Recombinant Staphylococcus aureus UPF0754 membrane protein SAOUHSC_01978 (SAOUHSC_01978)

What expression systems are recommended for recombinant production of SAOUHSC_01978?

For recombinant production of SAOUHSC_01978, E. coli expression systems are most commonly employed in research settings. When expressing this membrane protein, several methodological considerations are important:

• Vector selection: Vectors containing strong inducible promoters (e.g., T7) with His-tag fusion capabilities are typically used

• Culture conditions: Growth at lower temperatures (16-25°C) after induction to allow proper folding

• Induction protocols: IPTG concentration optimization (0.1-1.0 mM) with extended expression times (16-24 hours)

• Cell lysis: Methods compatible with membrane proteins, such as French press or sonication with detergent-containing buffers

This approach has been demonstrated to yield functional protein suitable for downstream applications including structural studies and functional assays .

How should SAOUHSC_01978 protein be stored to maintain stability and activity?

SAOUHSC_01978 requires specific storage conditions to maintain structural integrity and function. The recommended protocol includes:

For optimal stability, it is recommended to reconstitute the lyophilized protein to a concentration of 0.1-1.0 mg/mL using deionized sterile water. Repeated freeze-thaw cycles should be strictly avoided as they significantly compromise protein integrity. For working aliquots, storage at 4°C is sufficient for up to one week .

What strategies are most effective for solubilization and purification of SAOUHSC_01978?

Solubilization and purification of membrane proteins like SAOUHSC_01978 present unique challenges requiring specialized approaches:

Membrane Extraction and Solubilization Protocol:

• Harvest cells expressing His-tagged SAOUHSC_01978 by centrifugation (5,000 × g, 15 min, 4°C)

• Resuspend in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, protease inhibitor cocktail)

• Disrupt cells via sonication or French press (at least 3 cycles)

• Remove unbroken cells and debris (10,000 × g, 20 min, 4°C)

• Ultracentrifuge supernatant (100,000 × g, 1 hour, 4°C) to isolate membrane fraction

• Solubilize membrane pellet using detergent screening approach:

• Perform affinity chromatography using Ni-NTA resin with step gradient elution (20-500 mM imidazole)

• Consider size exclusion chromatography as a polishing step

This methodological approach typically yields >90% pure protein as determined by SDS-PAGE, suitable for structural and functional studies .

What experimental approaches are recommended for studying SAOUHSC_01978 membrane topology and orientation?

Determining the membrane topology and orientation of SAOUHSC_01978 requires multiple complementary experimental approaches:

Computational Prediction:
First employ hydropathy analysis and topology prediction algorithms (TMHMM, Phobius, TopPred) to generate initial models of transmembrane regions.

Experimental Validation Methods:

• Cysteine Scanning Mutagenesis:

  • Create single-cysteine variants throughout protein sequence

  • Perform membrane-impermeant/permeant thiol labeling assays

  • Quantify accessibility to determine cytoplasmic vs. periplasmic exposure

• Fluorescence Protease Protection Assay:

  • Generate GFP fusion constructs at N and C termini

  • Treat intact and permeabilized cells with proteases

  • Monitor fluorescence loss to determine protection by membrane

• SCAM (Substituted Cysteine Accessibility Method):

  • Substitute residues with cysteines incrementally

  • Apply membrane-permeable and impermeable thiol reagents

  • Detect modification patterns by mass spectrometry

Integrating data from these approaches provides a comprehensive model of SAOUHSC_01978 topology, essential for understanding structure-function relationships and designing targeted functional studies .

How does the lipid environment affect SAOUHSC_01978 folding and function?

The influence of lipid environment on SAOUHSC_01978 folding and function can be investigated through systematic lipid composition studies:

• Reconstitution in Defined Lipid Systems:

  • Extract purified protein in detergent micelles

  • Reconstitute into proteoliposomes with varying lipid compositions:

    • Phospholipid headgroup variation (PC, PE, PG, PS)

    • Acyl chain length and saturation

    • Cholesterol/ergosterol content

    • Bacterial-specific lipids (cardiolipin)

• Biophysical Characterization Methods:

  • Circular dichroism (CD) to assess secondary structure

  • Fluorescence spectroscopy for tertiary structure changes

  • Differential scanning calorimetry (DSC) for thermal stability

  • Atomic force microscopy (AFM) for membrane organization

• Functional Activity Assessment:

  • Develop specific activity assays based on predicted function

  • Compare activity metrics across lipid environments

Research in membrane protein folding indicates that matching the native lipid environment of S. aureus membranes (higher proportion of PG, cardiolipin, and branched-chain fatty acids) significantly improves stability and functional properties of bacterial membrane proteins compared to standard phosphatidylcholine-based systems .

What crystallization techniques have been successful for structural determination of proteins similar to SAOUHSC_01978?

Structural determination of membrane proteins like SAOUHSC_01978 requires specialized crystallization approaches:

Crystallization Strategies for SAOUHSC_01978 and Similar Membrane Proteins:

• Detergent-Based Methods:

  • Vapor diffusion with detergent-solubilized protein (sitting or hanging drop)

  • Systematic screening of:

    • Detergent types (maltosides, glucosides, neopentyl glycols)

    • Detergent concentrations slightly above CMC

    • Precipitants compatible with detergents (PEGs, ammonium sulfate)

    • pH range 5.5-8.5

• Lipidic Cubic Phase (LCP) Crystallization:

  • Mix protein-detergent solution with monoolein or related lipids

  • Form LCP matrix through which protein can organize

  • Screen precipitant solutions for crystal formation

  • Optimize temperature (typically 18-22°C)

• Crystallization Enhancers:

  • Antibody fragments (Fab, nanobody) to increase polar surface area

  • Fusion partners (T4 lysozyme, BRIL) to aid crystal contacts

  • Thermostabilizing mutations to reduce conformational heterogeneity

Optimization Strategies:

• Additive screening (small molecules, ions, lipids)

• Controlled dehydration protocols

• Seeding techniques (micro- and macroseeding)

These approaches have yielded successful structures for bacterial membrane proteins with similar properties to SAOUHSC_01978, though each protein typically requires customized optimization .

How can functional characterization of SAOUHSC_01978 be performed to determine its role in S. aureus pathogenicity?

To determine the functional role of SAOUHSC_01978 in S. aureus pathogenicity, a multi-pronged experimental approach is recommended:

• Genetic Manipulation Studies:

  • Generate precise deletion mutants (Δsaouhsc_01978) in S. aureus

  • Create complementation strains with wild-type and mutant variants

  • Perform phenotypic assays:

    • Growth curves under various stress conditions

    • Biofilm formation quantification

    • Antibiotic susceptibility testing

    • Host cell adhesion and invasion assays

• Protein-Protein Interaction Analysis:

  • Bacterial two-hybrid screening

  • Pull-down assays with purified His-tagged SAOUHSC_01978

  • Mass spectrometry identification of binding partners

  • Co-immunoprecipitation from native membrane extracts

• Transcriptomic and Proteomic Comparisons:

  • RNA-seq analysis of wild-type vs. deletion strains

  • Quantitative proteomics to identify affected pathways

  • Metabolomic profiling to detect physiological changes

• Infection Models:

  • Cell culture infection assays with wild-type and mutant strains

  • Animal infection models to assess virulence differences

  • Immune response profiling during infection

This comprehensive functional characterization workflow would provide insights into the molecular mechanisms by which SAOUHSC_01978 contributes to S. aureus biology and pathogenicity, potentially revealing new therapeutic targets .

What controls should be included when studying membrane protein interactions involving SAOUHSC_01978?

When investigating potential protein-protein interactions involving SAOUHSC_01978, implementing robust controls is essential for data interpretation:

Essential Controls for Interaction Studies:

• Negative Controls:

  • Empty vector/tag-only controls to identify non-specific binding

  • Irrelevant membrane protein from same organism with similar properties

  • Heat-denatured SAOUHSC_01978 to distinguish specific from non-specific interactions

  • Competitive binding with excess unlabeled protein

• Positive Controls:

  • Known membrane protein interaction pairs from S. aureus

  • Artificially engineered interacting domains as technical validation

• Detergent/Lipid Environment Controls:

  • Parallel experiments with multiple detergent types/concentrations

  • Reconstitution in proteoliposomes of varying composition

  • Native membrane extract validations

Data Validation Approaches:

Implementation of these controls ensures that observed interactions are physiologically relevant rather than artifacts of the experimental system .

How can researchers address challenges in expressing and purifying sufficient quantities of SAOUHSC_01978 for structural studies?

Obtaining sufficient quantities of properly folded SAOUHSC_01978 for structural studies requires optimized expression and purification strategies:

Enhanced Expression Approaches:

• Expression System Optimization:

  • Test multiple E. coli strains (BL21(DE3), C41/C43, Rosetta, Lemo21)

  • Evaluate alternative expression hosts (insect cells, yeast)

  • Develop cell-free expression systems with preformed nanodiscs

• Construct Engineering:

  • Codon optimization for expression host

  • N- and C-terminal truncations to remove flexible regions

  • Fusion partners to enhance solubility (MBP, SUMO, Mistic)

  • Introduce thermostabilizing mutations based on homology modeling

• Culture Condition Optimization:

Purification Enhancements:

• Tandem affinity tags (His6-MBP or His6-FLAG) for improved purity

• On-column detergent exchange during affinity chromatography

• Size exclusion chromatography with scattering detection (SEC-MALS)

• Lipid nanodisc or amphipol exchange for improved stability

These strategies have been successfully applied to challenging membrane proteins and can significantly improve yields (10-20 fold) and homogeneity of SAOUHSC_01978 preparations suitable for structural studies .

What computational approaches can predict the function of SAOUHSC_01978 based on its sequence and structural features?

Computational prediction of SAOUHSC_01978 function can be accomplished through a multi-level bioinformatics approach:

• Sequence-Based Analysis:

  • PSI-BLAST and HHpred for remote homology detection

  • Conserved domain identification (InterPro, Pfam, CDD)

  • Motif scanning for functional signatures (PROSITE, PRINTS)

  • Multiple sequence alignment with UPF0754 family members

  • Evolutionary analysis (conservation patterns, selective pressure)

• Structural Prediction:

  • Ab initio and template-based 3D structure prediction (AlphaFold2, RoseTTAFold)

  • Transmembrane topology prediction (TMHMM, TOPCONS)

  • Binding site prediction (SiteMap, FTMap)

  • Molecular dynamics simulations in membrane environment

  • Electrostatic potential mapping to identify functional regions

• Integrative Approaches:

  • Genomic context analysis (operon structure, co-regulation patterns)

  • Phylogenetic profiling to identify co-evolving partners

  • Gene expression correlation analysis from S. aureus transcriptome data

  • Protein-protein interaction network prediction

  • Pathway enrichment analysis of potential interactors

The integration of these computational approaches provides testable hypotheses about SAOUHSC_01978 function, which can then be validated through targeted experimental approaches. Recent advances in machine learning methods for protein function prediction have significantly improved the accuracy of such computational pipelines .

What are the common challenges and solutions in purifying membrane proteins like SAOUHSC_01978?

Purification of membrane proteins like SAOUHSC_01978 presents several technical challenges that researchers frequently encounter:

Common Challenges and Solutions:

• Low Expression Yields:

  • Challenge: Membrane protein overexpression often leads to toxicity and inclusion body formation

  • Solutions:

    • Use specialized expression strains (C41/C43)

    • Employ weaker promoters or leaky expression

    • Lower induction temperature (16-20°C)

    • Add chemical chaperones (5% glycerol, 0.5M sorbitol) to culture medium

• Protein Aggregation During Solubilization:

  • Challenge: Ineffective detergent extraction leads to protein aggregation

  • Solutions:

    • Systematic detergent screening (start with DDM, LMNG, GDN)

    • Include stabilizing additives (glycerol, cholesterol hemisuccinate)

    • Adjust ionic strength and pH of extraction buffer

    • Consider using mild extraction (native nanodiscs)

• Detergent-Induced Destabilization:

  • Challenge: Loss of activity or structure during purification

  • Solutions:

    • Perform activity assays at each purification step

    • Transition to more stable environments (amphipols, nanodiscs)

    • Include specific lipids essential for stability

    • Minimize purification duration and keep samples at 4°C

• Heterogeneity in Final Preparations:

  • Challenge: Multiple oligomeric states or conformations

  • Solutions:

    • Employ analytical SEC and SEC-MALS to assess homogeneity

    • Use thermal stability assays to optimize buffer conditions

    • Employ GraFix (gradient fixation) for structural studies

    • Consider mutagenesis to stabilize preferred conformations

These troubleshooting approaches can significantly improve the quality and quantity of purified SAOUHSC_01978, supporting downstream structural and functional studies .

How can isotope labeling of SAOUHSC_01978 be achieved for NMR structural studies?

Isotope labeling of SAOUHSC_01978 for NMR studies requires specialized protocols to achieve sufficient incorporation while maintaining protein folding:

Comprehensive Isotope Labeling Strategy:

• Uniform Labeling Approach:

  • Grow E. coli in M9 minimal media containing:

    • 15NH4Cl as sole nitrogen source (1-2 g/L)

    • 13C-glucose as carbon source (2-4 g/L)

    • Trace metal supplement (including zinc, iron, manganese)

  • Implement extended growth protocols (lower temperature, longer incubation)

  • Typically yields 30-50% of rich media expression levels

• Selective Amino Acid Labeling:

  • Use auxotrophic E. coli strains or metabolic inhibition

  • Supplement minimal media with specific labeled amino acids

  • Particularly useful for methyl-directed studies (Ile, Leu, Val)

  • Implementation of SAIL (Stereo-Array Isotope Labeling) for side chain analysis

• Segmental Labeling for Large Proteins:

  • Employ split-intein approaches for domain-specific labeling

  • Express domains separately with compatible intein fragments

  • Reconstitute full-length protein through trans-splicing reaction

Sample Preparation Considerations:

• Use deuterated detergents to reduce background signals

• Consider nanodiscs with deuterated lipids for native-like environment

• Optimize protein concentration (typically 0.3-0.8 mM)

• Develop stabilization protocols for multi-day NMR experiments

These methodologies enable detailed structural characterization of membrane proteins like SAOUHSC_01978 using solution NMR approaches, providing insights into dynamics and ligand interactions not accessible through crystallography .

What strategies can differentiate the functional roles of SAOUHSC_01978 from related membrane proteins in S. aureus?

Distinguishing the specific functions of SAOUHSC_01978 from related membrane proteins requires a systematic comparative approach:

• Comprehensive Phylogenetic Profiling:

  • Construct detailed phylogenetic trees of UPF0754 family members

  • Analyze co-evolution patterns with other functional partners

  • Identify conserved vs. variable regions across bacterial species

  • Map evolutionary changes to protein structural elements

• Differential Expression Analysis:

  • Perform RNA-seq under various growth conditions and stressors

  • Compare expression patterns of SAOUHSC_01978 with related proteins

  • Identify condition-specific regulation suggesting specialized functions

  • Analyze promoter regions for unique regulatory elements

• Cross-Complementation Studies:

  • Generate deletion mutants of SAOUHSC_01978 and related proteins

  • Perform reciprocal complementation with heterologous expression

  • Assess restoration of phenotypes to identify unique vs. redundant functions

  • Test chimeric proteins with domain swapping between family members

• Interactome Mapping:

  • Conduct parallel interaction studies (BioID, proximity labeling)

  • Compare binding partners using quantitative proteomics

  • Identify unique vs. shared protein-protein interactions

  • Validate specific interactions through direct binding studies

• Localization and Dynamics Comparison:

  • Perform fluorescent protein tagging of family members

  • Observe subcellular localization during different growth phases

  • Assess membrane dynamics using FRAP and single-molecule tracking

  • Correlate localization patterns with cell division or specialized structures

These comparative approaches can effectively delineate the specific functions of SAOUHSC_01978 from those of related membrane proteins, revealing its unique contribution to S. aureus physiology and potentially uncovering novel therapeutic targets .

How can cryo-electron microscopy be optimized for structural studies of SAOUHSC_01978?

Cryo-electron microscopy (cryo-EM) offers unique advantages for membrane protein structural studies but requires optimization for proteins like SAOUHSC_01978:

Cryo-EM Optimization Strategy:

• Sample Preparation Refinement:

  • Test multiple membrane mimetics:

    • Detergent micelles (LMNG, GDN)

    • Nanodiscs with varied lipid compositions

    • Amphipols (A8-35, PMAL-C8)

    • Reconstitution in liposomes for subtomogram averaging

  • Optimize protein concentration (typically 1-5 mg/mL)

  • Screen grid types (Quantifoil, C-flat) and hole sizes

  • Evaluate glow discharge vs. plasma cleaning parameters

• Vitrification Parameter Optimization:

• Particle Enhancement Strategies:

  • Addition of Fab fragments to increase protein size

  • Chemical crosslinking to stabilize complexes

  • Incorporation of fiducial markers for subtomogram averaging

  • GraFix method for increased stability and contrast

• Data Collection Optimization:

  • Detector settings (counting vs. super-resolution modes)

  • Defocus range determination (-0.8 to -3.0 μm)

  • Exposure rate optimization (beam-induced motion management)

  • Tilt series parameters for tomography applications

These optimizations can significantly improve the resolution and reliability of cryo-EM structures for challenging membrane proteins like SAOUHSC_01978, potentially revealing functional mechanisms and interaction interfaces crucial for understanding its biological role .

What NMR experiments are most informative for studying membrane protein dynamics of SAOUHSC_01978?

NMR spectroscopy offers unique insights into membrane protein dynamics that complement static structural methods. For SAOUHSC_01978, the following NMR experiments are particularly informative:

• Backbone Dynamics Assessment:

  • 15N relaxation measurements (T1, T2, heteronuclear NOE)

  • CPMG relaxation dispersion for μs-ms timescale motions

  • Hydrogen-deuterium exchange for solvent accessibility

  • TROSY-based experiments for improved sensitivity

• Side Chain Dynamics Analysis:

  • Methyl-TROSY for dynamics of Ile, Leu, Val residues

  • Deuteration with selective protonation strategies

  • 13C relaxation measurements of aromatic side chains

  • 2H quadrupolar relaxation for methyl group dynamics

• Membrane Topology Studies:

  • Paramagnetic relaxation enhancement with spin labels

  • Solvent PRE effects for surface mapping

  • Transferred cross-saturation for interaction interfaces

  • Solid-state NMR approaches for reconstituted samples

• Real-time Kinetic Studies:

These NMR methods provide detailed information about the conformational landscape and dynamic behavior of SAOUHSC_01978, offering insights into its functional mechanisms that may not be apparent from static structural studies. Integration of multiple NMR approaches allows mapping of dynamics across different timescales relevant to biological function .

What are the most promising research directions for understanding SAOUHSC_01978's role in antibiotic resistance mechanisms?

Understanding SAOUHSC_01978's potential role in antibiotic resistance mechanisms represents an important research frontier with several promising directions:

• Resistance Phenotype Correlation:

  • Compare expression levels across antibiotic-resistant S. aureus clinical isolates

  • Conduct systematic gene knockout studies followed by MIC determination

  • Perform overexpression analysis to identify potential resistance enhancement

  • Correlate structural variations with resistance profiles across strains

• Membrane Permeability Studies:

  • Investigate SAOUHSC_01978's impact on membrane composition and organization

  • Measure antibiotic penetration rates in wildtype vs. mutant strains

  • Assess changes in membrane potential and electrochemical gradients

  • Examine interactions with membrane-targeting antibiotics

• Stress Response Integration:

  • Analyze transcriptional co-regulation with known resistance determinants

  • Investigate post-translational modifications under antibiotic stress

  • Assess SAOUHSC_01978 localization changes during antibiotic challenge

  • Determine contribution to cell envelope stress response pathways

• Efflux System Interaction:

  • Evaluate physical and functional coupling with known efflux transporters

  • Measure antibiotic accumulation in presence/absence of SAOUHSC_01978

  • Investigate energetic contributions to efflux activity

  • Assess effects on proton motive force maintenance

These research directions, pursued through integrated multidisciplinary approaches, hold significant promise for uncovering SAOUHSC_01978's role in antibiotic resistance and potentially identifying novel therapeutic targets to address the growing challenge of resistant S. aureus infections .

How might high-throughput screening approaches identify small molecule modulators of SAOUHSC_01978 function?

Development of high-throughput screening (HTS) approaches for SAOUHSC_01978 modulators requires specialized methodologies for membrane protein targets:

HTS Strategy Development:

• Assay Development Considerations:

  • Target-based vs. phenotypic screening approaches

  • Development of function-specific biochemical assays

  • Reporter systems for indirect activity measurement

  • Thermal shift assays for binding detection

• Screening Platform Design:

• Compound Library Considerations:

  • Fragment-based approaches for membrane protein targets

  • Natural product collections enriched in membrane-active compounds

  • Focused libraries based on bacterial physiology

  • Diversity-oriented synthesis collections for novel scaffolds

• Validation Pipeline Development:

  • Secondary assays for mechanism confirmation

  • Counter-screening against related proteins

  • Resistance development assessment

  • Structure-activity relationship studies

  • Medicinal chemistry optimization pathway