Recombinant Saccharomyces cerevisiae UPF0479 membrane protein YER190C-B (YER190C-B)

What expression systems are most effective for producing recombinant YER190C-B?

E. coli expression systems have demonstrated successful production of recombinant YER190C-B with N-terminal His-tags, as evidenced by commercially available preparations . When designing expression constructs, researchers should consider:

• Codon optimization for the expression host

• Signal sequence selection (if targeting different cellular compartments)

• Tag placement (N-terminal tags are documented to work well)

• Induction conditions optimization

For membrane proteins like YER190C-B, expression in E. coli often requires careful optimization of growth temperature (typically lowering to 16-20°C during induction), inducer concentration, and media composition to prevent formation of inclusion bodies. While E. coli is documented to work for YER190C-B, researchers facing difficulties might consider alternative expression systems such as yeast (the native host), insect cells, or cell-free systems for producing functional protein .

How should recombinant YER190C-B be properly stored and reconstituted?

Recombinant YER190C-B is typically supplied as a lyophilized powder and requires proper reconstitution and storage to maintain functionality. The recommended protocol is:

Storage:

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

• For reconstituted protein, store at -20°C/-80°C with 5-50% glycerol (with 50% being optimal)

• Aliquot to avoid repeated freeze-thaw cycles

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

Reconstitution Protocol:

• Briefly centrifuge the vial to bring contents to the bottom

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

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

• Aliquot for long-term storage

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

What methods are optimal for solubilizing and stabilizing YER190C-B for structural and functional studies?

Membrane proteins like YER190C-B require special handling to maintain their native structure outside the lipid bilayer. Several approaches can be considered:

Detergent-Based Methods:

• DDM (n-Dodecyl β-D-maltoside) has been successfully used for solubilizing membrane proteins similar to YER190C-B

• LDAO (Lauryldimethylamine oxide) is another effective detergent for membrane protein extraction

• Triton X-100 may be used for initial extraction from membranes

Peptidisc Method:
The peptidisc approach represents a promising alternative to traditional detergent-based methods:

• Mix purified YER190C-B (in detergent) with the NSP (Nanodisc Scaffold Protein) peptide

• Allow brief incubation (1-2 minutes) at room temperature

• Dilute the mixture to reduce detergent concentration below its critical micelle concentration (CMC)

• Analyze by CN-PAGE to confirm successful incorporation

The peptidisc method offers several advantages:

• No requirement for exogenous lipids

• Simple and rapid protocol

• Maintains protein in a near-native membrane environment

• Enables structural and functional studies without detergent interference

How can I optimize purification protocols for YER190C-B to achieve high purity and yield?

The purification of YER190C-B requires a strategic approach to overcome challenges associated with membrane proteins:

Recommended Purification Strategy:

• Membrane Preparation:

  • Isolate membrane fractions from expression host

  • Solubilize membranes with appropriate detergent (1% DDM has been documented)

  • Clarify by ultracentrifugation (100,000 × g, 1 hour, 4°C)

• Initial Capture:

  • Apply solubilized material to Ni2+-NTA resin pre-equilibrated with Buffer A + 0.02% DDM

  • Incubate for 1 hour at 4°C with gentle agitation

  • Wash with Buffer B + 0.02% DDM (typically 50 mM Tris-HCl pH 8, 200 mM NaCl, 15 mM imidazole, 10% glycerol)

• Elution and Further Purification:

  • Elute with Buffer C containing higher imidazole (400 mM) and 0.02% DDM

  • Consider size exclusion chromatography on Superdex 200 for final polishing

  • Monitor purity by SDS-PAGE (>90% purity is achievable)

The purification can be assessed using SDS-PAGE, with expected purity greater than 90% for properly optimized protocols .

What analytical methods are most effective for characterizing YER190C-B structure and function?

Multiple complementary techniques can be employed to comprehensively characterize YER190C-B:

Structural Characterization:

• Circular Dichroism (CD): Assess secondary structure composition and thermal stability

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): Determine oligomeric state and homogeneity

• Cryo-Electron Microscopy: For high-resolution structural determination

• Native Mass Spectrometry: Analyze protein-lipid interactions and oligomeric states

Functional Analysis:

• Proteoliposome Reconstitution: Study transport or channel activity if applicable

• Thermal Shift Assays: Assess stability and ligand binding

• Surface Plasmon Resonance (SPR): Measure interaction with potential binding partners

Biophysical Techniques:

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map flexible and dynamic regions

• Microscale Thermophoresis (MST): Study molecular interactions in solution

These methods should be adapted based on the specific research questions regarding YER190C-B function, which remains to be fully characterized in the literature .

How does YER190C-B expression data compare across different growth conditions and what implications does this have?

According to the Saccharomyces Genome Database (SGD), there is currently no expression data available for YER190C-B in their database . This lack of expression data presents both a challenge and an opportunity for researchers:

Research Opportunities:

• Characterize expression patterns under various growth conditions

• Investigate regulation mechanisms of YER190C-B

• Compare expression with homologs or proteins in the same pathway

Experimental Approach:

• RT-qPCR: Quantify mRNA expression levels under different conditions

• Western Blotting: Measure protein expression with specific antibodies

• RNA-Seq: Perform transcriptomic analysis to identify co-regulated genes

• Proteomics: Use mass spectrometry to quantify protein abundance

This gap in knowledge about YER190C-B expression patterns highlights the need for researchers to perform systematic expression studies under different physiological conditions to better understand its biological role in Saccharomyces cerevisiae .

What are the most effective methods for studying YER190C-B interactions with other proteins or biomolecules?

Identifying interaction partners is crucial for understanding YER190C-B function. Several complementary approaches can be employed:

In Vitro Methods:

• Pull-down Assays: Using purified His-tagged YER190C-B as bait

• Surface Plasmon Resonance (SPR): Quantify binding kinetics and affinities

• Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of interactions

• Microscale Thermophoresis (MST): Detect interactions in solution with minimal protein consumption

In Vivo Methods:

• Yeast Two-Hybrid: Modified membrane yeast two-hybrid systems for membrane proteins

• Co-immunoprecipitation: From native Saccharomyces cerevisiae

• Proximity Labeling: BioID or APEX2 approaches to identify proximal proteins

• Fluorescence Resonance Energy Transfer (FRET): Study interactions in living cells

When stabilizing YER190C-B for interaction studies, the peptidisc method may offer advantages over detergent-based approaches by maintaining a more native-like membrane environment that preserves physiologically relevant interactions .

How can I effectively use the peptidisc method to stabilize YER190C-B for functional studies?

The peptidisc method represents an innovative approach for stabilizing membrane proteins like YER190C-B without conventional detergents. This method uses multiple copies of a unique amphipathic peptide to wrap around the hydrophobic surface of membrane proteins.

Detailed Protocol for Peptidisc Reconstitution:

• Preparation:

  • Purify YER190C-B in mild detergent (0.02% DDM recommended)

  • Prepare NSP peptide (Nanodisc Scaffold Protein) in assembly buffer

• Reconstitution Mixture:

  • Mix YER190C-B (~300 μg) with NSP peptide (480 μg) in a 1:1.6 protein:peptide ratio

  • Total volume: 100 μL

• Size Exclusion Chromatography:

  • Immediately inject the mixture onto a Superdex 200 column

  • Run at 0.4 mL/min in buffer containing 50 mM Tris-HCl, pH 8, 100 mM NaCl

  • Collect fractions containing the peptidisc-stabilized protein

• Concentration and Storage:

  • Pool fractions containing YER190C-B-peptidisc complexes

  • Concentrate using a 100 kDa molecular weight cutoff filter

  • Store at 4°C for short-term use

Optimization Tips:

• The optimal protein:peptide ratio may require empirical determination

• CN-PAGE can be used to verify successful peptidisc formation

• For membrane proteins like YER190C-B, monitor activity before and after peptidisc formation to ensure function is preserved

What experimental design considerations are important when studying the function of YER190C-B?

When designing experiments to investigate YER190C-B function, several key considerations should be addressed:

Experimental Controls:

• Positive Controls: Include well-characterized membrane proteins from the same family

• Negative Controls: Empty vectors or inactive mutants

• System Validation: Verify expression system performance with known membrane proteins

Experimental Variables to Consider:

Functional Assays to Consider:

• Binding assays for potential ligands

• Transport assays if YER190C-B is suspected to be a transporter

• Structural changes upon substrate binding

• Oligomerization studies under different conditions

Given the limited functional data available for YER190C-B, a combination of bioinformatic predictions and experimental validation should guide hypothesis generation.

How can I design effective mutagenesis studies to probe YER190C-B structure-function relationships?

Strategic mutagenesis can provide valuable insights into YER190C-B function and structure-function relationships:

Mutagenesis Strategy Planning:

• Bioinformatic Analysis:

  • Perform sequence alignment with homologs

  • Identify conserved residues across UPF0479 family members

  • Predict transmembrane regions and functional domains

  • Use the known amino acid sequence (MMPAKLQLDVLRTLQSSARHGTQTLKNSNFLERFHKDRIVFCLPFFPALFFVPVQKVLQHLCLRFTQVAPYFIIQLFDLPSRHAENLAPLLASCRIQYTNCFSSSSNGQVPSIISLYLRVDLSPFYAKIFQISYRVPMIWLDVFQVFFVFLVISQHSLHS) for structural prediction

• Target Selection:

  • Conserved residues in predicted functional domains

  • Charged residues in transmembrane regions

  • Potential post-translational modification sites

  • Residues at predicted protein-protein interaction interfaces

• Mutation Types:

  • Alanine scanning of functional regions

  • Conservative substitutions to probe chemical requirements

  • Cysteine substitutions for crosslinking studies

  • Domain swapping with homologs

Experimental Validation:

• Express mutants in the same system as wild-type

• Compare expression levels, stability, and localization

• Assess functional impact using established assays

• Consider using thermostability assays to detect structural perturbations

Creating a systematic mutation library of YER190C-B will provide valuable structure-function insights that can guide further mechanistic studies of this poorly characterized membrane protein.

How should I approach data analysis when characterizing YER190C-B for the first time?

When characterizing a poorly studied protein like YER190C-B, robust data analysis approaches are essential:

Data Integration Strategy:

• Multi-omics Integration:

  • Compare transcriptomic, proteomic, and metabolomic data

  • Use databases like SGD to identify patterns across datasets

  • Look for co-expression patterns with known proteins

• Structural Bioinformatics:

  • Use homology modeling based on related UPF0479 family members

  • Validate models with experimental data (CD spectroscopy, limited proteolysis)

  • Predict functional sites using conservation analysis

• Statistical Approaches:

  • Use appropriate controls and replicates (minimum n=3)

  • Apply statistical tests suitable for your data distribution

  • Consider Bayesian approaches for integrating multiple data types

Data Visualization:

• Create standardized plots for comparing wild-type and mutant properties

• Use structural mapping of data onto predicted models

• Develop interactive visualizations for complex datasets

Since YER190C-B lacks extensive characterization, initial data analysis should focus on establishing baseline parameters (expression levels, stability, purification yields) that can serve as references for future studies .

What are the common pitfalls when working with recombinant YER190C-B and how can they be addressed?

Membrane proteins like YER190C-B present specific challenges that researchers should anticipate:

Common Challenges and Solutions:

Quality Control Measures:

• Implement rigorous purity assessment (>90% by SDS-PAGE)

• Verify proper folding using circular dichroism

• Confirm homogeneity by size exclusion chromatography

• Establish functional assays for batch validation

For YER190C-B specifically, the reconstitution process from lyophilized powder is critical - following the recommended protocol with proper trehalose-containing buffer at pH 8.0 and adding the suggested 5-50% glycerol for storage can significantly improve stability and reduce variability between experiments .

What are promising research directions for further characterizing YER190C-B function?

YER190C-B remains poorly characterized, opening several promising research avenues:

Future Research Opportunities:

• Functional Genomics:

  • CRISPR-based knockout/knockdown studies in Saccharomyces cerevisiae

  • Phenotypic analysis under various stress conditions

  • Synthetic genetic array analysis to identify genetic interactions

• Structural Biology:

  • Cryo-EM structure determination of YER190C-B in peptidisc

  • X-ray crystallography of stable domains

  • NMR studies of dynamic regions

• Systems Biology:

  • Integration of YER190C-B into yeast metabolic and signaling networks

  • Identification of condition-specific expression patterns

  • Comparative analysis across fungal species

• Translational Potential:

  • Exploration of YER190C-B homologs in pathogenic fungi

  • Assessment as potential antifungal target

  • Development of tools for membrane protein research

Given the current lack of expression data in public databases , comprehensive transcriptomic and proteomic profiling of YER190C-B under various conditions would provide valuable insights into its biological role and regulation in Saccharomyces cerevisiae.

How can computational approaches enhance our understanding of YER190C-B?

Computational methods offer powerful tools for generating hypotheses about YER190C-B function:

Computational Approaches:

• Sequence-Based Analysis:

  • Hidden Markov Models to identify remote homologs

  • Evolutionary analysis to identify conserved functional residues

  • Prediction of post-translational modification sites

• Structural Bioinformatics:

  • Ab initio structure prediction using AlphaFold or RoseTTAFold

  • Molecular dynamics simulations in membrane environments

  • Protein-protein docking with predicted interactors

• Systems-Level Modeling:

  • Integration into genome-scale metabolic models of yeast

  • Network analysis to predict functional associations

  • Machine learning to identify patterns across diverse datasets

• Predictive Functional Analysis:

  • Based on the amino acid sequence (MMPAKLQLDVLRTLQSSARHGTQTLKNSNFLERFHKDRIVFCLPFFPALFFVPVQKVLQHLCLRFTQVAPYFIIQLFDLPSRHAENLAPLLASCRIQYTNCFSSSSNGQVPSIISLYLRVDLSPFYAKIFQISYRVPMIWLDVFQVFFVFLVISQHSLHS), perform transmembrane topology prediction

  • Identify potential ligand binding pockets

  • Predict subcellular localization signals

These computational approaches can generate testable hypotheses about YER190C-B function that guide experimental design and accelerate functional characterization.