Recombinant UPF0410 protein yeaQ (yeaQ)

What is UPF0410 protein yeaQ and where is it found?

UPF0410 protein yeaQ is a small bacterial protein consisting of 82 amino acids found in Escherichia coli, including pathogenic strains such as E. coli O157:H7. It belongs to the UPF (Uncharacterized Protein Family) 0410 class, indicating that its precise biological function remains to be fully elucidated. The protein is encoded by the yeaQ gene, also known by synonyms Z2837 and ECs2504 in certain E. coli strains . This protein represents an interesting target for basic research into bacterial membrane biology and protein structure-function relationships.

What structural features characterize the yeaQ protein?

Based on the amino acid sequence, yeaQ appears to be a predominantly hydrophobic protein with multiple potential membrane-spanning regions. The abundance of glycine residues (9 in total) suggests flexibility in certain regions of the protein structure. The C-terminal region contains several charged residues (RKIKS), which may be important for protein-protein interactions or membrane orientation. While detailed structural characterization through X-ray crystallography or NMR spectroscopy is not evident in the provided data, computational predictions would suggest a membrane protein with multiple transmembrane helices .

What expression systems are optimal for recombinant yeaQ production?

Multiple expression systems have been successfully employed for recombinant yeaQ production:

When designing expression systems for yeaQ, researchers should consider that E. coli-based systems have been demonstrated to successfully produce the full-length protein with N-terminal His tags while maintaining high purity (>90% by SDS-PAGE) .

What are the recommended protocols for reconstitution and storage of recombinant yeaQ?

For optimal handling of recombinant yeaQ:

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to opening

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

  • Add glycerol to a final concentration of 5-50% (50% is recommended by manufacturers)

  • Prepare working aliquots to avoid freeze-thaw cycles

• Storage conditions:

  • Long-term storage: -20°C to -80°C in aliquots

  • Working aliquots: 4°C for up to one week

  • Avoid repeated freeze-thaw cycles

  • Lyophilized form has longer shelf life (approximately 12 months) compared to liquid preparations (approximately 6 months)

The protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during storage .

What purification methods yield the highest purity recombinant yeaQ?

Although specific purification protocols are not detailed in the provided data, the commercially available recombinant yeaQ proteins consistently achieve >85-90% purity as determined by SDS-PAGE analysis . For His-tagged versions, standard immobilized metal affinity chromatography (IMAC) is likely employed, followed by potential additional purification steps such as size exclusion chromatography. Researchers working with membrane proteins like yeaQ should consider the challenges of maintaining proper folding during purification, potentially requiring the use of detergents or membrane-mimetic systems to preserve native structure.

What is the current understanding of yeaQ's biological function?

The biological function of yeaQ remains incompletely characterized, as indicated by its classification in the "UPF" (Uncharacterized Protein Family) category. Based on its sequence characteristics with multiple hydrophobic regions, it likely functions as a membrane protein, potentially involved in membrane structure, transport, or signaling pathways. The search results do not provide definitive information on confirmed functions, highlighting an area requiring further research .

How can researchers design experiments to elucidate yeaQ function?

To investigate the functional role of yeaQ, researchers might consider:

• Gene knockout/knockdown studies:

  • Generate yeaQ deletion mutants in E. coli

  • Conduct comprehensive phenotypic characterization under various growth conditions

  • Perform transcriptomic and proteomic analyses to identify affected pathways

• Protein localization experiments:

  • Use fluorescently tagged versions to determine subcellular localization

  • Employ cellular fractionation followed by Western blotting

  • Analyze membrane topology using protease accessibility assays

• Interaction studies:

  • Conduct pull-down assays using tagged recombinant yeaQ

  • Perform bacterial two-hybrid screens

  • Use crosslinking approaches to identify transient interactions

• Structural biology approaches:

  • Conduct structural studies using techniques suitable for membrane proteins

  • Compare with proteins of known function using structural alignment tools

What research applications benefit from recombinant yeaQ?

Recombinant yeaQ can serve multiple research applications:

• Structural biology:

  • Template for protein design studies

  • Model system for small membrane protein crystallization

  • Training dataset for computational prediction algorithms

• Antibody production:

  • Generation of specific antibodies for detection and localization studies

  • Development of tools for studying pathogenic E. coli strains

• Protein engineering:

  • Starting template for designing novel membrane proteins

  • Study of protein folding and stability in membrane environments

What are the major challenges in studying membrane proteins like yeaQ?

Membrane proteins like yeaQ present several research challenges:

• Expression and purification obstacles:

  • Toxicity when overexpressed

  • Proper membrane insertion in heterologous systems

  • Maintaining native conformation during solubilization

  • Prevention of aggregation during purification

• Structural analysis limitations:

  • Difficulties in obtaining crystals suitable for X-ray diffraction

  • Challenges in solution NMR due to size constraints and detergent micelles

  • Need for membrane-mimetic environments

• Functional characterization complexities:

  • Requirement for lipid environments for proper function

  • Challenges in reconstituting physiological interactions

  • Potential for functionally redundant proteins masking phenotypes

How can computational approaches complement experimental studies of yeaQ?

Computational methods offer valuable tools for yeaQ research:

• Structural prediction and analysis:

  • Homology modeling using related proteins with known structures

  • Ab initio structure prediction with specialized membrane protein algorithms

  • Molecular dynamics simulations in membrane environments

• Evolutionary analysis:

  • Identification of conserved residues suggesting functional importance

  • Comparative genomics to identify co-evolved genes

  • Phylogenetic profiling to predict functional associations

• Systems biology approaches:

  • Integration of transcriptomic and proteomic data to identify functional networks

  • Flux balance analysis to predict metabolic impact

  • Protein-protein interaction network analysis

What considerations are important when using recombinant yeaQ for protein design studies?

When using yeaQ in protein design research:

• Sequence-structure-function relationships:

  • Analysis of hydrophobic patterning and its impact on membrane integration

  • Role of conserved glycine residues in structural flexibility

  • Importance of charged C-terminal residues in function

• Experimental design factors:

  • Choice of expression system impacts folding and activity

  • Selection of appropriate membrane-mimetic environments

  • Consideration of tag position and its effect on protein properties

• Validation approaches:

  • Multiple biophysical techniques to confirm structural integrity

  • Functional assays to verify designed properties

  • In vivo testing to confirm biological relevance

What are common troubleshooting strategies for low yields of recombinant yeaQ?

When encountering low yields of recombinant yeaQ:

• Expression optimization:

  • Adjust induction conditions (temperature, inducer concentration, time)

  • Test different E. coli expression strains (BL21, C41/C43 for membrane proteins)

  • Modify codon usage for the expression system

  • Consider fusion partners that enhance solubility

• Purification refinement:

  • Optimize detergent selection and concentration

  • Adjust buffer composition and pH

  • Modify imidazole concentration gradient for His-tagged proteins

  • Implement on-column refolding strategies for inclusion bodies

• Stability enhancement:

  • Test different buffer systems and additives

  • Optimize protein concentration during handling

  • Consider lipid addition during purification

  • Evaluate alternative tag positions or cleavage strategies

How can researchers differentiate between properly folded and misfolded recombinant yeaQ?

Assessing proper folding of recombinant yeaQ:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to monitor tertiary structure

  • Size-exclusion chromatography to evaluate oligomeric state

  • Thermal shift assays to measure stability

• Functional assessment:

  • Membrane integration assays

  • Ligand binding studies (if ligands are known)

  • Activity assays (when function is established)

• Structural integrity evaluation:

  • Limited proteolysis to probe accessible regions

  • Detergent resistance as indicator of proper folding

  • NMR spectroscopy for structural assessment

What methodological approaches can improve structural studies of yeaQ?

To enhance structural characterization of yeaQ:

• Crystallization strategies:

  • Screen specialized detergents for membrane proteins

  • Utilize lipidic cubic phase crystallization

  • Consider antibody fragment co-crystallization

  • Test fusion partners that facilitate crystal contacts

• NMR optimization:

  • Selective isotope labeling strategies

  • Detergent screening for optimal spectral quality

  • Nanodiscs or amphipols as membrane mimetics

• Cryo-EM approaches:

  • Reconstitution in nanodiscs or amphipols

  • Use of Fab fragments to increase particle size

  • Implementation of advanced image processing algorithms

• Hybrid methods:

  • Integrate low-resolution structural data with computational modeling

  • Combine multiple structural techniques for comprehensive characterization

  • Use crosslinking mass spectrometry to obtain distance constraints