Recombinant Pseudomonas putida UPF0276 protein PP_0992 (PP_0992)

What is UPF0276 protein PP_0992 and which organism does it originate from?

PP_0992 is a protein that belongs to the UPF0276 family (Uncharacterized Protein Family) and originates from Pseudomonas putida (strain ATCC 47054/DSM 6125/NCIMB 11950/KT2440) . P. putida is a gram-negative, rod-shaped soil bacterium that has emerged as an important microbial laboratory workhorse with applications in biotechnology and natural product biosynthesis .

For researchers investigating this protein, methodological approaches should include:

• Sequence conservation analysis among homologs in related Pseudomonas species

• Genomic context examination of the pp_0992 gene to identify potentially functionally related neighboring genes

• Transcriptomic analysis under various growth conditions to determine expression patterns

These approaches can provide initial insights into the biological context and potential functions of this uncharacterized protein.

How does PP_0992 compare to other proteins in the UPF0276 family?

To comprehensively compare PP_0992 with other UPF0276 family members, researchers should implement several comparative analytical approaches:

Sequence-Based Comparisons:

• Multiple sequence alignment of UPF0276 family proteins from diverse bacterial species to identify:

  • Highly conserved residues that may be functionally or structurally critical

  • Variable regions that might confer species-specific functions

  • Conservation patterns that correlate with taxonomic relationships

Evolutionary Analysis:

• Phylogenetic tree construction to understand:

  • The evolutionary history of the UPF0276 protein family

  • Potential functional divergence across different bacterial lineages

  • Co-evolutionary patterns with other proteins or metabolic pathways

Genomic Context Analysis:

• Examination of neighboring genes across species, which often provides functional clues

• Identification of conserved operons or gene clusters containing UPF0276 family genes

• Analysis of regulatory elements associated with these genes in different organisms

This comparative approach would help determine whether PP_0992 possesses unique features or shares common characteristics with other UPF0276 family members, providing insights into potentially conserved functions across bacterial species.

What expression systems are most effective for recombinant production of PP_0992?

For optimal recombinant production of PP_0992, researchers should consider several expression systems, each with distinct advantages:

Bacterial Expression Systems:

• Escherichia coli-based systems:

  • Advantages: Rapid growth, high yield, ease of genetic manipulation

  • Recommended strains: BL21(DE3) for high expression; Rosetta for rare codon optimization

  • Vector considerations: pET series vectors with T7 promoter for strong, inducible expression

• Pseudomonas putida-based systems:

  • Advantages: Native cellular environment for P. putida proteins; elaborated techniques for cultivation and genetic manipulation available

  • Particularly valuable for maintaining proper folding and potential post-translational modifications

Optimization Parameters Table:

Methodologically, researchers should implement a systematic optimization approach, beginning with small-scale expression trials to identify optimal conditions before scaling up production. The selection of expression system should be guided by the intended application, with considerations for protein solubility, post-translational modifications, and required yield.

What are the optimal conditions for purifying recombinant PP_0992?

Purification of recombinant PP_0992 requires a methodical approach tailored to the expression system used and the protein's properties. A comprehensive purification strategy would include:

Initial Processing:

• Cell lysis optimization:

  • Mechanical methods: Sonication, French press, or homogenization

  • Chemical/enzymatic methods: Lysozyme treatment with detergents

  • Buffer composition: pH 7.0-8.0 phosphate or Tris buffer with protease inhibitors

• Clarification:

  • High-speed centrifugation (20,000-30,000 × g for 30-45 minutes)

  • Filtration through 0.45 or 0.22 μm filters

Chromatographic Purification Sequence:

• Primary capture:

  • Immobilized Metal Affinity Chromatography (IMAC) if His-tagged (similar to methods described for other recombinant proteins)

  • Gradient elution with imidazole (20-300 mM)

• Intermediate purification:

  • Ion exchange chromatography based on PP_0992's theoretical isoelectric point

  • Salt gradient elution (typically 0-1 M NaCl)

• Polishing step:

  • Size exclusion chromatography to ensure monodispersity

  • Buffer exchange into final storage buffer

Buffer Optimization Considerations:

• pH screening (typically pH 6.0-8.5)

• Salt concentration optimization (50-500 mM NaCl)

• Addition of stabilizing agents (5-10% glycerol, 1-5 mM DTT or TCEP)

• Testing various buffering agents (phosphate, Tris, HEPES)

Researchers should implement analytical quality control at each purification stage, including SDS-PAGE, Western blotting, and activity assays if available. For long-term storage, stability tests under various conditions (temperature, buffer composition) should be conducted to maintain structural integrity and activity.

How can researchers verify the structural integrity of recombinant PP_0992 after purification?

Verifying the structural integrity of purified recombinant PP_0992 requires a multi-analytical approach to ensure that the protein maintains its native conformation:

Spectroscopic Methods:

• Circular Dichroism (CD) spectroscopy:

  • Far-UV (190-250 nm) analysis for secondary structure content

  • Near-UV (250-350 nm) analysis for tertiary structure fingerprinting

  • Thermal denaturation profiles to assess stability

• Fluorescence spectroscopy:

  • Intrinsic tryptophan/tyrosine fluorescence to assess tertiary structure

  • ANS binding to detect exposed hydrophobic regions

  • Quenching studies to examine accessibility of fluorophores

Hydrodynamic Characterization:

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determination of absolute molecular weight

  • Assessment of oligomeric state and homogeneity

• Analytical Ultracentrifugation (AUC):

  • Sedimentation velocity analysis for shape parameters and heterogeneity

  • Sedimentation equilibrium for molecular weight determination

Thermal Stability Analysis:

• Differential Scanning Calorimetry (DSC):

  • Thermodynamic parameters of unfolding

  • Identification of domain-specific transitions

• Thermal shift assays:

  • Buffer optimization screening

  • Ligand binding effects on thermal stability

Functional Validation:
While specific functional assays for PP_0992 are not described in the available literature, general approaches might include:

• DNA binding assays if sequence homology suggests nucleic acid interaction

• Enzymatic activity assays based on structural predictions

• Protein-protein interaction studies with predicted partners

Integration of these complementary techniques provides a comprehensive assessment of the recombinant protein's structural integrity, essential for subsequent functional studies.

What is the current understanding of PP_0992's biological function in P. putida?

While the specific biological function of PP_0992 remains uncharacterized based on available information, researchers can implement a systematic functional discovery approach:

Genomic Context Analysis:

• Examination of genes adjacent to PP_0992 in the P. putida genome

• Determination of operon structure through transcriptional analysis

• Identification of conserved gene neighborhoods across related bacterial species

Transcriptomic Profiling:

• RNA-Seq analysis under various environmental conditions:

  • Different carbon sources

  • Stress conditions (oxidative, osmotic, temperature)

  • Growth phases

• Co-expression network analysis to identify functionally related genes

Genetic Manipulation Studies:

• Creation of knock-out mutants:

  • CRISPR-Cas9 gene deletion

  • Transposon mutagenesis approaches

  • Phenotypic characterization including:

    • Growth rates in various media

    • Stress tolerance

    • Metabolic capabilities

    • Biofilm formation

• Complementation studies:

  • Reintroduction of PP_0992 to confirm phenotype restoration

  • Domain-specific complementation to identify functional regions

Comparative Genomics:

• Analysis of PP_0992 conservation across Pseudomonas species

• Correlation between presence/absence and specific phenotypic traits

• Identification of co-evolving genes suggesting functional relationships

P. putida's versatile metabolism and enzymatic capabilities suggest that PP_0992 might contribute to these metabolic networks or stress response systems, particularly if it's conserved across related species with similar ecological adaptations.

Are there known binding partners or interaction networks for PP_0992?

To identify potential binding partners and establish interaction networks for PP_0992, researchers should implement complementary experimental and computational approaches:

Affinity-Based Protein Interaction Methods:

• Pull-down assays:

  • Immobilization of tagged PP_0992 on appropriate resin

  • Incubation with P. putida cell lysate

  • Mass spectrometry analysis of co-purified proteins

• Co-immunoprecipitation:

  • Generation of specific antibodies against PP_0992

  • Precipitation from native cellular environment

  • Western blot or mass spectrometry identification of co-precipitated proteins

• Cross-linking coupled with mass spectrometry:

  • In vivo or in vitro cross-linking to capture transient interactions

  • Enrichment of cross-linked complexes

  • MS/MS analysis with specialized software for cross-link identification

Library Screening Approaches:

• Bacterial two-hybrid systems:

  • Construction of P. putida genomic library

  • Screening for positive interactors

  • Validation of identified interactions

• Protein array screening:

  • Probing of proteome arrays with labeled PP_0992

  • Detection of binding events

  • Quantitative analysis of binding affinities

Biophysical Validation Methods:

• Surface Plasmon Resonance (SPR):

  • Quantitative binding kinetics

  • Determination of KD values for specific interactions

• Isothermal Titration Calorimetry (ITC):

  • Thermodynamic parameters of binding

  • Stoichiometry determination

Computational Prediction Approaches:

• Protein-protein interaction prediction algorithms

• Co-evolution analysis of potential interaction partners

• Structural docking simulations

While HupB and HupN are examples of functionally characterized proteins in P. putida that exhibit DNA-binding capabilities , specific interactions with PP_0992 have not been established in the available literature. Systematic application of these methods would help elucidate PP_0992's potential role within cellular protein networks.

How might PP_0992 contribute to P. putida's metabolic versatility or stress response?

P. putida is renowned for its metabolic versatility and remarkable tolerance to various stressors . To investigate PP_0992's potential contributions to these capabilities, researchers should implement a multi-faceted approach:

Stress Response Analysis:

• Expression profiling:

  • qRT-PCR analysis of PP_0992 under various stress conditions

  • Promoter-reporter fusion studies to visualize expression patterns

  • Western blot analysis of protein levels during stress response

• Phenotypic characterization of PP_0992 mutants under stress:

  • Oxidative stress (H₂O₂, paraquat)

  • Heavy metal exposure

  • Organic solvent tolerance

  • Temperature stress

  • Nutrient limitation

Metabolic Profiling:

• Comparative growth analysis:

  • Wild-type vs. PP_0992 mutant strains

  • Diverse carbon and nitrogen sources

  • Quantitative fitness measurements

• Metabolomics:

  • Targeted and untargeted metabolite analysis

  • Stable isotope labeling to track metabolic flux

  • Identification of altered metabolic pathways

Integration with Known Systems:

• Investigation of potential roles in established P. putida capabilities:

  • Aromatic compound degradation pathways

  • Secondary metabolite production

  • Biofilm formation and cell-surface interactions

• Protein localization studies:

  • Fluorescent protein fusions

  • Subcellular fractionation

  • Immunolocalization under different conditions

Comparative Analysis:

• Correlation between PP_0992 conservation and specific metabolic traits across Pseudomonas strains

• Identification of co-occurring genes in genomic islands associated with specific adaptations

While HupB and HupN have been characterized as DNA-bending proteins essential for certain cellular functions in P. putida , PP_0992's potential role may involve different aspects of cellular physiology. Integration of these experimental approaches would help position PP_0992 within P. putida's complex adaptive response networks.

How can recombinant PP_0992 be used in structural biology studies?

Recombinant PP_0992 provides an excellent substrate for structural biology investigations using multiple complementary approaches:

X-ray Crystallography:

• Crystallization optimization:

  • Systematic screening of conditions (pH, salt, precipitants)

  • Surface entropy reduction mutations if needed

  • Crystallization with potential ligands or binding partners

• Data collection and analysis:

  • Synchrotron radiation for high-resolution data

  • Phase determination strategies

  • Structure refinement and validation

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Sample preparation:

  • Expression in minimal media with ¹⁵N, ¹³C, and/or ²H isotopic labeling

  • Optimization of buffer conditions for spectral quality

  • Concentration determination for optimal signal-to-noise

• Structural studies:

  • Backbone and side-chain assignment

  • NOE-based distance restraints

  • Residual dipolar coupling for orientation information

  • Dynamic studies to identify flexible regions

Cryo-Electron Microscopy:

• Particularly valuable if PP_0992 forms larger complexes

• Sample preparation on holey carbon grids

• Single-particle analysis workflow

• 3D reconstruction and model building

Complementary Biophysical Techniques:

• Small-Angle X-ray Scattering (SAXS):

  • Low-resolution envelope determination

  • Analysis of conformational changes

  • Validation of high-resolution structures in solution

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Mapping solvent accessibility

  • Identification of dynamic regions

  • Detection of ligand-binding interfaces

These structural studies can provide invaluable insights into PP_0992's molecular architecture, potential binding sites, and structural dynamics, particularly important for an uncharacterized protein family like UPF0276. The structural data would guide subsequent functional characterization and potentially reveal unexpected relationships to proteins of known function.

What potential roles might PP_0992 play in biotechnological applications of P. putida?

P. putida has emerged as a valuable host for the production of natural products due to its metabolic versatility and xenobiotic tolerance . Although the specific function of PP_0992 is not fully characterized, several biotechnological applications could be explored:

Metabolic Engineering Applications:

• Pathway optimization:

  • If PP_0992 is involved in metabolic regulation, it might be manipulated to:

    • Enhance flux through valuable metabolic pathways

    • Reduce flux through competing pathways

    • Improve precursor availability for heterologous products

• Stress tolerance engineering:

  • If involved in stress response, PP_0992 modification might:

    • Improve survival during industrial fermentation

    • Enhance tolerance to product toxicity

    • Increase robustness in bioremediation applications

Bioremediation Applications:

• Xenobiotic degradation:

  • Investigation of PP_0992's potential role in:

    • Aromatic compound metabolism

    • Xenobiotic tolerance mechanisms

    • Biofilm formation for immobilized bioremediation

• Heavy metal resistance:

  • Potential contributions to metal binding or efflux

  • Applications in environmental detoxification

Recombinant Protein Production:

• If PP_0992 affects protein folding or stress response:

  • Engineering to improve recombinant protein yields

  • Enhancing solubility of heterologous proteins

  • Reducing proteolytic degradation

Synthetic Biology Applications:

• Biosensor development:

  • Potential use of PP_0992 regulatory elements for sensing specific conditions

  • Integration into synthetic genetic circuits

• Chassis optimization:

  • Contribution to P. putida's suitability as a synthetic biology platform

  • Potential for cell-free systems optimization

Understanding PP_0992's role would require systematic characterization through knockout/overexpression studies, followed by targeted engineering approaches to enhance beneficial properties or integrate it into synthetic pathways for biotechnological applications.

How might studying PP_0992 contribute to understanding bacterial protein family evolution?

The UPF0276 family to which PP_0992 belongs represents an uncharacterized protein family, making it valuable for studying protein evolution and functional diversification across bacterial species:

Phylogenetic Analysis Framework:

• Comprehensive sequence collection:

  • Identification of all UPF0276 family members across bacterial phyla

  • Multiple sequence alignment construction

  • Phylogenetic tree building using maximum likelihood or Bayesian methods

• Evolutionary rate analysis:

  • Calculation of synonymous vs. non-synonymous substitution rates (dN/dS)

  • Identification of positively selected residues

  • Correlation with potential functional sites

Structural Evolution Assessment:

• Comparative structural analysis:

  • Identification of conserved structural elements vs. variable regions

  • Mapping of conservation patterns onto 3D structures

  • Identification of potential functional divergence sites

• Ancestral sequence reconstruction:

  • Computational inference of ancestral UPF0276 sequences

  • Laboratory resurrection of ancestral proteins

  • Functional comparison between ancestral and extant proteins

Genomic Context Evolution:

• Comparative genomics approach:

  • Analysis of operon structure evolution

  • Identification of synteny conservation or rearrangements

  • Detection of horizontal gene transfer events

Functional Diversification Analysis:

• Experimental comparison of orthologs:

  • Complementation studies across species

  • Cross-species activity assays

  • Identification of species-specific functions

This evolutionary perspective would provide insights into how bacterial protein families acquire new functions, adapt to different ecological niches, and contribute to the metabolic diversity observed across bacterial species. Understanding PP_0992's evolutionary trajectory could reveal important principles of bacterial adaptation and protein functional evolution.

What techniques are most effective for studying PP_0992's role in vivo?

To comprehensively investigate PP_0992's in vivo function, researchers should implement state-of-the-art genetic, molecular, and systems biology approaches:

Advanced Genetic Manipulation Strategies:

• CRISPR-Cas9 genome editing:

  • Generation of complete gene deletions

  • Introduction of point mutations in conserved residues

  • Domain-specific modifications

  • Promoter replacements for expression control

• Conditional expression systems:

  • Inducible promoters for temporal control

  • Temperature-sensitive alleles

  • Degron-based protein depletion systems

In Vivo Protein Characterization:

• Protein localization and dynamics:

  • Fluorescent protein fusions (ensuring functionality retention)

  • Time-lapse microscopy during growth cycle or stress response

  • Super-resolution microscopy for detailed subcellular localization

  • Fluorescence Correlation Spectroscopy (FCS) for mobility analysis

• Protein-protein interactions:

  • Bacterial two-hybrid systems

  • Bimolecular Fluorescence Complementation (BiFC)

  • Förster Resonance Energy Transfer (FRET)

  • In vivo crosslinking followed by mass spectrometry

Systems-Level Analysis:

• Multi-omics integration:

  • Transcriptomics (RNA-Seq) of PP_0992 mutants

  • Proteomics to identify abundance changes in interacting partners

  • Metabolomics to detect altered metabolic profiles

  • Integration of datasets to identify affected pathways

• Network analysis:

  • Construction of gene/protein interaction networks

  • Identification of network perturbations in PP_0992 mutants

  • Comparison with other Pseudomonas species

Phenotypic Characterization:

• High-throughput phenotypic screening:

  • Biolog phenotype microarrays for metabolic capabilities

  • Growth profiling under diverse stress conditions

  • Biofilm formation assays

  • Motility and chemotaxis analysis

Similar approaches have been successfully used to characterize the DNA-bending proteins HupB and HupN in P. putida , demonstrating the value of integrating multiple methodologies for functional characterization of bacterial proteins.

How might post-translational modifications affect PP_0992 function?

Post-translational modifications (PTMs) can significantly influence protein function, localization, and interactions. For PP_0992, a systematic investigation of potential PTMs would include:

Computational Prediction and Analysis:

• PTM site prediction:

  • Scanning for conserved motifs recognized by modification enzymes

  • Machine learning algorithms trained on bacterial PTM datasets

  • Structural accessibility assessment of predicted sites

• Comparative analysis:

  • Conservation of predicted PTM sites across UPF0276 family members

  • Correlation with functional regions from structural analysis

Experimental Identification of PTMs:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics with enrichment for specific PTMs:

    • Phosphopeptide enrichment (TiO₂, IMAC)

    • Glycopeptide enrichment (lectins, hydrazide chemistry)

    • Enrichment of other modifications (acetylation, methylation)

  • Top-down proteomics for intact protein analysis:

    • Characterization of proteoforms

    • Quantification of modification stoichiometry

    • Identification of PTM combinations

• Site-specific validation:

  • Generation of modification-specific antibodies

  • Chemical labeling strategies for specific PTMs

  • Site-directed mutagenesis of modified residues

Functional Impact Assessment:

• Mutational analysis:

  • Phosphomimetic mutations (e.g., Ser→Asp)

  • Phosphoablative mutations (e.g., Ser→Ala)

  • Analysis of resulting phenotypes

• Temporal PTM dynamics:

  • Pulse-chase experiments

  • Stimulation time-course studies

  • Correlation with functional changes

PTM Enzymes Identification:

• Co-purification approaches to identify modifying enzymes

• Inhibitor studies to assess modification importance

• Heterologous expression with or without modification enzymes

While specific PTMs for PP_0992 are not described in the available literature, this methodological framework provides a comprehensive approach to investigate potential modifications and their functional consequences, which could be critical for understanding the protein's regulation and activity.

What are the challenges in characterizing proteins from the UPF0276 family?

Characterizing proteins from uncharacterized families like UPF0276 presents unique challenges requiring specialized approaches:

Functional Annotation Challenges:

• Limited homology to characterized proteins:

  • Absence of close homologs with known functions

  • Difficulty in transferring functional annotations

  • Need for experimental function determination

• Potential novel functions:

  • Possibility of enzyme activities not represented in current databases

  • Requirement for unbiased activity screening approaches

  • Challenges in designing appropriate functional assays

Structural Characterization Obstacles:

• Structural novelty:

  • Limited templates for homology modeling

  • Potential for novel folds or domains

  • Difficulties in phase determination for crystallography

• Expression and stability issues:

  • Uncertain stability properties

  • Unknown cofactor requirements

  • Potential toxicity when overexpressed

Methodological Approaches to Address These Challenges:

Integrative Strategy Recommendations:

• Multi-omics approach:

  • Integration of transcriptomics, proteomics, and metabolomics data

  • Network-based analysis to predict functional relationships

  • Comparative genomics across diverse bacterial species

• High-throughput functional screening:

  • Activity-based assays against diverse substrate libraries

  • Phenotypic screening of knockout mutants under various conditions

  • Suppressor mutant analysis to identify genetic interactions

• Advanced structural biology techniques:

  • Integrative structural biology combining multiple data types

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Chemical cross-linking for interaction mapping

Understanding the biological role of UPF0276 family proteins like PP_0992 requires creative experimental designs and integration of diverse data types, with constant refinement of hypotheses based on accumulated evidence.