Recombinant Saccharomyces cerevisiae UPF0041 protein YHR162W (YHR162W)

What is the fundamental structure and composition of the Recombinant Saccharomyces cerevisiae UPF0041 protein YHR162W?

The YHR162W protein is a full-length protein (129 amino acids) from Saccharomyces cerevisiae, also known as baker's yeast. The complete amino acid sequence is: MSTSSVRFAFRRFWQSETGPKTVHFWAPTLKWGLVFAGFSDMKRPVEKISGAQNLSLLSTALIWTRWSFVIKPRNILLASVNSFLCLTAGYQLGRIANYRIRNGDSISQLCSYILSGADESKKEITTGR . The protein is classified as a UPF0041 family protein and is also known as MPC2 (Mitochondrial pyruvate carrier 2) . For research applications, the protein is commonly produced as a recombinant protein with an N-terminal His-tag, expressed in E. coli expression systems, which facilitates purification and downstream applications .

What are the recommended storage and handling conditions for YHR162W recombinant protein?

For optimal stability and activity of the YHR162W recombinant protein, specific storage and handling protocols should be followed. The protein is typically supplied as a lyophilized powder and should be stored at -20°C or -80°C upon receipt . For working solutions, it is recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) to prevent freeze-thaw damage . For short-term use, working aliquots can be stored at 4°C for up to one week . It is crucial to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity . When preparing aliquots, brief centrifugation is recommended prior to opening the vial to bring the contents to the bottom .

How does YHR162W relate to the broader context of Saccharomyces cerevisiae as a model organism?

Saccharomyces cerevisiae is one of the best-studied eukaryotic organisms, serving as a valuable model for understanding fundamental biological processes. The genome of S. cerevisiae has been completely sequenced and contains approximately 6000 genes, with 5570 predicted to be protein-encoding . YHR162W, as one of these genes, exists within this well-characterized genomic context. S. cerevisiae's value as a model organism stems from its unicellular nature, which simplifies experimental design, while still possessing most biological functions found in higher eukaryotes . Unlike other model organisms, S. cerevisiae has dual significance as both a research tool and an organism with extensive biotechnological applications, particularly in fermentation processes . Understanding YHR162W within this context allows researchers to leverage the extensive knowledge base and genetic tools available for this organism.

What are the optimal expression and purification protocols for generating high-yield, functional YHR162W recombinant protein?

Expression System Selection:
For optimal expression of YHR162W, E. coli-based systems have proven most effective, particularly when the protein is fused to an N-terminal His-tag for purification purposes . The complete coding sequence (1-129 amino acids) should be cloned into an appropriate expression vector containing a strong inducible promoter such as T7.

Purification Protocol:

• Culture E. coli harboring the YHR162W expression construct in appropriate media with antibiotic selection

• Induce protein expression at mid-log phase (OD₆₀₀ ≈ 0.6-0.8) with IPTG

• Harvest cells by centrifugation (6,000 × g, 15 minutes, 4°C)

• Lyse cells using sonication or mechanical disruption in a buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 10 mM imidazole

  • Protease inhibitor cocktail

• Clarify lysate by centrifugation (15,000 × g, 30 minutes, 4°C)

• Apply supernatant to Ni-NTA affinity column pre-equilibrated with lysis buffer

• Wash with buffer containing increasing concentrations of imidazole (20-50 mM)

• Elute purified protein with elution buffer containing 250-300 mM imidazole

• Analyze purity by SDS-PAGE (should be >90%)

• Perform buffer exchange to remove imidazole and concentrate protein using ultrafiltration

Post-Purification Processing:
For long-term storage, lyophilize the purified protein or store in Tris/PBS-based buffer with 50% glycerol at -20°C/-80°C .

What analytical techniques are most appropriate for characterizing the structural properties of YHR162W?

Primary Structure Analysis:

• Mass Spectrometry (MS):

  • Electrospray ionization MS (ESI-MS) for intact mass determination

  • Tandem MS following tryptic digestion for sequence verification and post-translational modification identification

• N-terminal Sequencing:

  • Edman degradation to confirm the N-terminal sequence, particularly important when using tagged constructs

Secondary and Tertiary Structure Characterization:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm) to estimate secondary structure content (α-helix, β-sheet)

  • Near-UV CD (250-350 nm) to examine tertiary structure fingerprint

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence to probe tertiary structure and conformational changes

  • Use of extrinsic fluorophores for surface hydrophobicity assessment

• Fourier Transform Infrared Spectroscopy (FTIR):

  • Complementary technique to CD for secondary structure estimation

• X-ray Crystallography or NMR:

  • For high-resolution structural determination, though these may require optimization of construct design and conditions

Quaternary Structure Analysis:

• Size Exclusion Chromatography (SEC):

  • To determine oligomeric state under native conditions

• Dynamic Light Scattering (DLS):

  • To assess homogeneity and hydrodynamic radius

• Analytical Ultracentrifugation (AUC):

  • Sedimentation velocity and equilibrium experiments for accurate molecular weight and shape determination

How can researchers effectively develop functional assays to study YHR162W activity, considering its potential role as a mitochondrial pyruvate carrier?

Mitochondrial Localization and Import Assays:

• Subcellular Fractionation:

  • Isolate mitochondria from yeast cells expressing tagged versions of YHR162W

  • Analyze protein distribution across fractions via Western blotting

• Fluorescence Microscopy:

  • Generate GFP fusion constructs to visualize localization

  • Co-stain with mitochondrial markers (e.g., MitoTracker dyes)

Transport Activity Assays:

• Reconstitution in Liposomes:

  • Incorporate purified YHR162W into liposomes

  • Measure pyruvate uptake using radiolabeled pyruvate (¹⁴C-pyruvate)

  • Monitor internal pH changes during transport using pH-sensitive fluorescent dyes

• Mitochondrial Pyruvate Uptake:

  • Isolate intact mitochondria from wild-type and YHR162W knockout strains

  • Measure pyruvate uptake rates using rapid filtration techniques

  • Assess inhibitor sensitivity to characterize transport mechanism

Functional Complementation:

• Yeast Growth Analysis:

  • Compare growth of wild-type, YHR162W knockout, and complemented strains under different carbon sources

  • Measure growth curves in media requiring mitochondrial pyruvate metabolism

• Metabolic Profiling:

  • Quantify intracellular metabolites in wild-type versus knockout strains

  • Focus on pyruvate and TCA cycle intermediates using HPLC-MS

Protein-Protein Interaction Studies:

• Co-immunoprecipitation:

  • Identify binding partners of YHR162W in mitochondrial extracts

  • Validate interactions using reciprocal pull-downs

• Crosslinking Mass Spectrometry:

  • Map interaction surfaces and complex architecture

  • Identify functional complexes related to pyruvate transport

How can genetic manipulation techniques be optimized for studying YHR162W function in vivo?

CRISPR-Cas9 Genome Editing Strategies:

Precise genetic manipulation of YHR162W can be achieved through CRISPR-Cas9 technology, which allows for targeted modifications within the S. cerevisiae genome. For successful implementation:

• Guide RNA Design:

  • Design sgRNAs targeting the YHR162W locus using tools optimized for yeast

  • Select targets with minimal off-target effects, preferably with ≥3 mismatches to other genomic regions

  • Verify PAM sequence availability near the desired modification site

• Homology-Directed Repair Templates:

  • For gene knockouts: Design repair templates with 40-60 bp homology arms flanking a selection marker

  • For point mutations: Include 500-1000 bp homology regions with the desired mutation in the center

  • For tagging: Design in-frame fusions with reporter genes or epitope tags

• Transformation Protocol:

  • Use lithium acetate/PEG method with single-stranded carrier DNA

  • Include a plasmid expressing Cas9 and the sgRNA under appropriate promoters

  • Co-transform with the repair template at high concentration (>1 μg)

• Verification Strategies:

  • PCR screening of colonies using primers outside the homology regions

  • Sequencing validation of the entire modified locus

  • Phenotypic confirmation through functional assays

Conditional Expression Systems:

To study essential functions or toxic effects of YHR162W mutations:

• Tetracycline-Regulated Expression:

  • Replace native promoter with tetO operator sequences

  • Co-express tetracycline transactivator (tTA) or reverse transactivator (rtTA)

  • Titrate expression levels with doxycycline concentrations

• Auxin-Inducible Degron System:

  • Fuse YHR162W with an auxin-inducible degron (AID) tag

  • Express TIR1 F-box protein for targeted protein degradation

  • Achieve rapid protein depletion by adding auxin to growth medium

• Temperature-Sensitive Alleles:

  • Generate libraries of random or directed mutations in YHR162W

  • Screen for conditional phenotypes at restrictive temperatures

  • Characterize structural basis of temperature sensitivity

What methodologies should be employed to investigate the evolutionary conservation and functional divergence of YHR162W across yeast species?

Comparative Genomic Analysis:

• Ortholog Identification:

  • Perform reciprocal BLAST searches against multiple yeast genomes

  • Apply synteny analysis to confirm true orthologous relationships

  • Construct a comprehensive dataset of UPF0041 family proteins across fungal lineages

• Sequence Conservation Analysis:

  • Calculate sequence identity/similarity percentages across species

  • Identify universally conserved residues as potential functional hotspots

  • Map conservation scores onto structural models to reveal functional domains

• Phylogenetic Analysis:

  • Construct multiple sequence alignments using MUSCLE or MAFFT algorithms

  • Build phylogenetic trees using maximum likelihood methods

  • Test alternative tree topologies to resolve evolutionary relationships

Table: Conservation of Key Functional Domains in YHR162W Orthologs

Note: This table represents hypothetical values based on typical conservation patterns in yeast proteins; specific research would provide actual values.

Functional Complementation Assays:

• Cross-Species Complementation:

  • Express YHR162W orthologs from different yeast species in S. cerevisiae YHR162W deletion strains

  • Assess rescue of phenotypic defects in growth, metabolism, or stress response

  • Quantify complementation efficiency through growth curve analysis

• Domain Swapping Experiments:

  • Generate chimeric proteins containing domains from YHR162W orthologs

  • Map functional domains responsible for species-specific activities

  • Identify critical residues that determine functional specificity

• Coevolution Analysis:

  • Identify coevolving residues within YHR162W using statistical coupling analysis

  • Map interaction networks of coevolving proteins across species

  • Reconstruct ancestral sequences to trace functional innovations

What integrative multi-omics approaches can be used to fully characterize the functional role of YHR162W within cellular metabolic networks?

Integrated Transcriptomics and Proteomics:

• Differential Expression Analysis:

  • Compare transcriptome profiles of wild-type and YHR162W deletion strains

  • Identify genes with altered expression using RNA-Seq

  • Validate key findings with RT-qPCR

• Proteome-wide Interaction Mapping:

  • Perform BioID or APEX2 proximity labeling with YHR162W as bait

  • Identify physical interactors using affinity purification-mass spectrometry (AP-MS)

  • Construct protein-protein interaction networks centered on YHR162W

• Post-translational Modification Profiling:

  • Map phosphorylation, acetylation, and other modifications on YHR162W

  • Identify condition-dependent modification patterns

  • Assess effects of modifications on protein function and interactions

Metabolomics Integration:

• Targeted Metabolite Analysis:

  • Quantify changes in pyruvate, lactate, acetyl-CoA, and TCA cycle intermediates

  • Compare metabolite levels in wild-type versus YHR162W-deficient cells

  • Trace flux through mitochondrial pathways using stable isotope labeling

• Metabolic Flux Analysis:

  • Perform ¹³C metabolic flux analysis with glucose or pyruvate as labeled substrate

  • Calculate flux distributions through central carbon metabolism

  • Identify metabolic bottlenecks in YHR162W mutants

• Integration with Genome-Scale Models:

  • Incorporate experimental data into genome-scale metabolic models

  • Predict systemic effects of YHR162W perturbation

  • Identify synthetic lethal interactions and potential compensatory pathways

Systems Biology Framework:

• Network Analysis:

  • Construct integrated networks incorporating transcriptomic, proteomic, and metabolomic data

  • Identify key regulatory modules affected by YHR162W function

  • Apply machine learning approaches to predict novel functional associations

• Phenotypic Profiling:

  • Perform high-throughput phenotypic analysis under diverse growth conditions

  • Assess genetic interactions through synthetic genetic array (SGA) analysis

  • Map condition-specific functional relationships

• Multi-omics Data Integration:

  • Apply multivariate statistical methods to integrate heterogeneous data types

  • Use dimensionality reduction techniques to visualize complex relationships

  • Develop predictive models of YHR162W function in cellular metabolism

What are the optimal strategies for crystallization and structural determination of YHR162W protein?

Protein Preparation for Crystallization:

• Construct Optimization:

  • Generate multiple constructs with varying N- and C-terminal boundaries

  • Remove flexible regions that may hinder crystal formation

  • Consider surface entropy reduction mutations (SER) to promote crystal contacts

• Protein Purification for Crystallography:

  • Implement rigorous size-exclusion chromatography as a final purification step

  • Verify monodispersity through dynamic light scattering (DLS)

  • Concentrate protein to 5-15 mg/mL in a crystallization-compatible buffer

• Stability Assessment:

  • Perform thermal shift assays to identify stabilizing buffer conditions

  • Use limited proteolysis to identify stable domains

  • Evaluate long-term stability at 4°C and room temperature

Crystallization Screening Approaches:

• Initial Screening:

  • Employ sparse matrix screening with commercial kits

  • Utilize sitting drop vapor diffusion at multiple protein concentrations

  • Screen at both 4°C and 20°C to identify temperature-dependent crystallization

• Optimization Strategies:

  • Fine-tune promising conditions by varying:

    • Precipitant concentration

    • pH

    • Protein:reservoir ratio

    • Additive screening

  • Implement seeding techniques for improving crystal quality

• Alternative Crystallization Methods:

  • Counter-diffusion in capillaries for slower equilibration

  • Lipidic cubic phase methods if membrane association is suspected

  • Micro-batch under oil for alternatives to vapor diffusion

Structure Determination Workflow:

• Data Collection:

  • Test multiple crystals for diffraction quality

  • Collect complete datasets at high resolution

  • Consider heavy atom derivatives for phase determination

• Phasing Strategies:

  • Molecular replacement using homologous structures

  • Experimental phasing using selenomethionine incorporation

  • Single or multiple anomalous dispersion methods if necessary

• Model Building and Refinement:

  • Iterative cycles of manual model building

  • Refinement against experimental data

  • Validation using MolProbity or similar tools

How can researchers effectively implement computational approaches to predict the structure-function relationship of YHR162W?

Homology Modeling and Threading:

• Template Identification:

  • Search structural databases for remote homologs of YHR162W

  • Evaluate template quality using sequence coverage and structural resolution

  • Consider multiple templates for different domains

• Model Construction:

  • Generate multiple models using tools such as I-TASSER, SWISS-MODEL, or Rosetta

  • Evaluate model quality using GMQE (Global Model Quality Estimation) scores

  • Refine models through energy minimization and molecular dynamics

• Model Validation:

  • Assess stereochemical quality using Ramachandran plots

  • Verify proper packing of hydrophobic cores

  • Compare models from different methods to identify consensus structural features

Molecular Dynamics Simulations:

• System Preparation:

  • Embed protein model in appropriate membrane environment if indicated

  • Solvate system with explicit water molecules

  • Add counterions to neutralize the system

• Simulation Protocols:

  • Perform energy minimization

  • Equilibrate system in multiple phases (NVT, NPT)

  • Run production simulations on microsecond timescales if possible

• Analysis Approaches:

  • Calculate root mean square deviation (RMSD) and fluctuation (RMSF)

  • Identify stable conformational states

  • Characterize potential transport pathways or binding sites

Functional Site Prediction:

• Active Site Identification:

  • Apply conservation mapping to structural models

  • Use geometric approaches to detect pockets and cavities

  • Implement machine learning algorithms trained on known carrier proteins

• Molecular Docking:

  • Perform docking studies with potential substrates (pyruvate)

  • Evaluate binding energies and interaction patterns

  • Identify key residues for substrate recognition

• Electrostatic Analysis:

  • Calculate electrostatic potential maps

  • Identify potential proton transfer pathways

  • Characterize transmembrane potential effects on protein conformation