Recombinant Saccharomyces cerevisiae Putative UPF0479 protein YML133W-B (YML133W-B)

What is YML133W-B protein and what are its key characteristics?

YML133W-B is a putative UPF0479 membrane protein from Saccharomyces cerevisiae (baker's yeast). The full-length protein consists of 160 amino acids and is categorized as a transmembrane protein. The protein's UniProt ID is P0CL35, and it can be recombinantly expressed with an N-terminal His tag in E. coli expression systems .

Similar to other UPF0479 family proteins like YEL077W-A (UniProt ID: P0CX95), YML133W-B is likely a membrane-associated protein involved in cellular processes not yet fully characterized . Current structural and functional knowledge of this protein is limited, making it an interesting target for exploratory research.

How does the structure of YML133W-B compare with other UPF0479 family proteins in yeast?

While the specific three-dimensional structure of YML133W-B hasn't been fully elucidated, comparative analysis with the related UPF0479 family member YEL077W-A can provide some insights. Both proteins are similar in length (160 amino acids) and share conserved structural features typical of membrane proteins .

To conduct a proper structural comparison, researchers should:

• Perform sequence alignment using tools like BLAST or Clustal Omega

• Identify conserved domains and motifs

• Generate homology models using software such as AlphaFold2 or RoseTTAFold

• Validate model quality through Ramachandran plots and other validation metrics

• Analyze predicted transmembrane regions using tools like TMHMM or Phobius

This comparative approach allows for the identification of conserved structural elements and potential functional regions in the absence of experimentally determined structures.

What is the optimal expression system for recombinant YML133W-B protein?

For recombinant expression of YML133W-B, E. coli-based systems have been successfully employed . When designing an expression strategy, researchers should consider:

Expression vector selection:

• Use vectors with strong inducible promoters (e.g., T7)

• Include appropriate purification tags (N-terminal His-tag has been validated)

• Consider codon optimization for E. coli expression

Expression conditions:

• Test multiple E. coli strains (BL21(DE3), Rosetta, C41/C43 for membrane proteins)

• Optimize induction conditions (temperature, IPTG concentration, duration)

• Consider low-temperature induction (16-18°C) to enhance proper folding

For membrane proteins like YML133W-B, specialized E. coli strains designed for membrane protein expression may yield better results. Alternatively, yeast-based expression systems might provide a more native environment for proper folding and post-translational modifications.

What purification strategy yields the highest purity and activity for YML133W-B?

The purification of YML133W-B can be achieved using a multi-step approach optimized for membrane proteins:

• Cell lysis and membrane fraction isolation:

  • Use mechanical disruption (sonication, French press) in the presence of protease inhibitors

  • Separate membrane fraction through ultracentrifugation

• Solubilization:

  • Test various detergents (DDM, LDAO, Triton X-100) at different concentrations

  • Optimize solubilization time and temperature

• Affinity chromatography:

  • Utilize Ni-NTA chromatography for His-tagged YML133W-B

  • Implement a stepwise imidazole gradient for elution

• Secondary purification:

  • Size exclusion chromatography for increased purity

  • Ion exchange chromatography if needed

• Quality assessment:

  • SDS-PAGE analysis

  • Western blotting using anti-His antibodies

  • Mass spectrometry for identity confirmation

It's essential to avoid repeated freeze-thaw cycles, as noted for similar proteins, to maintain protein integrity and activity .

How can researchers determine the subcellular localization of YML133W-B in yeast cells?

To determine the subcellular localization of YML133W-B, researchers can employ multiple complementary approaches:

Fluorescent protein tagging:

• Create C- or N-terminal GFP/mCherry fusion constructs

• Express in S. cerevisiae under native or controlled promoters

• Visualize using confocal microscopy

• Co-localize with known organelle markers

Subcellular fractionation:

• Isolate different cellular compartments (membrane, cytosol, nucleus)

• Detect YML133W-B in fractions via Western blotting

• Validate fraction purity using known compartment markers

Immunofluorescence:

• Generate specific antibodies against YML133W-B or use anti-tag antibodies

• Fix and permeabilize yeast cells

• Perform immunostaining and microscopy analysis

By combining these approaches, researchers can reliably determine the subcellular localization of YML133W-B and gain insights into its potential function within the cell.

What experimental approaches can identify potential interacting partners of YML133W-B?

Identifying protein-protein interactions is crucial for understanding YML133W-B function. Several methodologies can be employed:

Affinity purification-mass spectrometry (AP-MS):

• Express tagged YML133W-B in yeast

• Perform pull-down assays under native conditions

• Identify co-purifying proteins via LC-MS/MS

• Validate hits using reciprocal pull-downs

Yeast two-hybrid screening:

• Use YML133W-B as bait against a yeast genomic library

• Screen for positive interactions

• Confirm using complementary methods

Proximity labeling approaches:

• Generate BioID or APEX2 fusions with YML133W-B

• Express in yeast to label proximal proteins

• Identify labeled proteins by streptavidin pull-down and MS

Cross-linking mass spectrometry:

• Apply protein cross-linkers to intact yeast cells

• Isolate YML133W-B complexes

• Identify cross-linked peptides using specialized MS methods

When analyzing potential interactors, careful attention should be paid to distinguishing specific interactions from background contaminants through appropriate controls and statistical analysis.

How can researchers apply deep learning approaches to predict YML133W-B structure and function?

Deep learning tools have revolutionized protein structure prediction and can be valuable for studying proteins like YML133W-B:

Structure prediction:

• Utilize AlphaFold2 or RoseTTAFold to generate high-confidence structure models

• Assess prediction quality through pLDDT scores and predicted alignment error

• Validate models through molecular dynamics simulations

Function prediction:

• Apply deep learning tools that predict protein function from sequence or structure

• Use models to identify potential binding sites or active sites

• Generate hypotheses about protein-protein interactions

Binding site analysis:

• Apply computational methods to identify potential ligand binding sites

• Use tools like DeepSite or P2Rank to predict binding pockets

• Validate through molecular docking simulations

As demonstrated in protein binder design research, AlphaFold2 and RoseTTAFold can assess the probability that a sequence adopts a specific structure, providing valuable insights for proteins with limited experimental data .

What are the challenges in determining the high-resolution structure of YML133W-B and how can they be addressed?

Membrane proteins like YML133W-B present unique challenges for structural determination:

Challenges:

• Low expression yields in heterologous systems

• Difficulties in purification and maintaining native conformation

• Limited stability in detergent solutions

• Challenges in crystallization for X-ray crystallography

Solutions:

• Expression optimization:

  • Test specialized expression systems designed for membrane proteins

  • Utilize fusion partners to enhance expression and solubility

  • Screen multiple detergents and stabilizing additives

• Cryo-EM approach:

  • Bypass crystallization requirements

  • Optimize sample preparation with appropriate detergents or nanodiscs

  • Apply focused refinement for flexible regions

• NMR spectroscopy:

  • Express isotope-labeled protein (15N, 13C)

  • Utilize detergent micelles or bicelles for sample preparation

  • Apply specialized pulse sequences for membrane proteins

• Hybrid approaches:

  • Combine computational predictions with limited experimental data

  • Use crosslinking mass spectrometry to validate structural models

  • Apply integrative modeling approaches

By addressing these challenges systematically, researchers can work toward determining the high-resolution structure of YML133W-B.

What methodologies can determine if YML133W-B is essential for yeast cellular function?

To determine if YML133W-B is essential for yeast function, researchers can employ genetic approaches similar to those used in studying other yeast genes:

Gene disruption:

• Create knockout strains using homologous recombination

• Replace YML133W-B with a selection marker

• Assess viability and growth rates under various conditions

Conditional expression systems:

• Generate strains with YML133W-B under a regulatable promoter

• Analyze phenotypic changes upon depletion

• Monitor growth in different media and stress conditions

Complementation assays:

• Reintroduce wild-type or mutant versions of YML133W-B

• Assess restoration of normal phenotype

• Identify essential functional domains

Similar approaches have been used to determine the essentiality of other yeast genes, such as MRP-L33, which was shown to be essential for mitochondrial function using gene disruption by insertion of a HIS3-containing fragment .

How can researchers investigate YML133W-B's potential role in membrane-related processes?

As a putative membrane protein, YML133W-B may participate in various membrane-related processes. To investigate these:

Membrane integrity assays:

• Compare wild-type and YML133W-B mutant strains using membrane-permeant dyes

• Assess sensitivity to membrane-disrupting agents

• Analyze membrane fluidity using appropriate fluorescent probes

Lipidomic analysis:

• Extract and analyze membrane lipids from wild-type and mutant strains

• Identify changes in lipid composition or distribution

• Correlate lipid changes with phenotypic observations

Membrane protein complex analysis:

• Perform blue native PAGE to identify native complexes containing YML133W-B

• Use co-immunoprecipitation to confirm interactions

• Apply dynamic light scattering to assess complex formation

Functional reconstitution:

• Purify YML133W-B and reconstitute into liposomes

• Measure specific activities (transport, enzymatic, etc.)

• Analyze how mutations affect reconstituted function

This multi-faceted approach can provide insights into the protein's role in membrane biology and cellular function.

How can researchers apply synthetic biology approaches to study YML133W-B function?

Synthetic biology offers powerful tools for understanding protein function:

Domain swapping:

• Create chimeric proteins by swapping domains between YML133W-B and related proteins

• Express in yeast and analyze functionality

• Identify essential functional domains

Minimal functional unit identification:

• Generate systematic truncations and internal deletions

• Test which constructs retain function

• Define the minimal functional unit

Synthetic genetic circuits:

• Place YML133W-B expression under synthetic regulatory networks

• Study effects of controlled expression timing and levels

• Identify genetic interactions through synthetic lethality screens

De novo design of YML133W-B variants:

• Apply computational design tools like ProteinMPNN for sequence design

• Generate libraries of designed variants

• Screen for altered or enhanced function

These approaches can provide unique insights into YML133W-B function that would be difficult to obtain through traditional methods alone.

What are the most effective approaches for studying YML133W-B in the context of cellular stress responses?

To investigate potential roles of YML133W-B in stress response:

Stress condition screening:

• Subject wild-type and YML133W-B mutant yeast to various stressors:

  • Temperature (heat shock, cold shock)

  • Oxidative stress (H₂O₂, menadione)

  • Osmotic stress (high salt, sorbitol)

  • ER stress (tunicamycin, DTT)

• Monitor growth rates, viability, and recovery

Transcriptional response analysis:

• Perform RNA-seq under normal and stress conditions

• Compare expression profiles between wild-type and mutant strains

• Identify differentially regulated pathways

Protein localization under stress:

• Track YML133W-B localization during stress using fluorescent tags

• Identify stress-induced changes in localization or abundance

• Monitor potential post-translational modifications

Quantitative fitness analysis:

• Generate a bar-coded YML133W-B deletion strain

• Compete in mixed populations under stress conditions

• Quantify relative fitness through next-generation sequencing

Similar approaches have been used to characterize the role of other yeast proteins in stress conditions, such as the temperature sensitivity observed in telomerase-related proteins at 37°C .

How can researchers address solubility issues when working with recombinant YML133W-B?

Membrane proteins like YML133W-B often present solubility challenges. Strategies to overcome these include:

Optimization of solubilization conditions:

Alternative solubilization approaches:

• Use nanodiscs or SMALPs for detergent-free extraction

• Try amphipols for improved stability after initial solubilization

• Consider fluorinated surfactants for challenging membrane proteins

Fusion tag strategies:

• Test solubility-enhancing tags (MBP, SUMO, Trx)

• Optimize tag position (N-terminal vs. C-terminal)

• Include adequate linkers between the tag and YML133W-B

Expression modifications:

• Reduce expression temperature to improve folding

• Co-express with chaperones

• Consider cell-free expression systems

These approaches, often used in combination, can significantly improve the solubility and stability of challenging membrane proteins like YML133W-B.

What strategies can overcome low expression yields of YML133W-B in heterologous systems?

Low expression yields are common with membrane proteins. Researchers can employ several strategies to improve YML133W-B expression:

Strain optimization:

• Test specialized E. coli strains (C41/C43, Lemo21)

• Consider eukaryotic expression systems (P. pastoris, insect cells)

• Use strains with rare codon supplementation

Vector and construct design:

• Optimize codon usage for the expression host

• Test different signal sequences or fusion partners

• Modify potential problematic regions (hydrophobic stretches)

Expression conditions optimization:

Scale-up considerations:

• Optimize oxygen transfer in bioreactors

• Implement fed-batch processes

• Monitor and control pH throughout growth

By systematically optimizing these parameters, researchers can significantly improve the expression yields of challenging membrane proteins like YML133W-B.

How can researchers utilize deep mutational scanning to understand YML133W-B structure-function relationships?

Deep mutational scanning provides comprehensive insights into protein function through systematic mutagenesis:

Library generation:

• Create a comprehensive library of YML133W-B variants using:

  • Error-prone PCR

  • Site-directed mutagenesis at every position

  • Saturation mutagenesis of key residues

• Incorporate unique barcodes for variant tracking

Functional selection:

• Express variant library in yeast lacking endogenous YML133W-B

• Apply appropriate selection pressure

• Sequence pre- and post-selection populations

Data analysis and interpretation:

• Calculate enrichment/depletion scores for each variant

• Generate mutational sensitivity profiles

• Map results onto structural models to identify:

  • Functionally critical residues

  • Tolerant regions

  • Potential allosteric networks

Validation of key variants:

• Select representative variants from different categories

• Perform in-depth functional characterization

• Validate predictions from mutational scanning

This approach provides a comprehensive map of functionally important regions and can guide future structure-function studies of YML133W-B.

What are the best approaches for analyzing potential post-translational modifications of YML133W-B?

Post-translational modifications (PTMs) can significantly impact protein function. To identify and characterize PTMs in YML133W-B:

Mass spectrometry-based approaches:

• Purify native YML133W-B from yeast cells

• Perform proteolytic digestion (trypsin, chymotrypsin)

• Analyze using high-resolution LC-MS/MS

• Implement enrichment strategies for specific PTMs:

  • Phosphopeptide enrichment (TiO₂, IMAC)

  • Glycopeptide enrichment (lectin affinity)

  • Ubiquitination detection (K-ε-GG antibodies)

Site-directed mutagenesis validation:

• Mutate identified modification sites

• Assess impact on function, localization, and interactions

• Generate phosphomimetic mutations (S/T to D/E) when appropriate

Temporal dynamics analysis:

• Monitor PTM changes under different conditions

• Analyze PTM patterns during cell cycle progression

• Study stress-induced modification changes

By comprehensively characterizing PTMs, researchers can gain deeper insights into the regulation and function of YML133W-B in various cellular contexts.