Recombinant Saccharomyces cerevisiae UPF0479 membrane protein YPR204C-A (YPR204C-A)

What is UPF0479 membrane protein YPR204C-A and what is its significance in research?

UPF0479 membrane protein YPR204C-A is a protein encoded by the YPR204C-A gene in Saccharomyces cerevisiae (Baker's yeast), specifically strain ATCC 204508/S288c . This protein belongs to the UPF0479 family of uncharacterized proteins, with "UPF" designating "uncharacterized protein family." The protein consists of 160 amino acid residues and is localized to cellular membranes. Its significance in research stems from its potential as a model for studying membrane protein integration, trafficking, and function in eukaryotic systems. The protein is relatively small compared to many membrane proteins, making it potentially valuable for structural studies and investigations into fundamental aspects of membrane protein biology in yeast, which serves as an important eukaryotic model organism.

What expression systems are most effective for producing recombinant YPR204C-A?

When using E. coli for YPR204C-A expression, consider these methodological approaches:

• Specialized E. coli strains: Use strains like BL21(DE3)pLysS or C41/C43(DE3) that are engineered for membrane protein expression

• Fusion partners: Employ solubility-enhancing tags such as MBP, SUMO, or Thioredoxin

• Codon optimization: Adapt the sequence for E. coli codon usage to enhance translation efficiency

For higher fidelity expression:

• Yeast systems: Pichia pastoris or native S. cerevisiae offer proper eukaryotic folding machinery

• Insect cell systems: Baculovirus-mediated expression provides advanced eukaryotic processing

The choice should be guided by research objectives - E. coli for high yield and initial characterization, yeast or insect cells for functional studies requiring proper folding and post-translational modifications.

What are the optimal storage conditions for maintaining stability of recombinant YPR204C-A?

For optimal stability of recombinant YPR204C-A, the protein should be stored at -20°C for routine use, and at -80°C for extended storage periods . The recommended storage buffer consists of a Tris-based buffer supplemented with 50% glycerol, which has been optimized specifically for this protein . This high glycerol concentration prevents ice crystal formation and maintains protein conformational stability during freeze-thaw cycles.

For working with the protein, it's advisable to prepare small aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing can lead to protein denaturation and loss of function. Working aliquots can be stored at 4°C for up to one week . When handling membrane proteins like YPR204C-A, the following additional considerations are important:

• Maintain a suitable detergent concentration above the critical micelle concentration (CMC) to prevent protein aggregation

• Include protease inhibitors to prevent degradation

• Consider adding reducing agents like DTT or β-mercaptoethanol if the protein contains cysteine residues

• Document protein concentration, buffer composition, and preparation date on each aliquot

These precautions will help ensure maximum retention of protein structure and function for experimental applications.

What experimental design approaches optimize the soluble expression of YPR204C-A?

Optimizing soluble expression of membrane proteins like YPR204C-A requires systematic evaluation of multiple variables through factorial experimental design. Based on established recombinant protein expression strategies, the following multivariate approach is recommended:

Table 1: Key Variables for Optimizing YPR204C-A Expression

*Significance levels based on similar membrane protein studies

Implement a fractional factorial design (2^8-4) with center point replicates to evaluate these variables simultaneously while minimizing experiment numbers . For YPR204C-A, focus particularly on:

• Lower induction temperatures (16-25°C): These typically enhance membrane protein folding by slowing synthesis rate

• Post-induction time: Limit to 4-6 hours to prevent formation of inclusion bodies

• Solubilization agents: Consider adding 0.5-1.0% glycerol or low concentrations of mild detergents

This systematic approach allows identification of statistically significant factors affecting YPR204C-A expression, enabling development of an optimized protocol that maximizes soluble protein yield while maintaining protein functionality .

How can membrane insertion efficiency of YPR204C-A be enhanced during recombinant expression?

Enhancing membrane insertion efficiency of YPR204C-A requires addressing the inherent challenges of membrane protein biogenesis. Research on type III membrane proteins suggests that translocation efficiency can be significantly improved through strategic modifications to targeting and insertion mechanisms .

One evidence-based approach involves modifying the protein's topogenic signals. Similar to studies with the human β2-adrenergic receptor (another membrane protein), conversion from type IIIb to type IIIa membrane protein structure by introducing an N-terminal cleavable signal sequence can enhance translocation across the endoplasmic reticulum membrane . For YPR204C-A, consider the following methods:

• Signal sequence engineering:

  • Introduce a well-characterized cleavable signal sequence (e.g., from Sec61α or prolactin) at the N-terminus

  • Optimize the hydrophobicity profile of transmembrane domains

  • Design constructs with modified N-terminal charge distribution

• Co-expression with membrane insertion facilitators:

  • Express with chaperones that assist membrane protein folding (e.g., DnaK/DnaJ/GrpE system)

  • Co-express components of the translocon complex

  • Include proteins that modify membrane lipid composition

• Systematic evaluation of expression conditions:

Table 2: Conditions for Optimizing Membrane Insertion

These modifications can significantly enhance the translocation efficiency of YPR204C-A, resulting in higher yields of properly folded and functionally inserted membrane protein .

What purification strategies yield the highest purity and retention of function for YPR204C-A?

Purifying membrane proteins while maintaining their native structure requires specialized approaches. For YPR204C-A, a multi-step purification strategy is recommended:

Stage 1: Membrane Preparation and Solubilization

• Harvest cells and disrupt by sonication or mechanical methods in buffer containing protease inhibitors

• Isolate membrane fraction through differential centrifugation (40,000-100,000 × g)

• Solubilize membranes using detergents optimized for YPR204C-A:

Table 3: Detergent Screening for YPR204C-A Solubilization

Stage 2: Chromatographic Purification
Implement a sequential purification scheme:

• IMAC (Immobilized Metal Affinity Chromatography): Using the recombinant protein's affinity tag

• Size-exclusion chromatography: To separate monomeric protein from aggregates

• Ion-exchange chromatography: For polishing and removing contaminants

Stage 3: Functional Validation
Assess protein quality at each purification step using:

• SDS-PAGE and Western blotting for purity assessment

• Circular dichroism to confirm secondary structure

• Thermal shift assays to evaluate stability

• Reconstitution into liposomes for functional tests

For structural biology applications, consider detergent exchange to more crystallization-friendly options like OG or DM during the final purification steps. This systematic approach typically yields protein with >90% purity while preserving the native folding necessary for functional studies.

How do different expression systems affect YPR204C-A folding and functional properties?

Expression systems significantly impact the folding and functional properties of membrane proteins like YPR204C-A due to differences in membrane composition, folding machinery, and post-translational modification capabilities. A comparative analysis reveals system-specific effects:

Table 4: Comparison of Expression Systems for YPR204C-A

To evaluate the impact of expression system on YPR204C-A function:

• Compare lipid binding properties across systems using fluorescence-based assays

• Assess thermal stability differences using differential scanning calorimetry

• Measure functional parameters in reconstituted systems using liposome-based assays

These comparisons can guide selection of the most appropriate expression system based on specific research objectives.

What structural analysis techniques are most informative for characterizing YPR204C-A?

Comprehensive structural characterization of YPR204C-A requires a multi-technique approach addressing different levels of structural organization. The following methodologies provide complementary insights:

Primary Structure Analysis:

• Mass spectrometry (MS) with techniques like MALDI-TOF for accurate mass determination

• MS/MS peptide mapping for sequence verification and post-translational modification identification

Secondary Structure Analysis:

• Circular Dichroism (CD) spectroscopy to quantify α-helical content expected in transmembrane domains

• Fourier-Transform Infrared Spectroscopy (FTIR) for complementary secondary structure information

Tertiary Structure Analysis:

Table 5: High-Resolution Structural Techniques for YPR204C-A

Membrane Topology Analysis:

• Limited proteolysis combined with MS to identify exposed regions

• Site-directed fluorescence labeling to map membrane-embedded domains

• Cysteine accessibility methods to determine transmembrane segment orientation

For YPR204C-A specifically, its relatively small size (160 amino acids) makes it amenable to solution NMR studies if sufficient quantities of isotope-labeled protein can be produced. Alternatively, recent advances in cryo-EM for smaller membrane proteins using specialized approaches like embedding in nanodiscs or amphipols could provide structural insights without crystallization challenges.

How can site-directed mutagenesis be applied to study structure-function relationships in YPR204C-A?

Site-directed mutagenesis provides powerful insights into structure-function relationships of membrane proteins like YPR204C-A. A systematic mutagenesis approach should target specific domains and conserved residues:

Strategic Mutation Design:

• Transmembrane domain mutations: Replace key hydrophobic residues to assess membrane anchoring

• Charged residue substitutions: Modify charged amino acids that may participate in electrostatic interactions

• Conserved motif alterations: Target any sequence patterns conserved across the UPF0479 family

Table 6: Priority Residues for YPR204C-A Mutagenesis

Methodological Approach:

• Generate mutations using overlap extension PCR or commercial site-directed mutagenesis kits

• Express wild-type and mutant proteins under identical conditions

• Compare expression levels, membrane integration, and stability

• Conduct functional assays to determine impact on biological activity

For validation, combine mutagenesis with crosslinking studies using photoactivatable amino acid analogs incorporated at specific positions. This approach can map interaction surfaces and identify critical residues for protein-protein or protein-lipid interactions, providing mechanistic insights into YPR204C-A function within the membrane environment.

What are the considerations for scaling up YPR204C-A production for structural studies?

Scaling up YPR204C-A production for structural studies requires systematic optimization of both upstream and downstream processes. Based on recombinant protein expression principles, the following approach is recommended:

Upstream Process Optimization:

• Bioreactor cultivation: Transition from shake flasks to controlled bioreactors (5-30L)

• Feed strategy development: Implement fed-batch cultivation with optimized carbon source feeding

• Induction optimization: Fine-tune induction parameters based on earlier factorial design results

Table 7: Bioreactor Parameters for Scaled YPR204C-A Production

Downstream Processing Considerations:

• Cell disruption scale-up: Transition from sonication to high-pressure homogenization

• Membrane preparation: Implement tangential flow filtration for membrane isolation

• Chromatography scale-up: Increase column dimensions while maintaining bed height:diameter ratios

• Detergent optimization: Balance between solubilization efficiency and downstream processing compatibility

Scale-up typically introduces challenges including:

• Oxygen transfer limitations affecting cell growth

• Heat transfer issues affecting protein folding

• Mixing inefficiencies leading to concentration gradients

• Increased mechanical stress potentially damaging membrane integrity

For structural biology applications, prioritize protein quality over quantity, as heterogeneous preparations severely impact crystallization success or cryo-EM data quality. Implement rigorous quality control at each step using analytical SEC, multi-angle light scattering, and functional assays to ensure consistency between batches.

How can advanced biophysical techniques be applied to study YPR204C-A interactions with lipids and other proteins?

Investigating YPR204C-A interactions with lipids and other proteins requires sophisticated biophysical approaches that can probe these associations under near-native conditions. The following methodologies are particularly informative:

Lipid Interaction Studies:

• Microscale Thermophoresis (MST): Measures binding affinities between YPR204C-A and fluorescently labeled lipids in solution, requiring minimal protein amounts (≤1 μg).

• Surface Plasmon Resonance (SPR): Provides real-time kinetic data for YPR204C-A interaction with immobilized lipid bilayers.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Maps regions of YPR204C-A that become protected upon lipid binding, indicating interaction interfaces.

Table 8: Comparative Analysis of Membrane Mimetics for YPR204C-A Studies

Protein-Protein Interaction Methods:

• Chemical Cross-linking coupled with MS: Identifies interaction partners and maps binding interfaces by covalently linking proximal proteins before MS analysis.

• FRET-based approaches: When combined with strategic fluorophore placement, can reveal dynamic interactions and conformational changes upon binding.

• Co-immunoprecipitation with quantitative proteomics: Identifies the interactome of YPR204C-A under different cellular conditions.

For comprehensive characterization, integrate multiple techniques to build a complete picture of YPR204C-A's interaction network. For example, initial screening using pull-down assays can identify potential interaction partners, followed by biophysical validation using SPR or isothermal titration calorimetry, and structural characterization of complexes using cryo-EM or X-ray crystallography.