematic testing is necessary to exclude potential contributions to plant-microbe interactions.

What approaches should I use to determine the three-dimensional structure of Psyr_4360?

For comprehensive structural characterization of Psyr_4360, implement a multi-method strategy:

• X-ray crystallography approach:

  • Initial crystallization screening (sparse matrix, 500-1000 conditions)

  • Optimization of promising conditions varying:

    • Protein concentration (5-15 mg/ml)

    • Precipitant type and concentration

    • pH range around pI (5.5-6.5)

    • Additives and detergents

  • Data collection at synchrotron facility

  • Structure determination using molecular replacement or experimental phasing

• NMR spectroscopy alternative:

  • Isotopic labeling with 15N and 13C

  • Collection of 2D and 3D spectra (HSQC, HNCA, HNCACB)

  • Chemical shift assignment and NOE analysis

  • Structure calculation using CYANA or ARIA software

• Complementary methods:

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Cryo-electron microscopy if the protein forms larger assemblies

For a 10 kDa protein like Psyr_4360 , NMR may be particularly suitable due to the favorable size for this technique. Structure determination would significantly advance functional hypotheses, particularly if structural homology is identified with proteins of known function.

How can I investigate potential ion channel or pore-forming activities of Psyr_4360?

Drawing from methodologies used to characterize the pore-forming activity of HrpZ(Psph) , implement the following experimental strategy to investigate Psyr_4360's potential membrane activity:

• Electrophysiological characterization:

  • Prepare planar lipid bilayers with defined lipid compositions

  • Add purified recombinant Psyr_4360 at nanomolar concentrations

  • Record ion currents under symmetric and asymmetric ionic conditions

  • Determine:

    • Unitary conductance

    • Ion selectivity (cation vs. anion preference)

    • Voltage dependency

    • Gating characteristics

• Liposome-based assays:

  • Prepare liposomes loaded with fluorescent dyes

  • Monitor dye release upon Psyr_4360 addition

  • Quantify size-dependent molecule release to estimate pore diameter

  • Test with liposomes of varying lipid compositions to determine specificity

• Structural verification:

  • Atomic force microscopy of protein-membrane complexes

  • Transmission electron microscopy with negative staining

  • Molecular dynamics simulations of membrane interaction

This systematic approach would determine whether Psyr_4360 forms pores similar to HrpZ(Psph), which creates cation-selective channels with a conductivity of 207 pS .

How might Psyr_4360 research benefit from integrating Google's "People Also Ask" data mining approaches?

Integrating Google's "People Also Ask" (PAA) data mining methodology into Psyr_4360 research offers significant advantages for understanding research trends and knowledge gaps:

• Implementation methodology:

  • Track PAA cascades specifically related to Pseudomonas proteins

  • Monitor evolving question patterns across specialized research domains

  • Apply natural language processing to extract semantic relationships between questions

• Research applications:

  • Identify understudied aspects of bacterial proteins like Psyr_4360

  • Discover connections between seemingly unrelated research questions

  • Map the conceptual landscape surrounding bacterial hypothetical proteins

• Strategic research planning:

  • Prioritize experiments based on frequency and complexity of questions

  • Identify potential collaborators through question pattern analysis

  • Develop targeted communication strategies for research findings

As demonstrated in SEO contexts, PAA data provides valuable insights into search behavior patterns and query relationships . For scientific research on poorly characterized proteins like Psyr_4360, this approach can reveal hidden associations between research questions and experimental approaches, potentially accelerating hypothesis generation.

What methodologies can effectively distinguish between the functions of Psyr_4360 and other UPF0250 family proteins?

To rigorously differentiate the functional characteristics of Psyr_4360 from other UPF0250 family members:

• Comparative functional genomics:

  • Generate knockout mutants across multiple bacterial species

  • Perform cross-complementation studies

  • Conduct phenotypic microarrays to identify condition-specific defects

  • Compare transcriptomic responses to environmental stressors

• Domain swapping and chimeric protein analysis:

  • Identify variable regions within the UPF0250 family

  • Create chimeric constructs between Psyr_4360 and distant homologs

  • Assess the function of each construct through complementation assays

  • Map species-specific functional domains

• Interactome comparison:

  • Perform pull-down assays coupled with mass spectrometry for each homolog

  • Construct protein-protein interaction networks

  • Identify common vs. species-specific interaction partners

  • Validate key interactions through reciprocal co-immunoprecipitation

This systematic approach would determine whether the function of Psyr_4360 is conserved across the 535 members of its ortholog group (POG001685) or if it has evolved species-specific functions despite sequence conservation.