Recombinant Escherichia coli Uncharacterized protein yebO (yebO)

What is the YebO protein and what is its known sequence information?

YebO is an uncharacterized protein from Escherichia coli with a UniProt ID of P64499. The protein consists of 95 amino acids with the following sequence:
MNEVVNSGVMNIASLVVSVVVLLIGLILWFFINRASSRTNEQIELLEALLDQQKRQNALLRRLCEANEPEKADKKTVESQKSVEDEDIIRLVAER

The protein is encoded by the yebO gene (also known as b1825 or JW1814) and appears to be a membrane-associated protein based on its amino acid composition, which includes a hydrophobic stretch characteristic of transmembrane domains . Despite being classified as "uncharacterized," preliminary analyses suggest it may function in membrane organization or cellular transport processes.

What expression systems are most effective for recombinant YebO production?

E. coli expression systems are the most commonly utilized for producing recombinant YebO protein. Based on available research protocols, the following expression systems have demonstrated successful results:

The choice of expression system should be determined by experimental requirements, with E. coli BL21(DE3) serving as an appropriate starting point for most applications .

How should recombinant YebO protein be stored and handled for optimal stability?

Recombinant YebO protein stability is significantly affected by storage conditions. For optimal results, the following guidelines should be followed:

• Store lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, add 5-50% glycerol (final concentration) before aliquoting for long-term storage at -20°C/-80°C

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Working aliquots may be stored at 4°C for up to one week

The protein appears to maintain stability in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which should be considered when designing experimental protocols .

What purification methods are most effective for His-tagged YebO protein?

Purification of His-tagged YebO protein typically follows standard immobilized metal affinity chromatography (IMAC) protocols with specific optimizations:

• Ni-NTA agarose columns perform well with binding buffers containing 20-50 mM imidazole to reduce non-specific binding

• A step gradient elution protocol using 100 mM, 250 mM, and 500 mM imidazole yields the highest purity

• Post-IMAC size exclusion chromatography may be necessary to achieve >95% purity

• For membrane-associated proteins like YebO, addition of mild detergents (0.1% DDM or 0.5% CHAPS) in purification buffers improves yield

Western blotting using anti-His antibodies can confirm successful purification, with expected bands at approximately 10-11 kDa for the YebO protein .

What structural characteristics of YebO influence its putative function in E. coli?

While YebO remains largely uncharacterized, bioinformatic analysis of its sequence reveals several structural features that may provide insight into its function:

• Hydrophobicity analysis indicates a transmembrane domain spanning residues 15-35, suggesting membrane integration

• The C-terminal region (residues 60-95) contains charged residues arranged in a pattern consistent with amphipathic helices, potentially mediating protein-protein interactions

• Secondary structure prediction algorithms suggest the presence of three α-helices and no β-sheets

These structural features place YebO in the category of small membrane proteins that may function in membrane organization, protein trafficking, or stress response pathways. Its relatively small size (95 amino acids) suggests a potential role as an accessory protein in larger complexes rather than as an enzymatic protein .

How can expression and purification of YebO be optimized for structural studies?

Optimizing YebO expression and purification for structural studies presents specific challenges due to its membrane association. The following methodological approaches have shown promise:

• Expression optimization:

  • Induction with 0.1-0.5 mM IPTG at lower temperatures (16-20°C) for 16-20 hours

  • Co-expression with molecular chaperones (GroEL/GroES) to improve folding

  • Use of specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression

• Purification refinements:

  • Inclusion of lipid nanodiscs or amphipols during purification to maintain native conformation

  • Detergent screening (DDM, LMNG, CHAPS) to identify optimal solubilization conditions

  • Buffer optimization with various salts (100-500 mM NaCl) and pH conditions (pH 6.5-8.5)

• Quality assessment metrics:

  • Size-exclusion chromatography profiles showing monodisperse peaks

  • Thermal shift assays indicating stable protein folding

  • Negative stain electron microscopy to confirm protein homogeneity

These methodological refinements significantly enhance the likelihood of obtaining protein preparations suitable for crystallography, cryo-EM, or NMR structural studies .

What approaches are most effective for functional characterization of uncharacterized proteins like YebO?

Functional characterization of uncharacterized proteins like YebO requires a multi-faceted approach:

• Protein-protein interaction studies:

  • Affinity purification coupled with mass spectrometry (AP-MS) to identify binding partners

  • Yeast two-hybrid screening against genomic libraries

  • Proximity labeling techniques (BioID, APEX) to identify neighboring proteins in vivo

• Localization studies:

  • Fluorescent protein tagging for live-cell microscopy

  • Immunogold electron microscopy for precise subcellular localization

  • Fractionation studies coupled with western blotting

• Phenotypic analysis:

  • Gene knockout/knockdown studies followed by comprehensive phenotypic assays

  • Complementation with mutant variants to identify critical residues

  • Growth under various stress conditions to identify conditional phenotypes

• Integrated omics approaches:

  • Transcriptomics of knockout strains

  • Quantitative proteomics to identify affected pathways

  • Metabolomics to detect metabolic alterations

These methodological approaches should be applied systematically to gradually build a functional profile of YebO .

How might YebO interact with other components of bacterial membrane systems?

Based on limited data and structural predictions, YebO may interact with bacterial membrane systems in several specific ways:

• Potential interactions with membrane protein complexes:

  • The conserved charged residues in the C-terminal domain might mediate electrostatic interactions with other membrane proteins

  • YebO may function as an accessory protein in larger complexes, similar to other small membrane proteins in E. coli

• Possible roles in membrane organization:

  • The transmembrane domain might participate in membrane microdomain formation

  • YebO could potentially influence membrane curvature or rigidity in specific cellular regions

• Interaction with the bacterial cytoskeleton:

  • Small membrane proteins often connect the membrane to cytoskeletal elements

  • YebO might serve as an anchor point for cytoskeletal proteins that maintain cell shape

Experimental approaches to test these hypotheses could include blue native PAGE, chemical crosslinking studies, and co-immunoprecipitation with candidate interacting proteins identified through bioinformatic predictions .

What is known about the regulation of yebO gene expression in different environmental conditions?

While specific data on yebO regulation is limited, uncharacterized proteins in E. coli often show condition-specific expression patterns. Based on general knowledge of bacterial gene regulation:

• Stress response regulation:

  • Many membrane proteins show altered expression under membrane stress conditions

  • Monitoring yebO expression using qRT-PCR under various stressors (temperature, pH, osmotic pressure) could reveal regulatory patterns

• Growth phase-dependent expression:

  • Small membrane proteins often show growth phase-dependent regulation

  • Analysis of yebO expression across different growth phases (lag, exponential, stationary) could provide functional insights

• Potential transcriptional regulators:

  • Bioinformatic analysis of the yebO promoter region might reveal binding sites for known transcription factors

  • Chromatin immunoprecipitation (ChIP) studies could identify factors binding to the yebO regulatory regions

A comprehensive gene expression analysis under various conditions would significantly advance understanding of yebO's physiological role in E. coli .

What controls should be included when studying YebO function?

When designing experiments to investigate YebO function, the following controls are essential:

• Genetic controls:

  • Clean deletion mutant (ΔyebO)

  • Complementation with wild-type yebO

  • Complementation with tagged versions of yebO to confirm functionality

  • Empty vector controls for complementation studies

• Protein expression controls:

  • Expression of unrelated membrane proteins of similar size

  • Expression of YebO with mutations in key predicted functional residues

  • Dose-dependent expression controls to differentiate between primary and secondary effects

• Localization controls:

  • Co-localization with known membrane compartment markers

  • Controls for antibody specificity in immunolocalization studies

  • Fractionation quality controls using established compartment markers

These controls will help distinguish genuine YebO-specific effects from artifacts and general consequences of membrane protein overexpression or deletion .

How can researchers effectively analyze the impact of YebO mutations on protein function?

Systematic analysis of YebO mutations requires careful experimental design:

• Mutation strategy:

  • Alanine scanning of conserved residues

  • Charge reversal mutations for charged residues

  • Conservative and non-conservative substitutions at key positions

  • Truncation analysis to identify functional domains

• Functional assessment approaches:

  • Complementation of knockout phenotypes

  • Protein stability and localization analysis

  • Interaction partner binding assays

  • In vitro activity assays (if applicable)

• Data analysis framework:

  • Quantitative phenotype scoring

  • Structure-function correlation

  • Conservation analysis across bacterial species

  • Molecular dynamics simulations to predict mutation effects

This systematic approach will generate a comprehensive mutational landscape that can reveal functionally important regions and residues within the YebO protein .

How should researchers interpret proteomic data involving YebO?

Interpreting proteomic data for uncharacterized proteins like YebO requires specialized analytical approaches:

• Differential abundance analysis:

  • Compare protein levels between wild-type and ΔyebO strains

  • Analyze affected pathways using enrichment analysis

  • Consider both direct and indirect effects based on magnitude and timing of changes

• Protein-protein interaction data:

  • Prioritize interactions detected across multiple experimental approaches

  • Evaluate interaction strength using quantitative metrics from AP-MS

  • Validate key interactions using orthogonal methods

• Post-translational modification analysis:

  • Identify modified residues in YebO under different conditions

  • Correlate modifications with functional changes

  • Compare modification patterns across growth conditions

These analytical approaches will help distinguish biologically relevant signals from experimental noise in complex proteomic datasets .

What bioinformatic approaches can predict YebO function based on sequence and structural information?

Several bioinformatic approaches can provide functional insights for YebO:

• Sequence-based approaches:

  • Hidden Markov Model profiling against domain databases

  • Remote homology detection using PSI-BLAST and HHpred

  • Genetic context analysis (neighboring genes, operon structure)

• Structure-based approaches:

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold

  • Structural comparison with characterized proteins

  • Binding site prediction based on surface properties

• Integrated approaches:

  • Co-evolutionary analysis to identify functionally linked residues

  • Phylogenetic profiling to identify co-occurring genes

  • Metabolic modeling to predict pathway associations

These computational approaches, when integrated with experimental data, can significantly narrow the functional hypothesis space for uncharacterized proteins like YebO .

What are the most promising research directions for understanding YebO function?

Based on current knowledge and available methodologies, several research directions show particular promise:

• Systematic interactome mapping:

  • Comprehensive identification of protein-protein interactions

  • Mapping of genetic interactions through synthetic genetic arrays

  • Characterization of condition-specific interaction networks

• Structural characterization:

  • High-resolution structure determination using cryo-EM or X-ray crystallography

  • Analysis of conformational dynamics using hydrogen-deuterium exchange mass spectrometry

  • In silico molecular dynamics simulations to predict functional motions

• Systems-level functional analysis:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Phenotypic profiling under diverse environmental conditions

  • Evolutionary analysis across diverse bacterial species

These complementary approaches will collectively advance understanding of YebO's functional role in E. coli physiology and potentially reveal new aspects of bacterial membrane biology .

How can researchers contribute to the collective understanding of uncharacterized proteins like YebO?

The scientific community can advance knowledge of uncharacterized proteins through several collaborative approaches:

• Data sharing and standardization:

  • Deposition of experimental data in public repositories

  • Adoption of standardized experimental protocols

  • Development of community resources for functional annotation

• Interdisciplinary collaboration:

  • Integration of structural biology, genetics, and systems biology approaches

  • Combining computational prediction with experimental validation

  • Engagement with researchers studying homologous proteins in diverse organisms

• Technological innovation:

  • Development of new methods for membrane protein characterization

  • Application of emerging technologies like single-cell proteomics

  • Implementation of machine learning approaches for functional prediction