Recombinant Staphylococcus aureus Probable CtpA-like serine protease (MW1310)

What is Recombinant Staphylococcus aureus Probable CtpA-like serine protease (MW1310)?

Recombinant Staphylococcus aureus Probable CtpA-like serine protease (MW1310) is a full-length (1-496 amino acids) bacterial protease with UniProt ID Q8NWR2, typically expressed with an N-terminal His-tag in E. coli expression systems for research purposes . This protease belongs to a novel class of serine proteases characterized by a Ser/Lys catalytic dyad instead of the more common Ser/His/Asp catalytic triad found in classical serine proteases . The CtpA-like protease shares functional similarities with other proteases involved in protein processing events, though its specific biological role in S. aureus requires further characterization.

How should researchers prepare and store recombinant MW1310 protease for optimal activity?

Proper preparation and storage of recombinant MW1310 protease is critical for maintaining enzymatic activity. The recommended protocol includes:

• Initial preparation: Centrifuge the vial briefly before opening to ensure all material is at the bottom.

• Reconstitution: Dissolve the lyophilized protein in deionized sterile water to a final concentration of 0.1-1.0 mg/mL.

• Storage preparation: Add glycerol to a final concentration of 5-50% (typically 50% is recommended) and aliquot for long-term storage.

• Storage conditions: Store at -20°C/-80°C for long-term preservation.

• Working conditions: When actively using the protein, store working aliquots at 4°C for up to one week.

• Avoid degradation: Repeated freeze-thaw cycles significantly reduce activity and should be strictly avoided .

This protocol ensures optimal stability of the protease's tertiary structure and preserves catalytic activity for experimental procedures. Buffer conditions can be adjusted based on specific experimental requirements, but care should be taken to maintain pH levels compatible with protein stability.

How does environmental pH affect the activity and substrate specificity of MW1310 protease?

The pH environment significantly influences the activity and substrate interaction profile of CtpA-like proteases such as MW1310. Research has revealed a notable difference in pH profile depending on the molecular environment:

• In solution (using synthetic peptide substrates): The enzyme exhibits a specific pH optimum that reflects its intrinsic catalytic properties.

• In membrane-associated contexts: When the substrate is embedded in a biological membrane (such as pD1 in thylakoid membranes), the pH dependency dramatically shifts, suggesting environmental modulation of activity .

This environmental sensitivity has important implications for experimental design when studying MW1310:

These differences likely reflect adaptations that allow the protease to respond to changing physiological conditions during bacterial growth and infection. When designing experiments, researchers should consider both solution-based and membrane-associated assays to fully characterize the enzyme's behavior .

What experimental approaches can be used to study MW1310 protease expression during bacterial infection?

Monitoring MW1310 protease expression during bacterial infection requires sophisticated approaches that capture dynamic changes in gene expression. Several effective methodologies include:

• mRNA quantification: Real-time quantitative PCR (RT-qPCR) can be used to measure mRNA levels during the course of infection, as demonstrated with other S. aureus proteases where expression increases over the infection timeline .

• Dual-reporter systems: Using a plasmid-based approach where the protease promoter drives expression of a fluorescent reporter (such as GFP), while a constitutive promoter drives expression of a different fluorescent protein (such as mRFP) for normalization. This allows for direct visualization and quantification of promoter activity during infection .

• Time-lapse microscopy: Combined with fluorescent reporters, this technique enables tracking of protease expression in real-time during infection progression, correlating expression with cytotoxic effects or other phenotypic changes .

• Western blotting: Using antibodies specific to MW1310 to detect the presence of both the pro-form and mature form of the protease in bacterial lysates or supernatants at different infection timepoints .

When implementing these approaches, researchers should include appropriate controls to account for background fluorescence, non-specific antibody binding, or variations in bacterial load that might confound interpretation of expression data.

How can structural homology modeling inform functional studies of MW1310 protease?

Structural homology modeling provides valuable insights into MW1310 protease function by predicting three-dimensional structure based on related proteases with known structures. This approach is particularly useful because:

• Catalytic mechanism prediction: CtpA-like proteases utilize a Ser/Lys catalytic dyad rather than the classical Ser/His/Asp triad. Homology modeling can identify the precise location of these catalytic residues and predict their spatial orientation during substrate binding and catalysis .

• Substrate binding pocket analysis: The model can reveal unique features of the substrate binding pocket that determine specificity. Unlike other Ser/Lys proteases that are sensitive to penem inhibitors, CtpA has shown insensitivity, suggesting a unique catalytic center configuration that can be explored through modeling .

• Target identification for mutagenesis: Homology models highlight conserved residues and structural elements that can be targeted for site-directed mutagenesis to test functional hypotheses.

• Protein-protein interaction prediction: Models can suggest potential interaction surfaces between MW1310 and other proteins or membranes that could modulate its activity in different cellular contexts.

When developing homology models, researchers should:

• Use multiple template structures (preferably other CtpA-like proteases)

• Validate models through energy minimization

• Assess model quality using Ramachandran plots and other validation tools

• Consider molecular dynamics simulations to explore conformational flexibility

The resulting structural insights can guide experimental design and interpretation, particularly for studies involving substrate specificity, inhibitor development, or investigating environmental effects on protease activity.

What is the relationship between MW1310 protease activity and bacterial pathogenicity?

The relationship between MW1310 protease activity and S. aureus pathogenicity involves several complex mechanisms that researchers should consider:

• Temporal expression patterns: Similar to other S. aureus proteases, MW1310 expression likely increases during intracellular infection phases, correlating with increased cytotoxicity. Studies of staphopain A (another S. aureus protease) have shown that mRNA levels increase over the course of intracellular infection, suggesting coordinated expression of virulence factors .

• Phagosomal escape mediation: Proteases can facilitate bacterial escape from host cell phagosomes, a critical step in the intracellular life cycle of S. aureus. Time-lapse microscopy studies have demonstrated that protease expression correlates with phagosomal escape events and subsequent host cell damage .

• Host cell morphological changes: Expression of active proteases within host cells induces distinctive morphological changes, beginning with pseudopodia retraction, followed by cell contraction and membrane protrusion formation. These changes are specifically observed in cells where S. aureus has escaped from the phagosome .

• Substrate specificity in host environments: The dramatically different pH profile and substrate affinity observed between solution and membrane environments suggest that MW1310 activity may be specifically modulated within host cells to target particular substrates relevant to pathogenesis .

Experimental approaches to investigate these relationships should include:

• Comparison of wild-type and protease-deficient mutants in infection models

• Time-course analysis of protease expression during different infection stages

• Identification of host protein substrates using proteomics approaches

• Correlation of protease activity with specific pathogenic outcomes

How can researchers differentiate between the activities of MW1310 and other S. aureus proteases in experimental systems?

Differentiating between the activities of MW1310 and other S. aureus proteases requires a multi-faceted approach:

• Specific substrate design: Develop synthetic peptide substrates that contain recognition sequences specific to MW1310. Based on homology with other CtpA-like proteases, which perform C-terminal processing of specific proteins, researchers can design substrates that mimic these natural targets .

• Selective inhibition profile: Unlike some other Ser/Lys proteases, CtpA-like proteases show insensitivity to penem inhibitors. This distinctive inhibition profile can be exploited to differentiate between protease activities. A panel of inhibitors with known specificities for different protease classes can be used to selectively inhibit other proteases while maintaining MW1310 activity .

• Genetic approaches: Construction of isogenic mutants lacking MW1310 but containing other proteases allows for direct comparison of phenotypes and biochemical activities. Complementation studies can confirm that observed differences are specifically due to MW1310 activity.

• Activity-based protein profiling: Using activity-based probes that covalently modify active site residues of specific protease classes can allow visualization and quantification of active MW1310 separate from other proteases.

• Expression system comparison: Expression of recombinant MW1310 in E. coli has been successful, suggesting that, unlike some other S. aureus proteases that may be involved in protein degradation systems like ssrA-tagging, MW1310 has distinct functional characteristics .

What control variables should be considered when designing experiments with MW1310 protease?

When designing experiments with MW1310 protease, researchers must rigorously account for several control variables to ensure valid and reproducible results:

• Buffer composition: The storage buffer contains Tris/PBS with 6% trehalose at pH 8.0, which may affect enzymatic activity. Control experiments should match these conditions or systematically vary them to determine optimal reaction conditions .

• Temperature: Enzymatic activity is temperature-dependent. Standardize all reactions at a consistent temperature, typically 25°C or 37°C, and include temperature controls when comparing different experimental conditions.

• Substrate concentration: Follow Michaelis-Menten kinetics by using a range of substrate concentrations to determine Km and Vmax values. This allows for proper comparison between different experimental conditions or enzyme variants.

• Enzyme concentration: Use consistent enzyme concentrations across experiments, with appropriate controls for enzyme stability and activity over time. When comparing different batches, normalize to specific activity rather than protein concentration.

• pH environment: As CtpA-like proteases show dramatically different pH profiles depending on the molecular environment, carefully control and monitor pH throughout experiments, especially when comparing solution-based versus membrane-associated activities .

• Membrane composition: When studying membrane-associated activities, the lipid composition can significantly affect enzyme-substrate interactions. Use defined membrane systems or native membranes with well-characterized compositions.

• Catalytically inactive controls: Include site-directed mutants that alter catalytic residues (e.g., Ser to Ala or Lys to Arg substitutions) as negative controls to distinguish between enzymatic and non-enzymatic effects.

These control variables should be explicitly defined in experimental protocols and reported in publications to ensure reproducibility and reliable interpretation of results .

How can researchers resolve data contradictions when studying MW1310 protease activity across different experimental systems?

Resolving data contradictions when studying MW1310 protease across different experimental systems requires a systematic approach:

• Standardize experimental conditions: Establish a core set of standardized conditions (buffer, temperature, pH, salt concentration) across all experimental platforms to eliminate these as sources of variation.

• Conduct parallel assays: When contradictory results emerge, perform parallel assays using multiple methodologies simultaneously with the same enzyme preparation to directly compare outcomes.

• Assess enzyme state: Verify that the enzyme is in the same state (pro-enzyme vs. mature form) across experimental systems. For instance, CtpA-like serine proteases may exist in both precursor and processed forms with different activities .

• Environmental context analysis: Consider the dramatic differences observed between solution-based and membrane-associated activities of CtpA-like proteases. These differences are not contradictions but reflect biological reality - the enzyme may have different pH dependencies and substrate affinities depending on environmental context .

• Substrate presentation: Evaluate how substrate accessibility differs between experimental systems. For membrane proteins, the orientation and exposure of cleavage sites may vary significantly depending on membrane composition and preparation methods.

• Time-resolved measurements: Collect time-course data rather than endpoint measurements to detect differences in reaction kinetics that might explain apparent contradictions in final outcomes.

• Computational modeling: Use structural models and molecular dynamics simulations to generate hypotheses that could explain seemingly contradictory results, particularly regarding substrate specificity or environmental effects .

By systematically exploring these potential sources of variation, researchers can often reconcile apparent contradictions and develop a more nuanced understanding of MW1310 protease function that accounts for its context-dependent activity.

What proteomics approaches can identify natural substrates of MW1310 protease in S. aureus?

Identifying the natural substrates of MW1310 protease requires sophisticated proteomics approaches that can detect specific protease-substrate interactions and cleavage events:

• Terminal amine isotopic labeling of substrates (TAILS): This negative selection approach enriches for N-terminal peptides, allowing identification of new protein N-termini generated by protease activity. By comparing samples with active versus inactive MW1310, researchers can identify specific cleavage events attributable to this protease.

• Stable isotope labeling with amino acids in cell culture (SILAC): Differential labeling of proteins in cultures with active versus inactive MW1310 allows for quantitative comparison of potential substrate degradation or processing.

• Protease-generated peptide enrichment: Using antibodies that recognize neo-epitopes created by specific cleavage events can enrich for peptides resulting from MW1310 activity.

• In vivo biotinylation proximity labeling: Fusing a promiscuous biotin ligase to MW1310 allows biotinylation of proteins in close proximity, potentially identifying transient enzyme-substrate interactions before cleavage occurs.

• Comparative secretome analysis: Since CtpA-like proteases are involved in protein processing and maturation, comparative analysis of secreted proteins from wild-type versus MW1310-deficient strains can reveal substrates that require this protease for proper processing.

When implementing these approaches, researchers should consider:

• Using multiple complementary methods to increase confidence in substrate identification

• Including appropriate controls such as catalytically inactive mutants

• Validating identified substrates through in vitro cleavage assays with purified components

• Correlating proteomic findings with phenotypic changes in S. aureus or host-pathogen interactions

How can crystallography and structural studies advance our understanding of MW1310 protease function?

Crystallography and structural studies of MW1310 protease would significantly advance our understanding of its function through several key insights:

• Catalytic mechanism elucidation: Crystal structures would definitively reveal the spatial arrangement of the Ser/Lys catalytic dyad, explaining how this unique configuration enables proteolytic activity without the typical His residue found in classical serine proteases .

• Substrate binding pocket characterization: Structural studies, particularly co-crystal structures with substrates or inhibitors, would reveal the detailed architecture of the substrate binding pocket, explaining the unique substrate specificity and the insensitivity to penem inhibitors that distinguish CtpA-like proteases .

• Conformational changes during catalysis: Using techniques like time-resolved crystallography or comparing structures in different states could reveal conformational changes associated with substrate binding and catalysis.

• Membrane interaction interfaces: Since MW1310 activity is significantly modulated in membrane environments, structural studies could identify potential membrane-interacting regions that explain this context-dependent behavior .

• Protein engineering opportunities: Detailed structural information would enable rational design of mutations to alter substrate specificity, enhance catalytic efficiency, or create specific inhibitors for research purposes.

Practical approaches for structural studies should include:

• Expression optimization to produce sufficient quantities of highly pure protein

• Screening multiple constructs with various truncations or tags to improve crystallization properties

• Exploring both apo-enzyme and enzyme-substrate complex crystallization

• Considering lipid cubic phase crystallization for capturing membrane-associated conformations

• Complementing crystallography with solution techniques like nuclear magnetic resonance (NMR) or cryo-electron microscopy for dynamic regions

The resulting structural information would provide a framework for interpreting biochemical data and guide future experiments investigating MW1310's role in S. aureus biology and pathogenesis.

What are the most promising applications of recombinant MW1310 protease in research?

Recombinant MW1310 protease offers several promising research applications that extend beyond basic characterization:

• Protein processing tool: Given its specific C-terminal processing activity (similar to other CtpA-like proteases), MW1310 could be developed as a biotechnological tool for precise C-terminal processing of recombinant proteins in research applications .

• Model system for environmental modulation: The dramatic differences in activity between solution and membrane environments make MW1310 an excellent model system for studying how molecular environments regulate enzyme function, with potential implications for understanding other context-dependent enzymes .

• Pathogenesis studies: As a potential virulence factor, MW1310 provides opportunities to investigate host-pathogen interactions and develop experimental systems to track S. aureus adaptation during infection .

• Structural biology platform: The unique Ser/Lys catalytic dyad presents an opportunity to investigate alternative catalytic mechanisms in proteases, potentially revealing evolutionary relationships between different protease families .

• Inhibitor development: The unique catalytic center that renders MW1310 insensitive to penem inhibitors provides an opportunity to develop novel, highly specific protease inhibitors that could serve as research tools or potential therapeutic leads .

These applications highlight how fundamental research on MW1310 can contribute to broader scientific advances while deepening our understanding of S. aureus biology and pathogenic mechanisms.

What experimental challenges remain in the full characterization of MW1310 protease?

Despite significant advances, several experimental challenges remain in fully characterizing MW1310 protease:

• Natural substrate identification: While homology with other CtpA-like proteases suggests C-terminal processing functions, the specific natural substrates in S. aureus remain largely unknown and require comprehensive proteomics approaches to identify.

• Structure determination: Obtaining high-resolution crystal structures remains challenging, particularly for membrane-associated conformations that may represent the physiologically relevant state of the enzyme .

• In vivo regulation: Understanding how MW1310 expression and activity are regulated during different phases of S. aureus growth and infection requires development of sensitive in vivo monitoring tools.

• Environmental modulation mechanisms: While dramatic differences in pH profile and substrate affinity have been observed between solution and membrane environments, the molecular mechanisms underlying these differences remain poorly understood .

• Host factor interactions: Identifying potential host factors that might modulate MW1310 activity during infection would provide important insights into host-pathogen interactions.

• Redundancy and compensation: Determining the degree of functional overlap between MW1310 and other S. aureus proteases requires sophisticated genetic approaches to create and analyze multiple protease-deficient strains.