Recombinant Bacillus cereus UPF0754 membrane protein BCE_0952 (BCE_0952)

What is the basic characterization of BCE_0952 protein?

BCE_0952 is a multi-pass membrane protein belonging to the UPF0754 family, found in Bacillus cereus (strain ATCC 10987). The protein consists of 378 amino acids with a molecular weight of approximately 42 kDa. Its amino acid sequence begins with MNIWLSMLTTTGLGAIIGGFTNHLAIKMLFRPHRPMYIGK and continues through a series of predominantly hydrophobic regions interspersed with charged residues . The protein's hydrophobicity profile suggests multiple transmembrane domains, consistent with its classification as a multi-pass membrane protein. For research applications, recombinant versions typically include an N-terminal His-tag to facilitate purification and detection.

What is known about the UPF0754 protein family?

The UPF0754 family represents a group of uncharacterized proteins (UncharacterizedProteinFamily0754) with conserved membrane-spanning domains found across various bacterial species. Current research indicates these proteins may have roles in membrane integrity, transport, or signaling. Comparative genomic analyses suggest the family maintains conserved structural elements despite sequence variations between species. Research methodologies for studying this family typically involve cross-species sequence alignment, hydrophobicity profiling, and topology prediction algorithms to identify functional domains.

How can researchers predict the structure of BCE_0952?

Structural prediction of BCE_0952 should employ a multi-faceted approach:

• Primary analysis: Use hydrophobicity plots and transmembrane domain prediction tools (TMHMM, HMMTOP) to identify membrane-spanning regions within the 378-amino acid sequence .

• Secondary structure prediction: Apply algorithms like PSIPRED or JPred to predict α-helical and β-sheet regions based on the amino acid sequence MNIWLSMLTTTGLGAIIGGFTNHLAIKMLFRPHR... etc.

• Homology modeling: Identify structural homologs using BLAST against proteins with known structures, even with low sequence identity, as membrane proteins often have structural conservation despite sequence divergence.

• Molecular dynamics simulations: Place predicted models in a simulated lipid bilayer to assess stability and potential conformational changes.

• Evolutionary coupling analysis: Use tools like EVfold to predict residue interactions based on evolutionary conservation patterns.

These methodologies provide a foundation for understanding BCE_0952 structure before undertaking challenging experimental approaches like X-ray crystallography or cryo-EM.

What expression systems are optimal for producing recombinant BCE_0952?

• Expression strain selection: BL21(DE3), C41(DE3), or C43(DE3) strains are preferable for membrane proteins, with the latter two specifically engineered for toxic membrane proteins.

• Vector design considerations:

  • Include an N-terminal His-tag for purification

  • Consider fusion partners (MBP, SUMO) to increase solubility

  • Incorporate precision protease cleavage sites for tag removal

• Induction optimization:

  • Lower temperatures (16-20°C) to slow expression and improve folding

  • Reduced IPTG concentrations (0.1-0.5 mM)

  • Extended induction periods (overnight)

• Membrane-specific additives:

  • Addition of glycerol (5-10%) to stabilize membrane proteins

  • Inclusion of specific lipids in growth media

E. coli has demonstrated effectiveness for BCE_0952 expression, but researchers examining protein-protein interactions or post-translational modifications might consider mammalian or insect cell systems for more native-like processing.

What purification strategies are most effective for BCE_0952?

Purification of recombinant BCE_0952 requires specialized approaches for membrane proteins:

• Membrane isolation and solubilization:

  • Cell lysis optimization (sonication, French press, or microfluidizer)

  • Differential centrifugation to isolate membrane fractions

  • Detergent screening (DDM, LDAO, OG) for optimal solubilization

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) utilizing the His-tag

  • Careful optimization of imidazole concentrations in wash and elution buffers

• Secondary purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography for additional purification

• Quality assessment:

  • SDS-PAGE analysis with Coomassie staining (target >90% purity)

  • Western blotting with anti-His antibodies

  • Mass spectrometry for molecular weight confirmation

Researchers should maintain detergent concentrations above critical micelle concentration throughout purification and consider protein stabilization with 6% trehalose as used in commercial preparations .

How can researchers verify proper folding of recombinant BCE_0952?

Verification of proper BCE_0952 folding presents unique challenges for membrane proteins:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Intrinsic tryptophan fluorescence to evaluate tertiary structure

  • Thermal shift assays to determine protein stability

• Functional validation:

  • Liposome reconstitution assays

  • Binding assays with potential interacting partners

  • Activity assays (if enzymatic function is known or predicted)

• Structural homogeneity assessment:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Dynamic light scattering (DLS) to evaluate monodispersity

  • Negative-stain electron microscopy for visual confirmation

• Protease resistance assays:

  • Limited proteolysis to identify stable, well-folded domains

  • Comparison of digestion patterns between detergent-solubilized and denatured protein

Researchers should note that working with membrane proteins often requires optimization of buffer conditions, including detergent type and concentration, pH, and ionic strength to maintain native-like folding.

What methodological approaches can be used to study BCE_0952 function?

Elucidating BCE_0952 function requires integrated experimental strategies:

• Genetic manipulation approaches:

  • Gene knockout or knockdown in B. cereus

  • Phenotypic analysis of mutants under various stress conditions

  • Complementation studies to confirm specificity of observed phenotypes

• Protein interaction studies:

  • Co-immunoprecipitation with potential interacting partners

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Cross-linking mass spectrometry to identify neighboring proteins

• Localization confirmation:

  • Immunofluorescence microscopy with BCE_0952-specific antibodies

  • GFP-fusion proteins with careful design to maintain functionality

  • Subcellular fractionation and Western blotting

• Comparative genomics:

  • Analysis of BCE_0952 conservation across B. cereus strains

  • Examination of gene neighborhood for functional clues

  • Identification of co-evolved genes suggesting functional relationships

• Transcriptomic and proteomic approaches:

  • RNA-Seq analysis comparing wild-type and BCE_0952 mutants

  • Proteomics to identify changes in protein expression or modification

  • Metabolomics to detect alterations in cellular metabolism

These methodologies should be applied systematically, with appropriate controls and replication to generate reliable insights into BCE_0952 function.

How might BCE_0952 contribute to B. cereus pathogenicity?

While direct evidence for BCE_0952's role in pathogenicity is limited, methodological approaches to investigate this connection include:

• Virulence assessment:

  • Comparison of BCE_0952 mutant and wild-type strains in infection models

  • Evaluation of adherence, invasion, and intracellular survival capabilities

  • Assessment of toxin production and secretion efficiency

• Host-pathogen interaction studies:

  • Infection of host cells with fluorescently labeled BCE_0952 mutants

  • Co-localization studies with host cellular components

  • Transcriptional responses of host cells to BCE_0952 mutants versus wild-type

• Analysis in the context of known B. cereus pathogenicity:

  • Evaluation of BCE_0952's relationship to established virulence factors

  • Assessment of BCE_0952 expression during different infection stages

  • Investigation of potential membrane protein clustering during infection

B. cereus is known to cause various infections, including food poisoning, respiratory tract infections, and nosocomial infections . Understanding BCE_0952's potential contribution to these pathogenic processes could provide insights into disease mechanisms and potential therapeutic targets.

How do membrane proteins like BCE_0952 impact bacterial stress responses?

Methodological approaches to investigate BCE_0952's role in stress responses include:

• Stress challenge experiments:

  • Expose BCE_0952 mutants to various stressors (pH, temperature, antimicrobials)

  • Monitor growth kinetics, survival rates, and morphological changes

  • Compare membrane integrity under stress conditions

• Transcriptional regulation analysis:

  • Identify promoter elements and potential regulatory proteins

  • Measure BCE_0952 expression under different environmental conditions

  • Determine if BCE_0952 is part of known stress response regulons

• Membrane property assessment:

  • Measure membrane fluidity changes in BCE_0952 mutants

  • Evaluate proton motive force maintenance

  • Assess lipid composition alterations

• Antimicrobial susceptibility testing:

  • Determine minimum inhibitory concentrations for various antimicrobials

  • Investigate BCE_0952's potential role in antimicrobial resistance mechanisms

  • Assess potential synergy between BCE_0952 inhibition and conventional antibiotics

Understanding BCE_0952's role in stress responses could be particularly relevant given B. cereus' ability to form spores resistant to cooking and pasteurization , potentially contributing to its persistence in food products and clinical environments.

What are the challenges in crystallizing BCE_0952 for structural studies?

Membrane protein crystallization presents specific methodological challenges:

• Protein preparation considerations:

  • Detergent selection is critical—screen multiple detergents (DDM, LDAO, etc.)

  • Consider lipid cubic phase (LCP) crystallization

  • Evaluate protein constructs with flexible terminal regions removed

• Crystallization optimization:

  • Extensive screening of conditions (pH, temperature, precipitants)

  • Addition of lipids to stabilize native conformation

  • Use of antibody fragments or nanobodies to provide crystal contacts

• Alternative approaches:

  • Cryo-electron microscopy for membrane proteins resistant to crystallization

  • Nuclear magnetic resonance (NMR) for smaller domains or fragments

  • Electron paramagnetic resonance (EPR) for distance measurements

• Quality assessment:

  • Pre-crystallization tests to evaluate sample homogeneity

  • Dynamic light scattering to monitor aggregation

  • Limited proteolysis to identify stable domains

The multi-pass nature of BCE_0952 suggests multiple transmembrane regions, which introduces additional challenges for maintaining proper folding and stability during purification and crystallization.

How can researchers perform protein-protein interaction studies with BCE_0952?

Several methodologies are suitable for identifying and characterizing BCE_0952 interactions:

• In vitro approaches:

  • Pull-down assays using His-tagged BCE_0952

  • Surface plasmon resonance (SPR) with immobilized BCE_0952

  • Microscale thermophoresis (MST) for quantitative binding analysis

  • Chemical cross-linking followed by mass spectrometry

• In vivo techniques:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Förster resonance energy transfer (FRET) with fluorescently tagged proteins

  • Bimolecular fluorescence complementation (BiFC)

  • Proximity-dependent biotin identification (BioID)

• Computational predictions:

  • Molecular docking simulations

  • Coevolution analysis to identify potential interacting partners

  • Network analysis based on genomic context

• Validation strategies:

  • Mutational analysis of predicted interaction interfaces

  • Competition assays with peptides derived from interaction regions

  • Reconstitution of protein complexes in artificial membrane systems

These techniques should be applied with appropriate controls, including negative controls (unrelated membrane proteins) and positive controls (known interacting partners from the same family or system).

How does B. cereus pathogenicity research inform BCE_0952 studies?

Understanding the broader context of B. cereus pathogenicity provides important frameworks for BCE_0952 research:

• Clinical relevance context:

  • B. cereus causes food poisoning and more severe non-dietary clinical infections

  • It has been implicated in nosocomial infections in immunocompromised patients

  • Some strains cause anthrax-like progressive pneumonia and fulminant sepsis

• Virulence mechanism investigation:

  • Determine if BCE_0952 is differentially expressed in clinical isolates

  • Investigate BCE_0952 conservation across strains with varying virulence

  • Assess BCE_0952 expression during infection processes

• Drug resistance considerations:

  • B. cereus produces β-lactamase conferring resistance to β-lactam antibiotics

  • Investigate BCE_0952's potential role in antimicrobial resistance

  • Evaluate BCE_0952 as a potential novel drug target

• Biofilm and persistence studies:

  • Examine BCE_0952's role in biofilm formation

  • Investigate its contribution to survival on hospital surfaces

  • Assess its involvement in spore formation or germination processes

Researchers should consider that B. cereus represents a group of 17 closely related species , and BCE_0952 homologs may have different roles across this spectrum, potentially contributing to the varying pathogenicity observed between strains.

What bioinformatic approaches can predict BCE_0952 function?

Computational methods provide valuable insights for directing experimental work:

• Sequence-based analysis:

  • Multiple sequence alignment with homologs across bacterial species

  • Identification of conserved domains and motifs

  • Prediction of functional sites using conservation mapping

• Structural bioinformatics:

  • Secondary structure prediction

  • Transmembrane topology modeling

  • Template-based threading for tertiary structure prediction

• Network-based approaches:

  • Gene neighborhood analysis

  • Co-expression network construction

  • Protein-protein interaction prediction

• Data integration strategies:

  • Literature-based discovery approaches

  • Integration of transcriptomic and proteomic data

  • Pathway enrichment analysis

These computational methodologies generate testable hypotheses about BCE_0952 function that can guide experimental design and interpretation, improving research efficiency.