Recombinant Pseudomonas putida UPF0042 nucleotide-binding protein PP_0949 (PP_0949)

Basic Research Questions

• What is the structural characterization of Recombinant Pseudomonas putida UPF0042 nucleotide-binding protein PP_0949?

  Recombinant UPF0042 nucleotide-binding protein PP_0949 is a protein from Pseudomonas putida characterized by a specific amino acid sequence. Based on structural analysis, the protein contains nucleotide-binding domains that enable it to interact with various nucleotides. The full-length protein typically contains 284 amino acids with a molecular weight of approximately 42.9 kDa . The protein's sequence begins with MRLIIVSGRS and contains distinctive structural elements including GTP-binding motifs that are critical for its functionality . The three-dimensional structure features alpha-helical regions interspersed with beta-sheets that form the nucleotide-binding pocket.

• Which expression systems are most effective for producing Recombinant PP_0949?

  Multiple expression systems have been evaluated for the production of Recombinant PP_0949, with varying degrees of success. E. coli and yeast expression systems typically offer the best yields and shorter turnaround times for production . The expression in bacterial systems like E. coli can reach >85% purity using SDS-PAGE analysis when coupled with appropriate purification techniques . For applications requiring post-translational modifications, expression in insect cells with baculovirus or mammalian cells can provide many of the modifications necessary for correct protein folding or retention of activity . The table below summarizes the comparative effectiveness of different expression systems:

  Expression System	Yield	Turnaround Time	Post-translational Modifications	Recommended Application
  E. coli	High	Short	Minimal	Basic structural studies
  Yeast	High	Short-Medium	Moderate	Functional studies
  Insect cells	Medium	Medium	Substantial	Activity-dependent research
  Mammalian cells	Low	Long	Extensive	In vivo modeling

• What purification methods are recommended for obtaining high-purity Recombinant PP_0949?

  For high-purity isolation of Recombinant PP_0949, a multi-step purification approach is recommended. The protein can be effectively purified using immobilized metal affinity chromatography (IMAC) when expressed with an N-terminal or C-terminal His-tag . After initial capture using Ni-NTA or similar matrices, further purification can be achieved through size exclusion chromatography to remove aggregates and ion exchange chromatography to eliminate contaminants with different charge properties. For optimal results, purification buffers typically contain 40 mM Tris-HCl (pH 8.0), 110 mM NaCl, 2.2 mM KCl, with elution using 200 mM imidazole . The addition of 20% glycerol in storage buffers helps maintain protein stability . These methods routinely achieve purity levels greater than 85% as determined by SDS-PAGE analysis.

Advanced Research Questions

• How does the UPF0042 nucleotide-binding protein PP_0949 function differ from other nucleotide-binding proteins in Pseudomonas species?

  UPF0042 nucleotide-binding protein PP_0949 belongs to a distinct class of nucleotide-binding proteins that differs from other well-characterized proteins in Pseudomonas species. Unlike formaldehyde dehydrogenase (FDH) from P. putida, which has a specific enzymatic function with a documented specific activity of ≥35 μmol/min/μg , the precise enzymatic activity of PP_0949 remains less characterized.

  PP_0949 also differs from immune-associated nucleotide-binding proteins like IAN9, which functions as a negative regulator of immunity in plant systems . While IAN9 is repressed upon pathogen infection and localizes to the plasma membrane , PP_0949 does not appear to have a direct role in immune regulation.

  The structural basis for these functional differences lies in specific binding domains and motifs. PP_0949 contains characteristic nucleotide-binding motifs that determine its specificity for certain nucleotides, which directly influences its biological role in P. putida metabolism.

• What experimental approaches are most effective for investigating PP_0949 binding kinetics with different nucleotides?

  To thoroughly investigate the binding kinetics of PP_0949 with different nucleotides, a multi-method approach is recommended. Surface Plasmon Resonance (SPR) provides real-time, label-free detection of binding interactions, allowing determination of association (kon) and dissociation (koff) rate constants. Isothermal Titration Calorimetry (ITC) complements SPR by providing thermodynamic parameters (ΔH, ΔS, and ΔG) of the binding interaction.

  For structural insights into nucleotide binding, X-ray crystallography of PP_0949 co-crystallized with various nucleotides can reveal the atomic details of the binding site. Additionally, Microscale Thermophoresis (MST) is valuable for measuring interactions in solution with minimal protein consumption.

  Data analysis should employ appropriate binding models—typically one-site or two-site binding models depending on the cooperativity observed. Following the experimental design principles outlined in scientific literature , researchers should include appropriate controls and technical replicates to ensure statistical validity. Data visualization using Scatchard or Hill plots can provide insights into binding cooperativity and mechanism.

• What role might PP_0949 play in the metabolic networks of Pseudomonas putida?

  PP_0949 likely serves as a regulatory node within P. putida's metabolic networks. Based on comparative analysis with other nucleotide-binding proteins, PP_0949 may participate in:

  1. Energy metabolism regulation through GTP/ATP binding and hydrolysis

  2. Signal transduction pathways responding to environmental stressors

  3. Regulation of cellular processes through protein-protein interactions

  Studies of similar proteins suggest that PP_0949 might interact with other cytoplasmic components to form functional complexes. Using approaches similar to those employed for analyzing other P. putida proteins , researchers could identify potential interaction partners through co-immunoprecipitation followed by mass spectrometry. This approach successfully identified the C3HC4-type RING-finger domain-containing protein (IAP1) as an interaction partner for another nucleotide-binding protein , suggesting similar methodologies could reveal PP_0949's interaction network.

  Integration of proteomics, metabolomics, and transcriptomics data would provide a comprehensive understanding of PP_0949's role within P. putida's complex metabolic landscape.

Data Analysis and Interpretation Questions

• What statistical approaches are most appropriate for analyzing PP_0949 functional assay data?

  The analysis of PP_0949 functional assay data requires robust statistical methods tailored to the specific experimental design. For quantitative analysis of enzymatic activity or binding kinetics, the following approaches are recommended:

  1. Descriptive Statistics: Calculate means, standard deviations, and confidence intervals to characterize the central tendency and dispersion of the data.

  2. Comparative Analysis:

     • For comparing two conditions: Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

     • For multiple conditions: One-way ANOVA followed by post-hoc tests (Tukey's HSD, Bonferroni)

     • For multiple factors: Two-way or three-way ANOVA with interaction terms

  3. Regression Analysis:

     • For enzyme kinetics: Non-linear regression to fit Michaelis-Menten, Hill, or other appropriate models

     • For binding data: Fit appropriate binding isotherms (one-site, two-site, cooperative models)

  4. Robust Statistical Methods:

     • Bootstrap resampling to generate confidence intervals without assuming normality

     • Permutation tests to assess significance without parametric assumptions

  When designing data analysis pipelines, researchers should implement validation strategies including cross-validation and bootstrapping to ensure the robustness of findings . For complex datasets, machine learning approaches similar to those used in other biological systems can be adapted for PP_0949 functional data . These methods can help identify patterns and relationships that might not be apparent with traditional statistical approaches.

• How can researchers troubleshoot expression and purification issues with Recombinant PP_0949?

  Troubleshooting expression and purification issues with Recombinant PP_0949 requires a systematic approach to identify and resolve specific challenges:

  Expression Issues:

  1. Low Expression Levels:

     • Verify plasmid sequence integrity

     • Test multiple expression strains (BL21(DE3), Rosetta, SHuffle)

     • Optimize codon usage for the expression host

     • Evaluate different promoter systems

  2. Inclusion Body Formation:

     • Reduce expression temperature to 15-20°C

     • Decrease inducer concentration

     • Co-express with molecular chaperones

     • Fuse with solubility-enhancing tags

  Purification Issues:

  1. Poor Binding to Affinity Resin:

     • Verify tag accessibility (N vs. C-terminal placement)

     • Optimize binding buffer composition (pH, salt concentration)

     • Ensure proper resin preparation and loading

  2. Contaminants:

     • Implement additional purification steps (ion exchange, size exclusion)

     • Optimize wash steps (imidazole gradient for His-tagged proteins)

     • Consider on-column refolding for proteins recovered from inclusion bodies

  3. Protein Instability:

     • Add stabilizing agents (glycerol, reducing agents)

     • Test different buffer systems and pH values

     • Minimize freeze-thaw cycles; store at appropriate temperature

  For each troubleshooting step, use analytical tools (SDS-PAGE, Western blot, activity assays) to assess the outcome. Document modifications to the protocol and their effects to guide future optimization efforts. Similar approaches have been successfully implemented for other recombinant proteins expressed in P. putida .

• What approaches can be used to analyze the evolutionary relationships between PP_0949 and related proteins in other bacterial species?

  To analyze evolutionary relationships between PP_0949 and related proteins in other bacterial species, researchers should implement a comprehensive phylogenetic analysis methodology:

  1. Sequence Retrieval and Alignment:

     • Identify homologs using BLAST/HMMER searches against protein databases

     • Create multiple sequence alignments using MUSCLE, MAFFT, or T-Coffee

     • Refine alignments to focus on conserved domains and remove poorly aligned regions

  2. Phylogenetic Tree Construction:

     • Maximum Likelihood methods (RAxML, PhyML)

     • Bayesian inference (MrBayes, BEAST)

     • Neighbor-Joining for initial rapid assessment

     • Select appropriate evolutionary models using tools like ProtTest

  3. Tree Evaluation and Visualization:

     • Assess node support using bootstrap analysis (1000 replicates)

     • Implement approximate likelihood ratio tests

     • Visualize trees using tools like FigTree or iTOL

  4. Comparative Analysis:

     • Identify conserved motifs using MEME or similar tools

     • Map functional domains to understand evolutionary conservation

     • Analyze selective pressure using dN/dS ratios to identify sites under selection

  5. Contextual Genomic Analysis:

     • Examine gene neighborhood conservation

     • Analyze synteny to identify genomic rearrangements

     • Look for co-evolution patterns with interacting partners

  These approaches can reveal the evolutionary history of PP_0949 and provide insights into functional conservation or divergence across bacterial species. Integration with structural data can further enhance understanding of structure-function relationships in this protein family.