Recombinant Staphylococcus aureus UPF0398 protein SAR1458 (SAR1458)

What is known about the structural features of SAR1458?

SAR1458 is a 187-amino acid protein classified as an UPF0398 family protein in Staphylococcus aureus subsp. aureus MRSA252. The protein's structure has been computationally modeled through AlphaFold DB (AF-Q6GGW3-F1), with the model released on December 9, 2021, and last modified on September 30, 2022. The computational model demonstrates exceptionally high confidence, with a global pLDDT score of 95.77, indicating very reliable structural predictions throughout most regions of the protein . While experimental verification of this structure is currently lacking, the high confidence scores suggest the predicted fold is likely accurate. Researchers should approach structural studies by combining this computational model with experimental validation techniques such as X-ray crystallography or cryo-EM.

How can researchers verify the AlphaFold predicted structure experimentally?

Experimental verification of the SAR1458 structure requires a multi-technique approach:

• X-ray Crystallography: Express recombinant SAR1458 with a cleavable His-tag, purify using nickel affinity chromatography followed by size exclusion chromatography, then screen crystallization conditions. Typical buffers include 20mM Tris-HCl pH 7.5, 150mM NaCl, with various precipitants.

• Circular Dichroism (CD) Spectroscopy: To confirm secondary structure elements, conduct CD scanning from 190-260nm at 20°C using purified protein at 0.1-0.5 mg/ml in phosphate buffer.

• Nuclear Magnetic Resonance (NMR): For regions with lower pLDDT scores, NMR can provide residue-level dynamics information using 15N-labeled protein.

The following table outlines recommended experimental validation methods based on confidence regions in the AlphaFold model:

What expression systems are most suitable for recombinant SAR1458 production?

For recombinant expression of SAR1458, E. coli-based systems typically provide the highest yield for structural and biochemical studies. The methodological approach should include:

• Vector Selection: pET-based vectors (particularly pET-28a with N-terminal His-tag) offer good expression control through T7 promoter.

• Expression Strains: BL21(DE3) represents the standard choice, while Rosetta strains can accommodate rare codons if sequence analysis indicates their presence in SAR1458.

• Expression Protocol:

  • Transform expression plasmid into chosen strain

  • Culture in LB or 2xYT media to OD600 of 0.6-0.8

  • Induce with 0.5mM IPTG

  • Express at 18°C overnight to minimize inclusion body formation

  • Harvest cells and lyse in buffer containing 50mM Tris-HCl pH 8.0, 300mM NaCl, 10mM imidazole, and protease inhibitors

For isotope labeling (for NMR studies), M9 minimal media supplemented with 15N-ammonium chloride and/or 13C-glucose should be used, with expression protocols adjusted to account for slower growth in minimal media .

What are the most effective experimental designs for studying SAR1458 function?

When investigating SAR1458 function, researchers should implement robust experimental designs that account for both direct and indirect effects. Based on established methods for characterizing proteins of unknown function in Staphylococcus aureus:

• Genetic Knockout Studies: Create SAR1458 deletion mutants using allelic replacement techniques. Implement a time-series experimental design to monitor phenotypic changes over multiple growth phases . This design allows detection of temporal effects that might be missed in single-timepoint experiments.

• Complementation Analysis: After knockout studies, complement with wild-type and mutant versions of SAR1458 to confirm phenotype specificity. Use a counterbalanced design to control for position effects if integrating at different genomic loci .

• Interactome Analysis: Implement pull-down experiments coupled with mass spectrometry to identify interaction partners. Use crosslinking techniques with controls for false positives through statistical validation.

• Transcriptomic Response: Employ RNA-seq to analyze global transcriptional changes in SAR1458 mutants under various conditions. Design should include biological triplicates with appropriate statistical analysis for differential expression .

For all experimental designs, implement appropriate controls based on the regression-discontinuity principle to identify causal relationships rather than mere associations .

How can researchers address contradictory data when studying SAR1458?

When faced with contradictory data in SAR1458 research, researchers should implement systematic approaches to identify and resolve contradictions:

• Data Contradiction Analysis Framework:

  • Self-contradictory Data: Identify internal inconsistencies within a single experimental dataset, such as conflicting phenotypes under seemingly identical conditions .

  • Pair Contradictions: Recognize conflicts between two separate experiments examining the same aspect of SAR1458 .

  • Conditional Contradictions: Address complex scenarios where data from one experiment creates a contradiction between two other experiments when interpreted together .

• Resolution Methodology:

  • Implement a controlled validation experiment targeting specifically the contradictory aspect

  • Employ orthogonal techniques to measure the same parameter

  • Analyze experimental conditions for subtle differences (media composition, growth phase, etc.)

  • Examine strain background effects, particularly in clinical versus laboratory S. aureus strains

• Documentation Protocol:

  • Record all contradictions in a structured format

  • Document resolution attempts and outcomes

  • Report both resolved and unresolved contradictions in publications to advance field knowledge

This systematic approach prevents confirmation bias and ensures research integrity when studying a protein with limited characterized functions .

What purification strategies yield the highest quality SAR1458 for structural studies?

For high-quality SAR1458 purification suitable for structural studies, implement this multi-step protocol:

• Initial Extraction:

  • Lyse cells in 50mM Tris-HCl pH 8.0, 300mM NaCl, 10mM imidazole, 1mM DTT, protease inhibitors

  • Use sonication (6×30s pulses) or high-pressure homogenization at 15,000 psi

  • Clarify lysate by centrifugation at 30,000×g for 45 minutes at 4°C

• Affinity Chromatography:

  • Apply clarified lysate to Ni-NTA column pre-equilibrated with lysis buffer

  • Wash extensively with 50mM Tris-HCl pH 8.0, 300mM NaCl, 20mM imidazole

  • Elute with 50mM Tris-HCl pH 8.0, 300mM NaCl, 250mM imidazole gradient

• Tag Removal:

  • Dialyze against 50mM Tris-HCl pH 8.0, 150mM NaCl, 1mM DTT

  • Add TEV protease at 1:50 ratio (protease:protein)

  • Incubate overnight at 4°C

  • Remove cleaved tag by reverse Ni-NTA chromatography

• Polishing Step:

  • Apply protein to Superdex 75 column in 20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM DTT

  • Collect fractions and analyze by SDS-PAGE

  • Pool fractions containing >95% pure SAR1458

• Quality Assessment:

  • Dynamic light scattering to confirm monodispersity

  • Thermofluor assay to determine optimal buffer conditions

  • Mass spectrometry to confirm intact mass

This protocol typically yields 10-15mg of purified protein per liter of culture, sufficient for crystallization trials or NMR studies .

What methods are most effective for determining SAR1458's role in S. aureus physiology?

To determine SAR1458's physiological role, employ a multi-faceted approach combining genetics, biochemistry, and systems biology:

• Conditional Expression Systems: Implement tetracycline-inducible or antisense RNA expression systems to create depletion strains when direct knockouts are lethal. Monitor growth parameters, morphology, and stress responses under varying expression levels.

• Metabolic Profiling: Conduct untargeted metabolomics comparing wild-type and SAR1458 mutant strains under different growth conditions. Look specifically for:

  • Changes in central carbon metabolism

  • Alterations in nucleotide pools

  • Differences in amino acid biosynthesis

  • Cell wall precursor abundance

• Transcriptional Regulation Analysis:

  • ChIP-seq to identify genomic binding sites if SAR1458 has DNA-binding domains

  • RNA-seq to identify differentially expressed genes in mutant strains

  • Quantitative RT-PCR validation of key targets

• Localization Studies: Employ fluorescent protein fusions or immunofluorescence to determine subcellular localization, which often provides functional clues.

• Phenotypic Microarrays: Test mutant and wild-type strains across hundreds of growth and stress conditions to identify specific sensitivities using Biolog plates or custom arrays .

This comprehensive approach can identify phenotypes that might be missed by single-method strategies, particularly for proteins like SAR1458 that may have condition-specific functions.

How can researchers investigate potential interactions between SAR1458 and other S. aureus proteins?

To investigate SAR1458's protein-protein interactions:

• In Vivo Approaches:

  • Bacterial Two-Hybrid (B2H): Clone SAR1458 into both bait and prey vectors of a B2H system (e.g., BACTH). Screen against an S. aureus genomic library to identify interaction partners.

  • Co-Immunoprecipitation: Express epitope-tagged SAR1458 in S. aureus, crosslink in vivo, then immunoprecipitate and identify binding partners via mass spectrometry.

  • Proximity-Dependent Biotin Identification (BioID): Fuse SAR1458 to a biotin ligase, express in S. aureus, and identify proximal proteins through streptavidin pulldown and mass spectrometry.

• In Vitro Approaches:

  • Pull-Down Assays: Use purified His-tagged SAR1458 as bait with S. aureus lysate, then identify bound proteins by mass spectrometry.

  • Surface Plasmon Resonance (SPR): Immobilize purified SAR1458 on a sensor chip and measure binding kinetics with candidate partner proteins.

  • Isothermal Titration Calorimetry (ITC): Measure binding thermodynamics between SAR1458 and putative interaction partners.

• Computational Predictions:

  • Use structure-based docking to predict interactions with other S. aureus proteins

  • Employ co-evolution analysis to identify potentially interacting proteins

  • Analyze genomic context for gene neighborhood conservation

• Validation Studies:

  • Mutagenesis of key residues to disrupt predicted interactions

  • Functional assays to determine biological significance of interactions

  • Co-crystal structure determination of protein complexes

These approaches should be used in combination, as each has specific strengths and limitations for detecting different types of protein interactions .

What techniques can identify post-translational modifications of SAR1458?

To comprehensively characterize post-translational modifications (PTMs) of SAR1458:

• Mass Spectrometry-Based Approaches:

  • Bottom-Up Proteomics: Digest purified SAR1458 with trypsin and analyze peptides by LC-MS/MS. Use neutral loss scanning for phosphorylation and precursor ion scanning for glycosylation.

  • Top-Down Proteomics: Analyze intact SAR1458 by high-resolution MS to determine exact masses of all proteoforms.

  • Targeted MS: Develop multiple reaction monitoring (MRM) assays for predicted modification sites based on motif analysis.

• Modification-Specific Enrichment:

  • Phosphorylation: Enrich phosphopeptides using TiO2 or immobilized metal affinity chromatography (IMAC)

  • Glycosylation: Use lectin affinity chromatography for glycopeptide enrichment

  • Acetylation: Immunoprecipitate with anti-acetyllysine antibodies

• Site-Specific Mutagenesis:

  • Mutate predicted modification sites to non-modifiable residues

  • Compare wild-type and mutant protein function in vivo

  • Assess impact on protein stability, localization, and interaction profile

• In Vitro Modification Assays:

  • Incubate purified SAR1458 with S. aureus kinases/acetyltransferases

  • Monitor modification by mobility shift or specific antibodies

  • Identify modifying enzymes through activity-based protein profiling

Researchers should be particularly attentive to condition-dependent modifications, as S. aureus proteins often show different modification patterns under stress conditions versus normal growth .

How can the AlphaFold structure of SAR1458 guide drug discovery efforts?

The high-confidence AlphaFold structure of SAR1458 (global pLDDT score of 95.77) provides an excellent starting point for structure-based drug discovery. Researchers should follow this methodological framework:

• Druggable Pocket Identification:

  • Use computational tools such as SiteMap, FTMap, or CryptoSite to identify potential binding pockets

  • Calculate pocket volumes, hydrophobicity, and evolutionary conservation

  • Prioritize pockets with high conservation and suitable physicochemical properties

• Virtual Screening Workflow:

  • Prepare a diverse compound library (consider ZINC, ChEMBL, or proprietary libraries)

  • Implement hierarchical screening: pharmacophore filtering → docking → molecular dynamics

  • Score compounds using consensus scoring with multiple force fields

• Experimental Validation Pipeline:

  • Thermal shift assays (TSA) to confirm direct binding

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for binding kinetics

  • Co-crystallization attempts with top hits to validate binding mode

• Optimization Strategy:

  • Structure-activity relationship studies based on validated hits

  • Fragment-based approaches for low-affinity but high-efficiency binders

  • Consider allosteric sites in addition to orthosteric pockets

When working with computational models rather than experimental structures, researchers should apply more stringent filters to account for potential inaccuracies, focusing primarily on regions with pLDDT scores above 90 .

What approaches can elucidate SAR1458's role in S. aureus pathogenesis and virulence?

To investigate SAR1458's potential role in pathogenesis:

• Infection Models:

  • In Vitro: Compare wild-type and SAR1458 mutant strains in:

    • Macrophage survival assays

    • Neutrophil killing assays

    • Epithelial cell adhesion and invasion models

  • In Vivo: Implement appropriate animal models based on infection site:

    • Skin infection model (subcutaneous infection)

    • Systemic infection model (tail vein injection)

    • Device-associated infection model (implanted catheters)

• Virulence Factor Expression Analysis:

  • Quantify major virulence factor expression via qRT-PCR

  • Measure toxin production through ELISA or functional hemolysis assays

  • Monitor global virulence regulator activity (Agr, SarA, etc.)

• Host-Pathogen Interaction Studies:

  • Assess host immune response to wild-type versus mutant strains

  • Measure cytokine/chemokine production by infected host cells

  • Analyze neutrophil extracellular trap (NET) formation and bacterial survival

• Comparative Analysis Under Infection-Relevant Conditions:

  • SAR1458 expression during different infection stages

  • Behavior under host-mimicking conditions (low pH, oxidative stress, nutrient limitation)

  • Response to antibiotic treatment in wild-type versus mutant strains

These approaches should be conducted with appropriate controls and statistical analysis, using the equivalent time-samples design or multiple time-series design for tracking infection progression .

How can researchers develop conditional knockout systems to study essential functions of SAR1458?

If SAR1458 proves essential for S. aureus viability, implement these conditional knockout strategies:

• Inducible Expression Systems:

  • Tetracycline-Regulated System:

    • Replace native promoter with tetO operator sequences

    • Express TetR repressor from a constitutive promoter

    • Add anhydrotetracycline (ATc) to repress SAR1458 expression

    • Monitor phenotypes at varying levels of depletion

  • IPTG-Inducible System:

    • Replace native promoter with Pspac

    • Express LacI repressor on the same construct

    • Titrate IPTG concentration to control expression levels

• Degron-Based Systems:

  • Fuse SAR1458 to SsrA degradation tag variants

  • Express modified SspB adaptor protein under inducible control

  • Induce degradation through adaptor protein expression

• CRISPRi Approach:

  • Express dCas9 under inducible promoter

  • Design multiple sgRNAs targeting SAR1458 coding sequence

  • Induce dCas9 to block transcription without genome editing

• Analysis Protocol:

  • Confirm depletion via Western blot and RT-qPCR

  • Monitor growth rate, morphology, and viability during depletion

  • Perform transcriptomics and metabolomics at defined depletion timepoints

  • Use time-series design to capture transition phenotypes before lethality

Each system has advantages and limitations; the optimal choice depends on experimental goals and resources. For non-lethal but severe phenotypes, the equivalent time-samples design allows for repeated measurements under controlled depletion conditions .

What statistical approaches are most appropriate for analyzing SAR1458 functional data?

For robust statistical analysis of SAR1458 functional data:

• Experimental Design Considerations:

  • Implement factorial designs to assess multiple factors simultaneously

  • Use nested classifications when analyzing hierarchical data (e.g., multiple strains, conditions)

  • Apply time-series experimental design for temporal phenotypes

  • Consider counterbalanced designs to control for order effects in sequential experiments

• Statistical Methods by Data Type:

  • Growth/Phenotypic Data:

    • ANOVA with appropriate post-hoc tests for parametric data

    • Kruskal-Wallis for non-parametric distributions

    • Mixed-effects models for repeated measures

  • Omics Data:

    • Differential expression analysis with multiple testing correction

    • Enrichment analysis for functional categorization

    • Network-based approaches for identifying affected pathways

  • Structural Data:

    • Bootstrap analysis for assessing model confidence

    • Geometric statistics for conformational analysis

    • Cluster analysis for identifying conformational states

• Addressing Specific Challenges:

  • Control for batch effects using appropriate normalization

  • Implement regression-discontinuity analysis for threshold-based phenomena

  • Use non-equivalent control group design when perfect randomization is impossible

• Validation Approaches:

  • Cross-validation for predictive models

  • Independent biological replicates (not just technical replicates)

  • Orthogonal techniques to confirm key findings

When reporting results, provide complete statistical details including test selection rationale, exact p-values, and effect sizes, not just significance indicators .

How should researchers approach the analysis of contradictory data in SAR1458 research?

When faced with contradictory data about SAR1458 function:

• Systematic Contradiction Analysis Framework:

  • Identification Phase: Categorize contradictions as self-contradictory, pairwise, or conditional

  • Evaluation Phase: Assess each contradiction's significance and potential impact

  • Resolution Phase: Implement systematic investigation of possible explanations

• Technical Investigation:

  • Examine methodological differences (buffers, tags, expression systems)

  • Assess protein quality and integrity across experiments

  • Evaluate instrument calibration and data processing pipelines

• Biological Investigation:

  • Consider strain background effects (laboratory vs. clinical isolates)

  • Evaluate growth conditions and stress responses

  • Assess potential context-dependent functions

• Statistical Reassessment:

  • Re-evaluate significance thresholds and multiple testing corrections

  • Implement more robust statistical methods where appropriate

  • Consider Bayesian approaches to incorporate prior knowledge

• Resolution Documentation:

  • Create a detailed record of contradiction investigation

  • Document hypotheses tested and outcomes

  • Present both resolved and unresolved contradictions transparently in publications

This systematic approach prevents selective reporting bias and enhances research reproducibility while advancing understanding of context-dependent protein functions .

What bioinformatic tools are most useful for predicting SAR1458 function from its sequence and structure?

To predict SAR1458 function through bioinformatics:

• Sequence-Based Analysis:

  • Homology Search: BLAST, HHpred, and HMMER against curated databases

  • Motif Analysis: PROSITE, MEME, and ScanProsite for functional motifs

  • Domain Identification: InterProScan, CDD, and SMART

  • Genomic Context: STRING database for conserved gene neighborhoods

• Structure-Based Approaches:

  • Structural Alignment: DALI, TM-align, and FATCAT against PDB database

  • Binding Site Prediction: SiteEngine, COFACTOR, and FTSite

  • Electrostatic Analysis: APBS for surface charge distribution

  • Molecular Dynamics: GROMACS or NAMD simulations to identify flexible regions

• Integrated Prediction Pipelines:

  • COFACTOR/COACH: Integrates sequence and structure for function prediction

  • I-TASSER suite: Combines multiple approaches for comprehensive annotation

  • ConSurf: Evolutionary conservation mapping onto structure

• Analysis Workflow:

  • Begin with basic sequence analysis (BLAST, domain prediction)

  • Progress to structure-based comparisons using the AlphaFold model

  • Apply specialized tools based on initial findings

  • Integrate results through scoring matrices for confident predictions

  • Validate top predictions experimentally

For the SAR1458 protein with its high-quality AlphaFold structure (pLDDT 95.77), structure-based methods may provide more specific functional insights than sequence-based approaches alone .

What are the most promising future research directions for SAR1458?

Based on current knowledge and methodological approaches, the most promising research directions for SAR1458 include:

• Comprehensive Functional Characterization:

  • Integration of transcriptomics, proteomics, and metabolomics data from SAR1458 mutants

  • Condition-specific phenotyping under various stress conditions

  • Investigation of potential regulatory roles in S. aureus physiology

• Structural Biology Advances:

  • Experimental validation of the AlphaFold structure through X-ray crystallography

  • Structure determination of SAR1458 in complex with interaction partners

  • Dynamic structural studies using NMR or hydrogen-deuterium exchange mass spectrometry

• Translational Applications:

  • Assessment of SAR1458 as a potential drug target or biomarker

  • Development of inhibitors targeting SAR1458 function

  • Evaluation of conservation across clinical S. aureus isolates

• Methodological Innovations:

  • Application of cryo-electron tomography for in situ visualization

  • Development of SAR1458-specific biosensors to monitor activity

  • Implementation of machine learning approaches to predict function from structure

• Integration with Systems Biology:

  • Placement of SAR1458 within S. aureus regulatory networks

  • Modeling of SAR1458's contribution to bacterial homeostasis

  • Comparative analysis across different staphylococcal species

These directions should be pursued using robust experimental designs and appropriate statistical approaches to ensure reproducible and meaningful advances in understanding this protein's role in S. aureus biology .

How can researchers effectively design experiments to resolve the current knowledge gaps about SAR1458?

To address knowledge gaps about SAR1458 function:

• Strategic Experimental Planning:

  • Implement factorial designs to test multiple hypotheses simultaneously

  • Use "equivalent time-samples design" for temporal studies

  • Apply counterbalanced designs when testing multiple conditions

  • Consider regression-discontinuity analysis for threshold-dependent processes

• Targeted Approaches for Specific Knowledge Gaps:

  • Biochemical Function: Combine structural predictions with targeted assays (e.g., testing predicted enzymatic activities)

  • Interaction Network: Implement systematic interactome mapping using complementary techniques

  • Regulatory Role: Combine ChIP-seq, RNA-seq, and promoter analysis if DNA-binding is predicted

  • Stress Response: Test mutant phenotypes under clinically relevant stress conditions

• Multi-Scale Investigation:

  • Single-cell level: Fluorescence microscopy for localization and dynamics

  • Population level: Growth and competition assays

  • Systems level: Multi-omics integration and network analysis

  • Host-pathogen interface: Infection models and immune response

• Validation Framework:

  • Independent validation of key findings through orthogonal methods

  • Genetic complementation with wild-type and mutant variants

  • Cross-species comparison with orthologs from related staphylococci

By implementing these systematic approaches with appropriate controls and statistical analyses, researchers can efficiently address knowledge gaps while minimizing experimental bias and maximizing reproducibility .

What methodological innovations might accelerate SAR1458 research in the coming years?

Emerging methodological innovations with high potential to accelerate SAR1458 research include:

• Advanced Structural Biology Techniques:

  • Cryo-Electron Tomography: For visualizing SAR1458 in its native cellular context

  • Integrative Structural Biology: Combining multiple experimental data types with computational modeling

  • Time-Resolved Structural Methods: Capturing structural dynamics during function

• Genome Engineering Advances:

  • CRISPR Interference (CRISPRi): For tunable gene repression without genome editing

  • Base Editing: For precise nucleotide substitutions without double-strand breaks

  • Multiplex Genome Engineering: For simultaneous modification of SAR1458 and potential partners

• Single-Cell Technologies:

  • Single-Cell RNA-Seq: For heterogeneity analysis in SAR1458 mutant populations

  • Single-Cell Proteomics: For protein-level phenotyping

  • Microfluidics-Based Approaches: For high-throughput phenotypic screening

• Artificial Intelligence Applications:

  • Deep Learning for Function Prediction: Beyond traditional bioinformatics approaches

  • Machine Learning for Experimental Design: To optimize conditions and reduce experimental iterations

  • AI-Augmented Data Analysis: For identifying subtle phenotypes and complex relationships

• Advanced Imaging Innovations:

  • Super-Resolution Microscopy: For detailed subcellular localization

  • Correlative Light and Electron Microscopy (CLEM): For connecting function to ultrastructure

  • Spatial Transcriptomics/Proteomics: For location-specific functional analysis