Recombinant Uncharacterized protein YPO1740/y2567/YP_1481 (YPO1740, y2567, YP_1481)

What is protein YPO1740/y2567/YP_1481 and what organism does it originate from?

YPO1740/y2567/YP_1481 is an uncharacterized protein found in Yersinia pestis, the bacterium responsible for bubonic plague . The protein has multiple identifiers (YPO1740, y2567, YP_1481) reflecting different naming conventions across databases. The "y" prefix in y2567 specifically denotes its status as a gene of unknown function, following the naming convention established for uncharacterized genes in bacterial genomes .

The protein consists of 91 amino acids, making it a relatively small protein with a molecular mass of approximately 10.2 kDa . Based on its amino acid sequence (MLDTNMSAFGVASIALPLLTVLFFLIVWFFLSRASVRANEQVRLLREIAEQQKRQTELLTALLENATGTRDGQNDSDTVSPLDFKGFIPER), hydrophobicity analysis suggests it likely contains transmembrane domains, indicating it may be a membrane-associated protein .

How are uncharacterized proteins classified in genomic databases?

Uncharacterized proteins like YPO1740 are classified using a hierarchical system in major genomic databases. Current classification schemes typically divide proteins into three or four categories based on their level of functional characterization . For example, EcoCyc (a comprehensive database for Escherichia coli) employs a three-tier system:

• Well-characterized: Proteins with experimental evidence for both molecular function and biological process involvement

• Partially characterized: Proteins with experimental evidence for either molecular function or biological process (but not both), or with strong computational evidence for function

• Uncharacterized: Proteins with minimal knowledge about function, often only predicted cellular location

The classification algorithm considers multiple factors, including:

• Product names containing keywords like "hypothetical" or "putative"

• Presence of experimental evidence codes in annotations

• GO term assignments with experimental validation

• Reactions catalyzed by the protein product

• Pathway associations with experimental support

What approaches can be used to predict potential functions of YPO1740 based on its sequence?

Predicting the function of YPO1740 requires a multi-faceted computational approach followed by experimental validation. The methodological workflow should include:

• Sequence-based analysis: Use algorithms like BLAST to identify homologous proteins in other organisms. Even distant homology can provide initial functional clues. For YPO1740, its sequence (MLDTNMSAFGVASIALPLLTVLFFLIVWFFLSRASVRANEQVRLLREIAEQQKRQTELLTALLENATGTRDGQNDSDTVSPLDFKGFIPER) should be analyzed against both characterized and uncharacterized protein databases .

• Domain identification: Search for conserved domains using tools like Pfam, InterPro, and SMART to identify functional modules. The presence of transmembrane regions in YPO1740 suggests membrane-associated functions that should be investigated with specialized tools like TMHMM or Phobius .

• Structural prediction: Use AlphaFold2 or RoseTTAFold to generate structural models, which can reveal functional sites not obvious from sequence alone. For small proteins like YPO1740 (91 amino acids), these methods can produce relatively reliable predictions.

• Genomic context analysis: Examine neighboring genes and operonic structures, as functionally related genes are often co-located in bacterial genomes. The search results indicate that uncharacterized genes may cluster in specific genomic regions, suggesting YPO1740 might be part of a functional unit with other uncharacterized proteins .

• Phylogenetic profiling: Determine which organisms contain YPO1740 homologs and correlate this distribution with ecological or metabolic characteristics to infer potential functions.

What experimental approaches are most effective for characterizing the function of YPO1740?

Characterizing an uncharacterized protein like YPO1740 requires a systematic experimental approach combining multiple techniques:

• Genetic manipulation and phenotypic analysis:

  • Generate knockout mutants using CRISPR-Cas9 or homologous recombination

  • Perform phenotypic screening under various stress conditions (pH, temperature, osmotic pressure)

  • Conduct fitness assays to identify growth defects in specific media or environments

  • Implement Tn-seq for high-throughput phenotypic screening

• Protein localization and interaction studies:

  • Express fluorescently tagged versions to determine subcellular localization

  • Perform immunoprecipitation followed by mass spectrometry (IP-MS) to identify interaction partners

  • Use bacterial two-hybrid assays to validate specific protein-protein interactions

  • Employ in vivo crosslinking to capture transient interactions

• Structural biology approaches:

  • Express and purify recombinant protein for X-ray crystallography or cryo-EM

  • Perform NMR spectroscopy for small proteins like YPO1740 (91 amino acids)

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions

• Biochemical activity testing:

  • Design activity assays based on predicted functions (e.g., membrane transport, signaling)

  • Screen for binding to various metabolites, nucleic acids, or lipids

  • Test enzymatic activities with substrate libraries

The integration of these approaches provides complementary data that can converge on functional hypotheses for testing.

How can researchers effectively express and purify recombinant YPO1740 for structural studies?

Recombinant expression and purification of membrane or membrane-associated proteins like YPO1740 presents specific challenges that require methodological optimization:

• Expression system selection:

  • For membrane proteins, specialized expression systems like C41(DE3) or C43(DE3) E. coli strains (Walker strains) often yield better results

  • Consider cell-free expression systems for toxic or highly hydrophobic proteins

  • Evaluate eukaryotic expression systems (yeast, insect cells) if bacterial expression fails

• Fusion tag strategies:

  • N-terminal tags: His6, MBP, or GST to enhance solubility and facilitate purification

  • C-terminal tags: Consider if N-terminal is functionally important

  • Cleavable tags: Include TEV or PreScission protease sites for tag removal

• Expression optimization:

  • Test multiple induction conditions (temperature, inducer concentration)

  • Include membrane-stabilizing additives in growth media

  • Consider co-expression with chaperones (GroEL/GroES) to aid folding

• Purification strategy for YPO1740:

  • Membrane extraction using mild detergents (DDM, LMNG, or amphipols)

  • Implement two-step purification (affinity chromatography followed by size exclusion)

  • Verify protein integrity through mass spectrometry

• Quality control measures:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to assess oligomeric state

  • Thermal shift assays to identify stabilizing conditions

The complete workflow should be adapted based on initial results, with particular attention to detergent selection for membrane protein extraction.

What challenges arise when designing experiments to determine if YPO1740 belongs to the "partially characterized" or "uncharacterized" category?

Categorizing YPO1740 definitively requires addressing specific experimental challenges:

• Evidence threshold definition:

  • According to the categorization framework used in genomic databases, a protein requires experimental evidence for both molecular function and biological process involvement to be considered "well-characterized"

  • For partial characterization, evidence of either function or process is sufficient

• Experimental design considerations:

  • Function determination requires direct biochemical assays (binding, catalysis)

  • Process involvement requires in vivo studies linking YPO1740 to specific pathways

  • Both aspects must be addressed with non-computational evidence codes

• Validation requirements:

  • Multiple independent experimental techniques should confirm function

  • Control experiments must rule out artifact interactions or activities

  • Physiological relevance must be demonstrated in the native organism

• Documentation challenges:

  • Results must be published with appropriate GO term annotations

  • Evidence codes must clearly indicate experimental validation

  • Database submissions should follow standardized formats

The key challenge is designing experiments that definitively connect molecular activities to biological contexts, as many proteins demonstrate activities in vitro that may not reflect their physiological roles.

How might researchers approach studying YPO1740 in the context of its genomic neighborhood?

Studying YPO1740 in its genomic context requires a systematic approach to uncover functional relationships:

• Operon structure analysis:

  • Identify potential operons containing YPO1740 using RNA-seq data

  • Map transcription start sites and terminators

  • Determine if YPO1740 is co-expressed with neighboring genes

• Genomic clustering investigation:

  • The search results indicate that uncharacterized genes may cluster in specific regions, forming "hotspots" of 5-8 genes

  • These clusters may represent functionally related gene sets or horizontally transferred elements

  • Map synteny across related bacterial species to assess conservation

• Co-expression network analysis:

  • Generate transcriptomic data under various conditions

  • Identify genes consistently co-regulated with YPO1740

  • Construct co-expression networks to visualize functional associations

• Multi-gene knockout studies:

  • Create deletion mutants of YPO1740 together with neighboring genes

  • Compare phenotypes of single vs. multiple gene knockouts

  • Identify synthetic lethality or suppressor interactions

This contextual approach recognizes that functionally related genes in bacteria are often physically clustered, providing valuable clues about YPO1740's role within the cellular system.

What safety protocols should researchers implement when working with proteins from Yersinia pestis?

Working with proteins from Yersinia pestis requires stringent safety measures due to its classification as a Tier 1 Select Agent:

• Biosafety level requirements:

  • Work with live Y. pestis requires BSL-3 containment

  • Recombinant proteins expressed in non-pathogenic hosts may be handled at BSL-2 with proper risk assessment

  • Institutional Biosafety Committee (IBC) approval is mandatory before initiating work

• Regulatory compliance:

  • Researchers must comply with export control regulations

  • Rigorous biosecurity screening is required when ordering recombinant Y. pestis proteins

  • Documentation and record-keeping are essential for compliance verification

• Laboratory protocols:

  • Use sealed centrifuge rotors and biosafety cabinets for all procedures

  • Implement validated decontamination procedures for all waste

  • Establish emergency response plans for potential exposures

  • Conduct regular safety training for all personnel

• Alternative approaches:

  • Consider working with attenuated strains or closely related non-pathogenic species

  • Use synthetic biology approaches with minimal gene segments rather than complete genes

  • Implement computational studies when possible to minimize handling of pathogenic material

These safety considerations should be integrated into experimental design from the earliest planning stages.

How can researchers determine if the uncharacterized status of YPO1740 impacts Yersinia pestis pathogenicity?

To investigate potential links between YPO1740 and Y. pestis pathogenicity, researchers should implement a systematic approach:

• Gene expression analysis during infection:

  • Measure YPO1740 expression levels during different stages of infection

  • Compare expression in virulent vs. attenuated strains

  • Assess expression under conditions mimicking host environments (temperature, pH, nutrient limitation)

• Genetic manipulation approaches:

  • Create clean deletions or conditional knockdowns of YPO1740

  • Perform complementation studies to verify phenotypes

  • Test attenuated strains in appropriate infection models

• Host response evaluation:

  • Monitor host immune responses to wild-type vs. YPO1740 mutants

  • Assess impact on key virulence phenotypes (phagocytosis resistance, cytotoxicity)

  • Examine effects on biofilm formation or intracellular survival

• Comparative genomics across Yersinia species:

  • Determine if YPO1740 is conserved in pathogenic and non-pathogenic Yersinia

  • Identify potential horizontal gene transfer events

  • Compare sequence conservation in highly virulent vs. attenuated isolates

The significance of uncharacterized proteins in pathogenicity is increasingly recognized, and systematic studies linking YPO1740 to virulence traits could provide valuable insights for both basic science and therapeutic development.

How might systems biology approaches contribute to understanding YPO1740's function?

Systems biology offers powerful approaches to contextualize YPO1740 within broader cellular networks:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify condition-specific regulation patterns

  • Map YPO1740's position in regulatory networks

• Network modeling approaches:

  • Construct protein-protein interaction networks

  • Develop metabolic models incorporating hypothetical functions

  • Use Bayesian networks to predict functional associations

• Evolutionary systems biology:

  • Compare system-level properties across species with and without YPO1740 homologs

  • Identify co-evolving gene sets that might share functions

  • Analyze selection pressures on YPO1740 across Yersinia lineages

• Machine learning applications:

  • Develop algorithms to predict function from integrated data

  • Identify patterns in experimental data not obvious through traditional analysis

  • Prioritize hypotheses for experimental validation

Systems approaches are particularly valuable for uncharacterized proteins like YPO1740, as they leverage diverse data types to generate testable hypotheses about function.

What are the most promising techniques for determining the 3D structure of YPO1740?

Determining the structure of small membrane-associated proteins like YPO1740 requires selecting appropriate techniques based on protein properties:

• Cryo-electron microscopy (cryo-EM):

  • Traditionally challenging for small proteins (<50 kDa)

  • Recent advances with Volta phase plates improve resolution for smaller proteins

  • Consider expressing YPO1740 as a fusion with a larger scaffold protein

• X-ray crystallography:

  • Optimize crystallization conditions for membrane proteins using lipidic cubic phase methods

  • Screen multiple detergents and lipid compositions

  • Consider antibody fragment co-crystallization to increase polar surfaces

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Particularly suitable for YPO1740 due to its small size (91 amino acids, 10.2 kDa)

  • Requires isotope labeling (15N, 13C) for structural determination

  • Can provide dynamics information not available from static techniques

• Integrative structural biology:

  • Combine multiple techniques (SAXS, HDX-MS, crosslinking)

  • Validate computational predictions with experimental constraints

  • Implement molecular dynamics simulations based on partial structural data

The choice of technique should balance resolution requirements with practical considerations of protein production and stability.

What criteria would need to be met to move YPO1740 from "uncharacterized" to "partially characterized" status?

Based on the classification framework described in the research literature, specific criteria must be met to reclassify YPO1740:

• Evidence requirements:

  • Experimental evidence for either molecular function OR biological process involvement

  • Evidence must use non-computational evidence codes (not predictions)

  • Multiple independent experimental approaches are preferable

• Functional annotation thresholds:

  • Function must be more specific than broad categories (e.g., "binding" is insufficient)

  • Process involvement must connect to established cellular pathways

  • Annotations should avoid ambiguous terms like "putative" or "predicted"

• Documentation standards:

  • Findings must be published in peer-reviewed literature

  • Results must be submitted to appropriate databases with correct evidence codes

  • GO term assignments should follow consortium guidelines

• Validation requirements:

  • In vivo confirmation of in vitro findings

  • Demonstration of physiological relevance

  • Replication by independent research groups

The reclassification of YPO1740 from uncharacterized to partially characterized represents an important contribution to the systematic characterization of bacterial proteomes and enhances our understanding of Yersinia pestis biology.

How does the study of uncharacterized proteins like YPO1740 contribute to broader understanding of bacterial biology?

The characterization of proteins like YPO1740 has far-reaching implications:

• Completion of functional genomics:

  • Approximately 15.5% of E. coli genes remain uncharacterized despite extensive study

  • The percentage is likely higher in less-studied pathogens like Y. pestis

  • Complete functional annotation is required for accurate metabolic and regulatory models

• Discovery of novel biological mechanisms:

  • Uncharacterized proteins often represent undiscovered cellular functions

  • Characterization frequently reveals unexpected biological processes

  • Novel protein families expand our understanding of protein structure-function relationships

• Therapeutic target identification:

  • Pathogen-specific uncharacterized proteins represent potential therapeutic targets

  • Essential uncharacterized proteins may offer new antibiotic development avenues

  • Understanding virulence-associated proteins enables targeted intervention strategies

• Evolutionary insights:

  • Distribution of uncharacterized proteins across species provides evolutionary context

  • Genomic clustering of uncharacterized genes suggests functional relationships

  • Conservation patterns reveal selective pressures and adaptation mechanisms