Recombinant Staphylococcus aureus UPF0154 protein SAR1353 (SAR1353), partial

What is the structural classification of Staphylococcus aureus UPF0154 protein SAR1353?

Staphylococcus aureus UPF0154 protein SAR1353 is a small protein of 80 amino acids belonging to the UPF0154 protein family. According to the SWISS-MODEL Repository, this protein has been identified in Staphylococcus aureus strain MRSA252 with the UniProt identification code Q6GH63 . Structurally, there are at least two existing models based on templates 7w2e.1.A and 2od5.1.A, both suggesting the protein exists as a monomer. The models demonstrate QMEAN scores of 0.58 and 0.52 respectively, indicating moderate to good model quality .

Which Staphylococcus strains have identical SAR1353 protein sequences?

The SAR1353 protein sequence appears to be highly conserved across multiple Staphylococcus strains. According to the SWISS-MODEL Repository, there are 10 identical sequences documented across various Staphylococcus aureus strains (Q5HG76, A7X1Z8, A6U1G3, Q6G9L5, Q2YXS4, P67292, P67291, P67290, A0A0D1GVP1) and Staphylococcus sp. 53017 (A0A9P3ZF38) . This high sequence conservation suggests the protein may serve an important function in Staphylococcus species.

What experimental approaches should researchers consider when planning initial characterization studies of SAR1353?

When designing initial characterization studies for SAR1353, researchers should adhere to key experimental design principles. As outlined in general experimental design frameworks, researchers must:

Methodologically, initial characterization should include sequence analysis, structural prediction validation, and preliminary functional assays based on bioinformatic predictions of potential functions.

What expression systems are optimal for recombinant production of SAR1353?

Based on current recombinant protein production practices, several expression systems may be suitable for SAR1353:

Prokaryotic Expression Systems:

• E. coli BL21(DE3) remains the first-choice expression host for small bacterial proteins like SAR1353 given its rapid growth and high yield potential

• For a protein originating from Staphylococcus, codon optimization may improve expression efficiency

• IPTG-inducible promoters (T7, tac) are recommended with induction preferably at OD600 0.6-0.8 at 18-25°C to enhance soluble protein production

Expression Optimization Parameters:

As this is a small bacterial protein (80 amino acids), bacterial expression systems are likely to produce sufficient yields for research purposes .

What purification strategy should be employed for obtaining research-grade SAR1353?

A multi-step purification strategy is recommended for obtaining high-purity SAR1353:

• Initial Capture: Affinity chromatography using His-tag (if recombinantly expressed with tag) or ion exchange chromatography based on the protein's theoretical pI

• Intermediate Purification: Size exclusion chromatography (SEC) to separate monomeric SAR1353 from aggregates or other proteins of different molecular weights

• Polishing Step: If necessary, a second ion exchange step or hydrophobic interaction chromatography

Quality Control Metrics:

• Purity assessment by SDS-PAGE (target >95%)

• Western blot confirmation of identity

• Mass spectrometry verification of intact mass

• Circular dichroism to confirm proper folding

• Dynamic light scattering to confirm monodispersity

When designing a purification protocol, researchers should implement a systematic experimental approach that considers multiple buffer conditions and purification parameters to identify optimal conditions .

What computational methods are recommended for analyzing SAR1353 structure?

For comprehensive structural analysis of SAR1353, researchers should employ multiple computational approaches:

• Template-Based Modeling: The SWISS-MODEL Repository indicates two existing models (based on templates 7w2e.1.A and 2od5.1.A) with QMEAN scores of 0.58 and 0.52 respectively . These models provide a starting point for structural investigation.

• Model Validation: Validation should include:

  • Ramachandran plot analysis

  • QMEAN scoring (as already performed for existing models)

  • MolProbity analysis for steric clashes

  • ProSA Z-score calculation

• Advanced Analysis:

  • Molecular dynamics simulations (10-100 ns) to assess stability and conformational flexibility

  • Virtual screening to identify potential ligand binding sites

  • Electrostatic surface potential mapping to predict interaction interfaces

Researchers should also consider that as of February 2025, new templates may have become available that could improve model quality beyond the two identified in the SWISS-MODEL Repository .

How can experimental structural data be obtained for SAR1353?

While computational models provide valuable insights, experimental structural data offers higher confidence. For SAR1353, consider these methods:

X-ray Crystallography Approach:

• Produce highly pure (>95%), concentrated (10-15 mg/mL) protein

• Screen crystallization conditions using commercial sparse matrix screens

• Optimize promising conditions varying precipitant concentration, pH, and additives

• Collect diffraction data and solve structure by molecular replacement using existing models

Nuclear Magnetic Resonance (NMR) Approach:

• Express isotopically labeled protein (15N, 13C)

• Collect standard triple-resonance datasets for backbone assignment

• Assign side chain resonances using TOCSY-based experiments

• Generate distance restraints from NOESY spectra

• Calculate structure using restrained molecular dynamics

Cryo-EM Considerations:

• At 80 amino acids (~9 kDa), SAR1353 is below the typical size limit for conventional cryo-EM

• Consider fusion to a larger protein partner or antibody fragment to increase molecular weight

The experimental approach selection should be guided by the research question, available resources, and expertise .

What approaches should be used to identify potential functions of SAR1353?

Given that UPF0154 family proteins are uncharacterized (as implied by the UPF designation - Uncharacterized Protein Family), a systematic approach to functional discovery is necessary:

• Bioinformatic Analysis:

  • Sequence similarity networks with characterized proteins

  • Gene neighborhood analysis to identify conserved genomic context

  • Protein-protein interaction predictions

  • Structural similarity to characterized proteins

• Experimental Approaches:

  • Gene knockout/knockdown studies in S. aureus

  • Transcriptomic analysis comparing wild-type and knockout strains

  • Pull-down assays coupled with mass spectrometry to identify interaction partners

  • Phenotypic assays focusing on stress responses, biofilm formation, and antibiotic resistance

• Biochemical Characterization:

  • Screen for enzymatic activities (kinase, phosphatase, protease, etc.)

  • Ligand binding assays with metabolites from central metabolism

  • DNA/RNA binding assays

Using this comprehensive approach allows researchers to formulate and test hypotheses regarding SAR1353 function in a systematic manner that minimizes bias and maximizes discovery potential.

How should researchers approach contradictory findings in SAR1353 functional studies?

When facing contradictory findings in SAR1353 functional studies, researchers should:

• Evaluate Methodological Differences:

  • Compare experimental conditions (temperature, pH, buffer composition)

  • Assess protein preparation methods (tags, purification approach)

  • Evaluate experimental design parameters (controls, replicates, statistical approaches)

• Consider Biological Context:

  • Different S. aureus strains may utilize the protein differently

  • Growth conditions may affect protein function

  • Post-translational modifications might be present in some studies but not others

• Resolution Strategies:

  • Design critical experiments that directly address the contradiction

  • Use multiple complementary techniques to study the same phenomenon

  • Consider collaborations with labs reporting contradictory findings

• Systematic Data Analysis:

  • Meta-analysis of published results

  • Bayesian approaches to weight evidence from different studies

  • Reanalysis of raw data when available

This systematic approach to resolving contradictions adheres to good experimental design principles by clearly defining variables, controlling for confounding factors, and implementing appropriate controls .

How might SAR1353 be involved in Staphylococcus aureus pathogenicity?

While direct evidence linking SAR1353 to pathogenicity is not established in the provided search results, researchers can investigate potential roles through:

• Comparative Genomics:

  • Analyze SAR1353 presence/absence and sequence conservation across virulent and avirulent S. aureus strains

  • Examine genomic context for proximity to known virulence factors

  • Survey expression data in infection models versus laboratory conditions

• Infection Models:

  • Compare wild-type versus SAR1353 knockout strains in infection models

  • Assess bacterial fitness, persistence, and virulence factor production

  • Evaluate host immune responses to both strains

• Protein-Host Interaction Studies:

  • Screen for interactions with host proteins using yeast two-hybrid or pull-down approaches

  • Assess effects on host cell signaling pathways

  • Evaluate potential immunomodulatory properties

• Stress Response Connection:

  • Investigate SAR1353 expression under infection-relevant stresses (oxidative stress, antimicrobial peptides, pH shifts)

  • Determine if SAR1353 contributes to survival under these conditions

The high conservation of SAR1353 across S. aureus strains (including MRSA252) suggests it may play an important role in bacterial physiology that could indirectly contribute to pathogenicity .

What approaches should be considered for structure-based inhibitor design targeting SAR1353?

For researchers considering SAR1353 as a potential therapeutic target, the following structure-based drug design approach is recommended:

• Target Validation:

  • Confirm essentiality through knockout studies and complementation

  • Assess conservation across clinically relevant strains

  • Evaluate effects of gene silencing on pathogenicity

• Druggability Assessment:

  • Analyze structural models for potential binding pockets

  • Calculate physicochemical properties of identified pockets

  • Compare to known druggable sites in related bacterial proteins

• Virtual Screening Strategy:

  • Prepare receptor by optimizing hydrogen bonding network

  • Select diverse compound libraries (fragment-based, focused antimicrobial libraries)

  • Implement consensus scoring from multiple docking algorithms

  • Cluster results to identify chemical scaffolds with promising binding modes

• Hit Validation Plan:

  • Thermal shift assays to confirm binding

  • Surface plasmon resonance for binding kinetics

  • Co-crystallization attempts for structural validation

  • Functional assays to confirm inhibitory activity

• Structure-Activity Relationship Development:

  • Medicinal chemistry optimization guided by computational predictions

  • Iterative testing in biochemical and cellular assays

  • ADMET property optimization

This systematic approach integrates computational and experimental methodologies to efficiently identify and develop potential inhibitors against SAR1353.

What statistical approaches are recommended for analyzing SAR1353 experimental data?

• Experimental Design Considerations:

  • Power analysis to determine appropriate sample sizes

  • Randomization and blinding where applicable

  • Appropriate control selection (positive, negative, vehicle)

  • Technical and biological replicates clearly distinguished

• Recommended Statistical Tests:

  • For comparing two groups: t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For dose-response relationships: regression analysis

  • For binding data: non-linear regression with appropriate models

• Advanced Analysis Techniques:

  • Principal component analysis for multivariate data

  • Bayesian approaches for integrating prior knowledge

  • Machine learning for pattern detection in complex datasets

• Reporting Standards:

  • Effect sizes with confidence intervals

  • Exact p-values rather than thresholds

  • Clear statement of statistical tests used

  • Data availability statement

This comprehensive statistical approach ensures findings regarding SAR1353 are robust and reproducible, aligning with principles of rigorous experimental design .

How can researchers ensure reproducibility in SAR1353 studies?

Reproducibility is a cornerstone of scientific research and particularly important for studies of uncharacterized proteins like SAR1353:

• Protocol Documentation:

  • Provide detailed methods including buffer compositions, incubation times, and temperatures

  • Document reagent sources, catalog numbers, and lot numbers

  • Specify equipment models and software versions with parameters

• Data Management:

  • Implement version control for analysis scripts

  • Use standardized file naming conventions

  • Maintain raw data alongside processed results

  • Consider using R's data.table package for efficient data manipulation and reproducible analysis

• Experimental Considerations:

  • Validate key reagents (antibodies, recombinant proteins)

  • Include internal controls for batch effects

  • Perform independent biological replicates

  • Consider blinded analysis where applicable

• Reporting Practices:

  • Follow field-specific reporting guidelines

  • Provide all necessary control experiments

  • Document failed approaches along with successful ones

  • Consider pre-registration for confirmatory studies

By implementing these practices, researchers studying SAR1353 can contribute to a more robust and reproducible body of knowledge about this uncharacterized protein.