Recombinant Staphylococcus aureus UPF0754 membrane protein SACOL1903 (SACOL1903)

What is currently known about the biological function of SACOL1903?

SACOL1903 is currently classified as a protein of unknown function (UPF), specifically in the UPF0754 family. While its precise biological role remains to be fully elucidated, structural analysis suggests it is an integral membrane protein that likely plays a role in membrane organization or transport.

Given the importance of membrane proteins in S. aureus pathogenicity and antibiotic resistance, SACOL1903 may be involved in processes such as nutrient acquisition, stress response, or cell envelope maintenance. Recent research on S. aureus membrane proteins has revealed their involvement in functional membrane microdomains (FMMs) that organize membrane complexes essential for bacterial survival and virulence .

What are the optimal expression systems for producing recombinant SACOL1903?

For expressing recombinant SACOL1903, researchers typically use one of the following systems:

• Cell-free expression systems: These are particularly useful for membrane proteins like SACOL1903 because they avoid toxicity issues often encountered with traditional cellular expression systems .

• E. coli expression systems with specific membrane protein vectors: When using E. coli, consider vectors designed for membrane protein expression that include:

  • C-terminal fusion tags for detection/purification

  • Inducible promoters for controlled expression

  • Signal sequences for proper membrane targeting

• Alternative hosts such as Lactococcus lactis or Bacillus subtilis may provide a more natural membrane environment for proper folding of Gram-positive bacterial proteins.

The choice of expression system should be determined by experimental requirements, particularly whether the protein will be used for structural studies, functional assays, or antibody production.

What purification strategies are most effective for isolating SACOL1903 while maintaining its native structure?

Purification of membrane proteins like SACOL1903 requires specialized approaches:

• Membrane isolation:

  • Harvest cells at mid-logarithmic phase

  • Disrupt cells using mechanical methods (sonication or French press)

  • Separate membranes by ultracentrifugation (100,000 × g for 1 hour)

• Solubilization:

  • Screen detergents (DDM, LDAO, or Triton X-100) at concentrations 2-3× their critical micelle concentration

  • Incubate at 4°C with gentle agitation for 1-2 hours

  • Remove insoluble material by ultracentrifugation

• Affinity chromatography:

  • Utilize the His-tag commonly incorporated in recombinant SACOL1903 for immobilized metal affinity chromatography (IMAC)

  • Use mild elution conditions (imidazole gradient 20-300 mM)

  • Include detergent in all purification buffers

• Size exclusion chromatography:

  • Final polishing step to separate monomeric protein from aggregates

  • Assess protein homogeneity and oligomeric state

For structural biology applications, consider using amphipols or nanodiscs to stabilize the protein in a lipid environment after purification.

How can researchers incorporate SACOL1903 into liposomes for functional studies?

Liposome reconstitution protocol for SACOL1903:

• Prepare lipid mixture:

  • Use a mixture of E. coli polar lipids and POPG (7:3 ratio)

  • Dissolve lipids in chloroform, evaporate to form a thin film

  • Rehydrate with buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl)

  • Sonicate to form unilamellar vesicles

• Protein incorporation:

  • Add purified SACOL1903 (protein:lipid ratio of 1:100 to 1:50)

  • Destabilize liposomes with detergent (0.1% Triton X-100)

  • Remove detergent using Bio-Beads SM-2 or dialysis

• Verification of incorporation:

  • Sucrose density gradient centrifugation to separate proteoliposomes

  • Western blot analysis to confirm protein presence

  • Freeze-fracture electron microscopy to visualize protein distribution

• Functional assays:

  • Measure changes in liposome properties (size, charge)

  • Perform transport assays if applicable

  • Analyze protein-lipid interactions via fluorescence spectroscopy

This methodology allows researchers to study SACOL1903 in a controlled membrane environment that mimics its natural context.

What analytical techniques are most suitable for studying SACOL1903 interactions with other S. aureus proteins?

Several techniques are particularly effective for investigating SACOL1903 interactions:

• Bacterial two-hybrid assays:

  • Similar to methods used to study FhuC-IsdF interactions in S. aureus

  • Can identify direct protein-protein interactions in a cellular context

  • Requires cloning SACOL1903 into appropriate vectors

• Pull-down assays with membrane fractions:

  • Immobilize purified SACOL1903 on affinity resin

  • Incubate with solubilized S. aureus membrane fractions

  • Identify binding partners via mass spectrometry

• Fluorescence microscopy with protein fusions:

  • Generate fluorescent protein fusions (e.g., GFP-SACOL1903)

  • Examine co-localization with other fluorescently tagged proteins

  • Particularly useful for studying association with functional membrane microdomains

• Cross-linking coupled with mass spectrometry:

  • Treat intact cells with membrane-permeable cross-linkers

  • Isolate SACOL1903 complexes

  • Identify cross-linked peptides via LC-MS/MS

• Surface plasmon resonance (SPR):

  • Immobilize purified SACOL1903 on sensor chips

  • Measure binding kinetics with potential interaction partners

  • Determine affinity constants for specific interactions

These methods can reveal whether SACOL1903 forms part of larger protein complexes involved in membrane organization, transport, or signaling pathways in S. aureus.

What are the challenges and solutions for determining the three-dimensional structure of SACOL1903?

Challenges:

• Membrane protein crystallization barriers:

  • Hydrophobic nature limits solubility

  • Detergent micelles create heterogeneous samples

  • Conformational flexibility reduces crystal formation

• NMR limitations:

  • Size constraints for traditional solution NMR

  • Complexity of membrane mimetics

Solutions:

• X-ray crystallography approaches:

  • Lipidic cubic phase (LCP) crystallization

  • Antibody fragment co-crystallization to increase polar surface area

  • Systematic detergent screening (vapor diffusion with detergent/lipid mixtures)

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis of purified protein in nanodiscs

  • Use of Volta phase plates to enhance contrast

  • Classification algorithms to address conformational heterogeneity

• Solution NMR strategies:

  • Selective isotope labeling of specific domains

  • TROSY techniques for large membrane proteins

  • Solid-state NMR for proteins in native-like lipid environments

• Hybrid approaches:

  • Combining computational modeling with low-resolution experimental data

  • Integrating hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics simulations

  • Cross-linking mass spectrometry to obtain distance constraints

Recent advances in membrane protein structural biology make it increasingly feasible to determine SACOL1903's structure, which would provide valuable insights into its function.

How can molecular dynamics simulations enhance our understanding of SACOL1903's membrane interactions?

Molecular dynamics (MD) simulations offer powerful insights into SACOL1903's behavior within the membrane environment:

• System preparation:

  • Build homology model based on related proteins if structure is unknown

  • Embed protein in phospholipid bilayer matching S. aureus membrane composition

  • Solvate system with explicit water and physiological ion concentrations

• Simulation protocols:

  • Equilibration phase (10-50 ns) with position restraints on protein

  • Production runs (minimum 500 ns) to observe conformational changes

  • Enhanced sampling techniques (metadynamics, umbrella sampling) for energy barriers

• Analysis approaches:

  • Protein-lipid interactions and preferential binding sites

  • Conformational flexibility and domain movements

  • Water/ion permeation through potential channels

  • Electrostatic potential maps across the membrane

• Specific phenomena to investigate:

  • Lipid sorting or domain formation around the protein

  • Interactions with functional membrane microdomain components like FloA

  • Conformational responses to membrane thickness or composition changes

  • Potential oligomerization interfaces

These simulations can generate testable hypotheses about SACOL1903's functional mechanisms and membrane interactions that can guide experimental design.

How might SACOL1903 contribute to membrane organization in S. aureus, and what experimental approaches can test these hypotheses?

Based on studies of other S. aureus membrane proteins, SACOL1903 may participate in functional membrane microdomains (FMMs) that organize protein complexes essential for bacterial processes . To investigate this possibility:

• Hypothesis testing for FMM association:

  • Create fluorescent protein fusions (SACOL1903-GFP)

  • Examine co-localization with known FMM markers like FloA

  • Use FRET to measure proximity to other FMM-associated proteins

  • Isolate detergent-resistant membrane fractions and identify SACOL1903 by immunoblotting

• Mutational analysis to identify critical domains:

  • Generate deletion or point mutations in potential interaction domains

  • Assess effects on protein localization and function

  • Identify residues required for FMM association

• Phenotypic characterization of knockout mutants:

  • Create SACOL1903 deletion strain

  • Compare growth under various stress conditions

  • Assess membrane permeability and antibiotic susceptibility

  • Evaluate virulence in infection models

• Proteomic analysis of protein complexes:

  • Perform immunoprecipitation of tagged SACOL1903

  • Identify co-precipitating proteins by mass spectrometry

  • Compare complexes under different growth conditions

These approaches can reveal whether SACOL1903 plays a role similar to other membrane proteins like IsdF, which requires proper FMM localization for function in iron acquisition .

What role might SACOL1903 play in S. aureus stress response and antibiotic resistance?

Membrane proteins often contribute to bacterial stress responses and antibiotic resistance. To investigate SACOL1903's potential role:

• Transcriptional regulation analysis:

  • Monitor SACOL1903 expression under various stresses (oxidative, pH, osmotic, antibiotic exposure)

  • Use qRT-PCR and reporter gene fusions

  • Identify transcription factors regulating expression

• Phenotypic characterization:

  • Compare wild-type and SACOL1903 mutant strains for:

    • Survival under membrane stress conditions

    • Minimal inhibitory concentrations (MICs) of various antibiotics

    • Ability to form biofilms

    • Membrane permeability changes using fluorescent dyes

• Membrane composition analysis:

  • Assess lipid profiles in wild-type vs. mutant strains

  • Examine changes in membrane fluidity using fluorescence anisotropy

  • Investigate membrane potential differences

• Potential mechanisms to investigate:

  • Role in membrane integrity maintenance

  • Contribution to proton motive force

  • Involvement in cell wall synthesis or remodeling

  • Function in detoxification or efflux systems

Understanding SACOL1903's role in stress response could reveal new targets for antimicrobial development against S. aureus.

How can SACOL1903 be engineered for use as a tool in vaccine development against S. aureus?

Engineering SACOL1903 for vaccine applications requires several strategic approaches:

• Immunogenicity enhancement:

  • Identify immunogenic epitopes using in silico prediction and epitope mapping

  • Create fusion constructs with known immunostimulatory proteins

  • Design truncated versions exposing key extracellular domains

• Delivery system development:

  • Incorporate into extracellular vesicles (EVs) as demonstrated with other S. aureus membrane proteins

  • Use detoxified EVs expressing modified SACOL1903 to stimulate immune response

  • Design nanoparticle formulations displaying SACOL1903 epitopes

• Adjuvant selection:

  • Test compatibility with aluminum-based adjuvants

  • Evaluate TLR agonists for enhanced immune stimulation

  • Consider combination with other S. aureus antigens for broader protection

• Immunological assessment:

  • Measure antibody titers against native and recombinant forms

  • Evaluate opsonophagocytic activity of induced antibodies

  • Assess T cell responses via cytokine profiling and proliferation assays

  • Test protective efficacy in animal infection models

Research on S. aureus EVs has demonstrated that engineered vesicles containing detoxified membrane proteins can elicit protective immune responses , suggesting a similar approach might be viable for SACOL1903-based vaccine development.

How can researchers investigate potential post-translational modifications of SACOL1903 and their functional significance?

Post-translational modifications (PTMs) can significantly impact membrane protein function. To investigate PTMs in SACOL1903:

• Mass spectrometry-based identification:

  • Purify native SACOL1903 from S. aureus membranes

  • Perform protease digestion with multiple enzymes for comprehensive coverage

  • Use high-resolution LC-MS/MS with ETD or ECD fragmentation

  • Search for common bacterial PTMs (phosphorylation, glycosylation, lipidation)

• Site-directed mutagenesis validation:

  • Create point mutations at identified PTM sites

  • Assess effects on protein localization, stability, and function

  • Compare wild-type and mutant phenotypes under various conditions

• Targeted analysis of specific modifications:

  • Phosphorylation: Pro-Q Diamond staining, phospho-specific antibodies

  • Glycosylation: Lectin blotting, PNGase F treatment

  • Lipidation: Click chemistry with lipid analogs, hydrophobicity analysis

• Regulatory enzyme identification:

  • Screen for kinases, phosphatases, or other modifying enzymes

  • Use co-immunoprecipitation to identify physical interactions

  • Perform in vitro modification assays with purified enzymes

Understanding PTMs can provide crucial insights into how SACOL1903's function is regulated in response to changing environmental conditions.

What are common challenges in working with recombinant SACOL1903 and strategies to overcome them?

Additional troubleshooting tips:

• Store protein in 50% glycerol at -20°C for extended stability

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

• Consider using nanodiscs or amphipols as alternatives to detergent micelles for maintaining native-like environment

How can researchers validate that recombinant SACOL1903 maintains its native conformation and functionality?

Validating the proper folding and function of recombinant SACOL1903 requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Fluorescence spectroscopy to assess tertiary structure through intrinsic tryptophan emission

  • Dynamic light scattering to verify monodispersity and absence of aggregation

  • Thermal shift assays to compare stability profiles of recombinant and native forms

• Functional validation:

  • Develop activity assays based on predicted function

  • Compare phenotypes of knockout strains complemented with recombinant vs. native gene

  • Assess membrane integration in proteoliposomes

  • Test for interaction with known binding partners via pull-down or SPR

• Epitope accessibility:

  • Generate antibodies against multiple domains

  • Compare antibody recognition patterns between native and recombinant forms

  • Use limited proteolysis to examine domain accessibility

• In vivo localization:

  • Express fluorescently tagged protein in S. aureus

  • Verify proper membrane localization

  • Assess co-localization with functional membrane microdomain markers