Recombinant Staphylococcus aureus UPF0298 protein SAS1056 (SAS1056)

What is the structure and function of Recombinant Staphylococcus aureus UPF0298 protein SAS1056?

SAS1056 belongs to the UPF0298 protein family found in Staphylococcus aureus strain MSSA476, with a UniProt accession number of Q6GA93 . The protein consists of 84 amino acids with a sequence that suggests specific structural properties important for its biological function. While the exact function remains under investigation, structural analysis indicates potential roles in bacterial cell processes based on its conserved domains. The UPF (Uncharacterized Protein Family) designation indicates that this protein's precise biological function has not been fully elucidated, making it an important target for foundational research.

Methodologically, researchers should approach functional characterization through a combination of computational prediction tools and experimental validation techniques, including gene knockout studies, interaction assays, and localization experiments to establish its role in bacterial cellular processes. Structural studies may employ X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy to determine three-dimensional conformation.

What expression systems are most effective for producing SAS1056 protein?

The baculovirus expression system has proven effective for recombinant production of SAS1056 . This eukaryotic system provides advantages for bacterial protein expression including proper folding and reduced toxicity compared to bacterial expression systems. The expression region spans amino acids 1-84, representing the full-length protein .

For methodological optimization, researchers should consider the following approaches:

• Expression vector selection with appropriate promoters and fusion tags

• Host cell optimization (insect cell lines such as Sf9 or High Five)

• Infection conditions (MOI, timing of harvest)

• Purification strategy based on fusion tags

• Validation of protein folding and activity

Alternative expression systems such as E. coli or yeast may be considered depending on research needs, though each presents distinct advantages and challenges that should be systematically evaluated through expression trials and purification optimization.

How should SAS1056 protein be stored and reconstituted for experimental use?

For optimal stability and reproducibility in experiments, SAS1056 should be handled following these methodological guidelines:

• Storage: The lyophilized form has a shelf life of approximately 12 months at -20°C/-80°C, while the liquid form remains stable for about 6 months under the same conditions .

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (with 50% being recommended) for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

• Working conditions:

  • Store working aliquots at 4°C for no more than one week

  • Avoid repeated freezing and thawing as this significantly reduces protein activity

These guidelines are essential for maintaining protein integrity and ensuring experimental reproducibility. Researchers should document storage conditions as variables in experimental design, as protein stability can significantly impact research outcomes.

What quality control methods should be employed when working with SAS1056?

Quality control for recombinant SAS1056 typically involves verification of purity (>85% by SDS-PAGE) and confirmation of identity. Methodologically, researchers should implement a comprehensive quality control workflow:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining

  • Size exclusion chromatography (SEC)

  • Reverse-phase HPLC

• Identity confirmation:

  • Western blotting with specific antibodies

  • Mass spectrometry analysis

  • N-terminal sequencing

• Functional verification:

  • Activity assays specific to predicted function

  • Binding studies with potential interaction partners

  • Circular dichroism to confirm proper folding

• Endotoxin testing:

  • LAL assay for endotoxin contamination if using in cell culture

These quality control steps should be documented as part of the experimental design phase to ensure that variable results are not attributable to differences in protein quality.

How can molecular dynamics simulations be applied to study SAS1056 protein behavior?

Molecular dynamics (MD) simulations provide valuable insights into protein behavior in simulated cellular environments. For SAS1056, researchers can apply similar approaches to those used in other protein studies with the following methodological considerations:

• Simulation setup:

  • Select appropriate force fields (AMBER, CHARMM, or GROMOS)

  • Define simulation box with explicit solvent molecules

  • Add counterions to neutralize the system

  • Energy minimization before production runs

• Simulation parameters:

  • Time step selection (typically 1-2 fs)

  • Temperature and pressure control (NPT ensemble)

  • Periodic boundary conditions

  • Long-range electrostatic interactions (PME method)

• Analysis approaches:

  • RMSD and RMSF calculations for stability and flexibility assessment

  • Hydrogen bond analysis for structural stability

  • Principal Component Analysis for conformational changes

  • Solvent accessible surface area calculations

  • Potential binding site identification

These simulations can reveal dynamic properties not observable in static structural studies, particularly regarding flexibility of specific regions and potential interaction sites. Similar to approaches used in studying disease-related proteins , researchers should run multiple independent simulations to ensure statistical significance of observed phenomena.

What methods can be used to analyze protein frustration in SAS1056?

Protein frustration analysis examines energetic conflicts within protein structures that can impact stability and function. While no specific frustration analysis has been reported for SAS1056, methodological approaches similar to those used for FUS protein variants can be applied :

• Frustration calculation methods:

  • Local frustration calculations using the Frustratometer algorithm

  • Identification of minimally and highly frustrated regions

  • Configuration frustration index computation

• Visualization and interpretation:

  • Map frustration patterns onto the three-dimensional structure

  • Correlate frustrated regions with functional domains

  • Analyze how mutations might alter frustration patterns

• Functional implications:

  • Connect frustration patterns to potential binding interfaces

  • Assess impact on protein stability and aggregation propensity

  • Evaluate potential chaperone requirements for proper folding

This approach can provide insights into which regions of SAS1056 might be energetically strained and how this might relate to its function or stability. As observed in FUS protein studies, mutations can significantly alter frustration patterns , suggesting similar analyses could be valuable for understanding variants of SAS1056.

How does SAS1056 compare with related proteins such as SAR1095?

Comparative analysis between SAS1056 and related proteins like SAR1095 requires a systematic methodological approach:

• Sequence comparison:

  • Pairwise sequence alignment to identify conserved residues

  • Multiple sequence alignment with other UPF0298 family members

  • Calculation of sequence identity and similarity percentages

  • Identification of signature motifs

• Structural comparison:

  • Superimposition of solved or predicted structures

  • Domain organization analysis

  • Comparison of electrostatic surface potentials

  • Analysis of secondary structure elements

• Evolutionary relationship:

  • Phylogenetic tree construction

  • Evolutionary rate analysis for different protein regions

  • Identification of adaptive versus conserved sites

• Functional comparison:

  • Analysis of expression patterns in different bacterial strains

  • Comparison of interaction partners

  • Assessment of phenotypic effects when each protein is altered

This comparative approach can reveal insights into functional divergence or conservation across Staphylococcus aureus strains and inform research directions focusing on strain-specific adaptations.

What experimental design considerations are critical when using SAS1056 in bacterial studies?

When designing experiments involving SAS1056, researchers should follow systematic experimental design principles with specific considerations for this protein:

• Variable identification and control:

  • Define independent variables (e.g., SAS1056 concentration, bacterial strain, environmental conditions)

  • Identify dependent variables to measure outcomes

  • Control extraneous variables that might confound results

• Hypothesis formulation:

  • Develop specific, testable hypotheses about SAS1056 function

  • Ensure hypotheses are falsifiable and operationally defined

• Treatment design:

  • Include appropriate negative and positive controls

  • Consider dose-response relationships

  • Account for potential batch effects in protein preparation

• Subject assignment:

  • Use randomization where appropriate

  • Consider between-subjects or within-subjects designs depending on the question

• Measurement planning:

  • Select appropriate quantitative methods for assessing outcomes

  • Ensure measurement validity and reliability

  • Plan for appropriate statistical analysis

How can evolutionary conservation analysis inform SAS1056 functional studies?

Evolutionary conservation analysis provides valuable insights into functionally important regions of proteins. Using approaches similar to those applied in the study of other proteins , researchers can:

• Methodological steps:

  • Identify homologous sequences across bacterial species

  • Perform multiple sequence alignment

  • Calculate conservation scores for each residue (e.g., using CONSURF server)

  • Map conservation onto structural models

• Interpretation framework:

  • Highly conserved residues (scores 7-9) likely indicate functional or structural importance

  • Conservation patterns can identify domain boundaries

  • Conserved surface patches often indicate interaction sites

  • Variable regions may reflect species-specific adaptations

• Functional implications:

  • Target highly conserved residues for site-directed mutagenesis

  • Design functional assays focused on conserved regions

  • Interpret experimental results in light of evolutionary constraints

This approach enables prediction of critical functional regions within SAS1056, comparable to analyses showing that nuclear localization signals tend to be highly conserved in proteins like FUS due to their functional importance .

What protein frustration indices are most relevant for SAS1056 analysis?

When analyzing protein frustration in SAS1056, researchers should consider multiple metrics to comprehensively assess energetic conflicts:

Similar to analyses performed for other proteins , researchers should interpret these metrics collectively rather than in isolation. For example, the R521C mutation in FUS protein showed a noticeable increase in frustration involving the mutated residue, indicating that cysteine was less favorable in that position . Similar analysis for SAS1056 variants could provide insights into stability determinants and potential functional alterations.

What approaches can be used to study potential post-translational modifications of SAS1056?

Post-translational modifications (PTMs) can significantly impact protein function. To investigate potential PTMs in SAS1056, researchers should employ:

• Computational prediction:

  • Use algorithms specific for different PTM types (phosphorylation, glycosylation, etc.)

  • Identify consensus sequences for modification enzymes

  • Consider bacterial-specific modification patterns

• Experimental detection:

  • Mass spectrometry-based proteomics (MS/MS)

  • Western blotting with modification-specific antibodies

  • Radioactive labeling with modification-specific precursors

  • Chemical reactivity assays for specific modifications

• Functional validation:

  • Site-directed mutagenesis of predicted modification sites

  • Comparison of native versus recombinant protein modification patterns

  • Temporal analysis of modifications under different conditions

• Structural impact assessment:

  • Molecular dynamics simulations comparing modified and unmodified forms

  • Binding assays to determine effects on protein-protein interactions

  • Stability studies to assess structural impacts

While the search results don't specifically mention PTMs for SAS1056, this methodological framework provides a comprehensive approach for investigating their presence and significance.