Recombinant Bacillus anthracis UPF0754 membrane protein BAMEG_3697 (BAMEG_3697)

What is BAMEG_3697 and what is its cellular localization?

BAMEG_3697 is a UPF0754 family membrane protein from Bacillus anthracis with 378 amino acids. It has the UniProt ID C3LE57 and is classified as a membrane protein based on its sequence characteristics . Analysis of its amino acid sequence reveals multiple hydrophobic regions consistent with transmembrane domains, particularly evident in the sequence "MNIWLSMLTTTGLGAIIGGFTNHLAIKMLFRPHRP" at the N-terminus and "LGGMIGIVQGLLLLFLK" at the C-terminus .

For determining cellular localization experimentally, researchers should consider:

• Membrane fractionation followed by Western blotting using anti-His antibodies for the recombinant protein

• Immunofluorescence microscopy with antibodies against the native protein or tag

• GFP-fusion protein expression for live-cell imaging

• Protease protection assays to determine membrane topology, similar to methods described for other membrane proteins

The hydrophobicity profile of BAMEG_3697 suggests multiple transmembrane regions, indicating it likely spans the bacterial membrane multiple times rather than being a peripheral membrane protein.

What expression systems are available for producing recombinant BAMEG_3697?

When expressing BAMEG_3697 in E. coli, codon optimization may be beneficial to account for the different codon usage between Bacillus and E. coli. The existing protocols have successfully yielded protein of greater than 90% purity as determined by SDS-PAGE .

What purification strategies are recommended for recombinant BAMEG_3697?

The purification of recombinant BAMEG_3697 requires careful consideration of its membrane protein nature. Based on available information and general membrane protein purification principles:

• Solubilization: Begin with appropriate detergent screening to solubilize BAMEG_3697 from membranes. Common detergents include:

  • n-Dodecyl β-D-maltoside (DDM)

  • n-Octyl β-D-glucopyranoside (OG)

  • Digitonin

  • CHAPS

• Affinity Chromatography: Utilize the N-terminal His tag for initial purification via Immobilized Metal Affinity Chromatography (IMAC)

  • Begin with Ni-NTA resin

  • Use increasing imidazole concentrations for washing and elution

  • Consider using low concentrations of detergent in all buffers to maintain protein solubility

• Additional Purification Steps:

  • Size Exclusion Chromatography (SEC) to separate monomeric from aggregated protein

  • Ion Exchange Chromatography for further purification if needed

• Quality Control:

  • SDS-PAGE to verify purity (>90% has been achieved previously)

  • Western blot to confirm identity

  • Dynamic Light Scattering to assess homogeneity

  • Mass Spectrometry to confirm molecular weight and integrity

For researchers working with the commercially available recombinant protein, it comes as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage .

How should recombinant BAMEG_3697 be stored to maintain activity?

Proper storage of recombinant BAMEG_3697 is crucial for maintaining its structural integrity and functional activity. According to the product information:

• Short-term storage:

  • Store working aliquots at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles which can lead to protein denaturation and aggregation

• Long-term storage:

  • Store at -20°C/-80°C upon receipt

  • Aliquoting is necessary for multiple use

  • Add glycerol (5-50% final concentration, with 50% being the default recommendation) before freezing

• Reconstitution procedure:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

  • Allow the protein to fully dissolve before making aliquots

• Stability considerations:

  • The protein is shipped as a lyophilized powder in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • The trehalose acts as a stabilizing agent, protecting protein structure during lyophilization and reconstitution

  • Maintain pH at approximately 8.0 for optimal stability

Researchers should perform activity assays after storage to verify that the protein has maintained its functional properties, especially when designing experiments to characterize its biological function.

What analytical methods are appropriate for characterizing recombinant BAMEG_3697?

Comprehensive characterization of recombinant BAMEG_3697 requires multiple complementary analytical approaches:

• Biochemical Characterization:

  • SDS-PAGE for purity assessment and molecular weight determination (~42 kDa expected)

  • Western blotting with anti-His antibodies for identity confirmation

  • Circular Dichroism (CD) spectroscopy for secondary structure analysis

  • Thermal shift assays to evaluate stability

• Structural Analysis:

  • Crystallography trials (challenging for membrane proteins)

  • Cryo-electron microscopy for 3D structure determination

  • NMR spectroscopy for dynamic structural information

  • Computational modeling based on the amino acid sequence: "MNIWLSMLTTTGLGAIIGGFTNHLAIKMLFRPHRPMYIGKFQVPFTPGLIPKRRDELAVQLGKMVVEHLLTPEGIGKKLTNEEFQKGLIHWAQVEVDKVITNEQSLRHMLGKWDVAHVEKEATEKIEQVITEKIQAFLEEYYTYTWEQALPHSVHEKIENAIPNVSAFILKRAIHFFESEEGKSRLSRMIDDFFASRGALLNLVGMFLGNVSVVDRVQPEVIKFLGQDGTKQLLTDVLQKEWEKLKGRDVKELETFVEKEMIVSSILSAVKVEETVSKFLNQSVQQVCEPVRETIIEKVVPNAVTKGLKWGGENVESILNNLHLAEIVQQEVSTFSTERLEDLVLSITKNELKMITYLGALLGGMIGIVQGLLLLFLK"

• Membrane Topology Analysis:

  • Protease protection assays coupled with mass spectrometry

  • Fluorescence quenching techniques

  • Cysteine scanning mutagenesis

  • Computational prediction using algorithms such as TMHMM, HMMTOP, and TopPred

• Mass Spectrometry Applications:

  • Intact mass analysis to confirm sequence integrity

  • Peptide mapping after protease digestion

  • Hydrogen-deuterium exchange (HDX) for accessible regions

  • Cross-linking MS for interaction studies

Combining these approaches will provide complementary information about the structural and biochemical properties of BAMEG_3697, essential for understanding its function in Bacillus anthracis.

What methodologies can be used to determine the membrane topology of BAMEG_3697?

Determining the membrane topology of BAMEG_3697 is crucial for understanding its function and interaction with other cellular components. Several complementary approaches can be employed:

• Computational Prediction and Analysis:

  • Hydrophobicity analysis of the amino acid sequence suggests multiple transmembrane regions

  • Transmembrane prediction algorithms (TMHMM, HMMTOP, PHDhtm)

  • Signal peptide prediction tools to identify potential cleavage sites

  • Positive-inside rule application for loop orientation prediction

• Experimental Approaches:

  • Protease Protection Assays: As described in search result , this technique allows for mapping the soluble domains of integral membrane proteins
    a. Isolate membrane fractions containing BAMEG_3697
    b. Treat with proteinase K under controlled conditions
    c. Analyze protected fragments using mass spectrometry
    d. Compare results between intact and disrupted membranes (high-pH treatment)

  • Cysteine Scanning Mutagenesis:
    a. Create a cysteine-less version of BAMEG_3697
    b. Introduce single cysteines at various positions
    c. Test accessibility using membrane-permeable and -impermeable thiol-reactive reagents
    d. Map accessible and inaccessible regions to determine topology

  • Fluorescence-Based Methods:
    a. GFP-fusion reporters at different predicted loop regions
    b. pH-sensitive fluorescent proteins to determine lumen vs. cytoplasmic localization
    c. FRET-based distance measurements between domains

• Combined Structural Methods:

  • Cryo-electron microscopy of membrane-embedded protein

  • X-ray crystallography of detergent-solubilized protein

  • Solid-state NMR for membrane-embedded structural determination

These approaches together can create a comprehensive topological map of BAMEG_3697, revealing which domains face the cytoplasm versus the periplasm/extracellular space, and how many times the protein traverses the membrane.

How can post-translational modifications of BAMEG_3697 be identified and characterized?

Post-translational modifications (PTMs) can significantly impact protein function and are particularly relevant for bacterial membrane proteins in pathogenesis. To identify and characterize PTMs in BAMEG_3697:

• Mass Spectrometry-Based Approaches:

  • Bottom-up Proteomics:
    a. Digest protein with multiple proteases (trypsin, chymotrypsin, proteinase K)
    b. Analyze resulting peptides by LC-MS/MS
    c. Search for mass shifts indicating modifications
    d. Use neutral loss scanning for specific modifications (e.g., phosphorylation)

  • Top-down Proteomics:
    a. Analyze intact protein by high-resolution MS
    b. Determine exact mass and compare to theoretical
    c. Fragment intact protein in MS (ECD/ETD) to localize modifications

  • Targeted Analysis:
    a. Multiple Reaction Monitoring (MRM) for known modifications
    b. Parallel Reaction Monitoring (PRM) for improved specificity
    c. SWATH-MS for comprehensive modification scanning

• Enrichment Strategies for Specific PTMs:

  • Phosphorylation: IMAC (Fe³⁺, Ti⁴⁺, Ga³⁺), titanium dioxide, phospho-antibodies

  • Glycosylation: Lectin affinity, hydrazide chemistry, HILIC

  • Lipidation: Click chemistry for fatty acid modifications

  • Methylation/acetylation: Specific antibodies, SILAC labeling

• Functional Validation of PTMs:

  • Site-directed mutagenesis of modified residues

  • Functional assays comparing wild-type and mutant proteins

  • In vitro modification with purified enzymes

  • Inhibitor studies targeting specific modification enzymes

A table summarizing potential PTMs in bacterial membrane proteins and their detection methods:

Given the overlapping peptides produced from digestion with proteinase K as mentioned in search result , this approach would be particularly suitable for identifying phosphorylation and methylation sites on BAMEG_3697.

What techniques are most effective for studying protein-protein interactions involving BAMEG_3697?

Investigating protein-protein interactions of membrane proteins like BAMEG_3697 presents unique challenges but is crucial for understanding their biological functions. Several complementary approaches can be employed:

• Affinity-Based Methods:

  • Co-immunoprecipitation (Co-IP):
    a. Use anti-His antibodies to pull down recombinant BAMEG_3697
    b. Identify co-precipitating proteins by MS or Western blotting
    c. Perform reciprocal Co-IP to confirm interactions
    d. Consider crosslinking prior to lysis to capture transient interactions

  • Pull-down Assays:
    a. Immobilize purified BAMEG_3697 on Ni-NTA or other affinity resin
    b. Incubate with bacterial lysate or purified candidate interactors
    c. Wash and elute complexes for analysis

  • Proximity-Dependent Labeling:
    a. BioID or TurboID fusion to BAMEG_3697
    b. Express in B. anthracis or reconstituted system
    c. Identify biotinylated proximity partners by MS

• Biophysical and Structural Methods:

  • Surface Plasmon Resonance (SPR):
    a. Immobilize BAMEG_3697 on sensor chip
    b. Measure real-time binding kinetics with potential partners
    c. Determine binding affinities and kinetic parameters

  • Microscale Thermophoresis (MST):
    a. Label BAMEG_3697 with fluorescent dye
    b. Measure changes in thermophoretic mobility upon binding
    c. Determine Kd values in solution

  • Cryo-EM and X-ray Crystallography:
    a. Obtain structural information of protein complexes
    b. Identify binding interfaces and structural changes upon interaction

• Genetic and In Vivo Approaches:

  • Bacterial Two-Hybrid:
    a. Fuse BAMEG_3697 to DNA-binding domain
    b. Screen against prey library fused to activation domain
    c. Identify positive interactions through reporter activation

  • Split GFP/Luciferase Complementation:
    a. Fuse fragments to BAMEG_3697 and candidate interactors
    b. Express in bacterial system
    c. Measure complementation signal indicating proximity

• Computational Prediction:

  • Homology-based interaction prediction

  • Co-evolution analysis to identify potential binding partners

  • Molecular docking simulations with candidate interactors

When designing these experiments, it's important to maintain the membrane environment or use appropriate detergents to preserve native protein conformation and interaction capability. The methods described for proteomic analysis of membrane proteins in search result could be adapted for studying BAMEG_3697 interactions, particularly the approach that preserves the lipid bilayer structure.

How does BAMEG_3697 compare structurally and functionally with homologous proteins in other bacterial species?

Comparative analysis of BAMEG_3697 with homologous proteins can provide valuable insights into its evolutionary conservation, functional importance, and potential roles in Bacillus anthracis biology. A systematic approach includes:

• Sequence-Based Comparative Analysis:

  • Homology Identification:
    a. BLAST/PSI-BLAST searches against bacterial protein databases
    b. HMM-based searches using HMMER to identify distant homologs
    c. Focus on both pathogenic and non-pathogenic bacterial species

  • Multiple Sequence Alignment:
    a. Align BAMEG_3697 with homologs using tools like MUSCLE, MAFFT, or T-Coffee
    b. Identify conserved motifs and residues
    c. Map conservation onto predicted transmembrane topology

  • Phylogenetic Analysis:
    a. Construct phylogenetic trees to understand evolutionary relationships
    b. Correlate with bacterial species phylogeny
    c. Identify potential horizontal gene transfer events

• Structural Comparison:

  • 3D Structure Prediction:
    a. Use AlphaFold2 or RoseTTAFold for BAMEG_3697 and homologs
    b. Compare predicted structures to identify conserved structural elements
    c. Focus on membrane-spanning regions and potential functional domains

  • Domain Architecture Analysis:
    a. Identify protein domains using tools like InterPro and Pfam
    b. Compare domain composition across homologs
    c. Identify unique domains in BAMEG_3697 vs. homologs

• Functional Comparative Analysis:

  • Genomic Context:
    a. Examine neighboring genes in B. anthracis and related species
    b. Identify potential operons and functional associations
    c. Compare gene neighborhood conservation across species

  • Expression Pattern Comparison:
    a. Analyze expression data for BAMEG_3697 and homologs
    b. Identify conditions triggering expression
    c. Compare regulation mechanisms across species

  • Experimental Functional Substitution:
    a. Express BAMEG_3697 in species with deleted homologs
    b. Test functional complementation
    c. Identify species-specific vs. conserved functions

Note: The percentages and specific homologs are estimated and would need to be determined through actual sequence analysis, as specific homology information was not provided in the search results.

What are the optimal experimental conditions for functional characterization of BAMEG_3697?

Functional characterization of a membrane protein with unknown function like BAMEG_3697 requires carefully designed experimental conditions. Based on its properties as a membrane protein from Bacillus anthracis, the following approaches are recommended:

• Reconstitution Systems:

  • Proteoliposomes:
    a. Purify BAMEG_3697 using methods described earlier
    b. Test different lipid compositions mimicking B. anthracis membrane
    c. Optimize protein:lipid ratios (typically 1:100 to 1:1000)
    d. Verify correct orientation using protease protection assays

  • Nanodiscs:
    a. Incorporate BAMEG_3697 into nanodiscs with MSP proteins
    b. Provide native-like membrane environment with defined size
    c. Optimize MSP:protein:lipid ratios for stability
    d. Confirm incorporation by size exclusion chromatography

  • Detergent Micelles:
    a. Screen detergents for optimal stability (DDM, LMNG, etc.)
    b. Monitor protein stability using thermal shift assays
    c. Optimize detergent concentration above CMC

• Functional Assays Based on Predicted Properties:

  • Transport Assays (if suspected to be a transporter):
    a. Fluorescent substrate uptake measurements
    b. Counterflow assays with radiolabeled substrates
    c. Liposome swelling assays for channel activity

  • Enzyme Activity Assays (if suspected to have enzymatic function):
    a. Screen potential substrates based on sequence analysis
    b. Develop coupled enzyme assays for activity detection
    c. Test pH, temperature, and ion dependencies

  • Binding Assays (if suspected to be a receptor):
    a. Screen potential ligands using thermal shift assays
    b. Measure binding affinities using ITC, SPR, or fluorescence-based methods
    c. Perform competition binding assays to identify specific binding sites

• Genetic and Cell-Based Approaches:

  • Gene Knockout/Knockdown:
    a. Generate BAMEG_3697 deletion mutants in B. anthracis
    b. Characterize phenotypic changes (growth, morphology, stress response)
    c. Perform complementation studies with wild-type and mutant variants

  • Overexpression Effects:
    a. Analyze consequences of BAMEG_3697 overexpression
    b. Monitor changes in membrane properties and cellular physiology
    c. Identify potential toxic effects indicating function

  • Reporter Fusion Assays:
    a. Generate transcriptional and translational fusions
    b. Identify conditions affecting expression
    c. Map regulatory networks controlling BAMEG_3697

• Buffer and Environmental Conditions:

  • pH range: Test pH 6.0-8.0 (physiological range for B. anthracis)

  • Temperature: 25-37°C (optimal growth temperature for B. anthracis)

  • Salt concentration: 100-300 mM NaCl

  • Additional components: Mg²⁺, Ca²⁺, and other physiologically relevant ions

  • Reducing conditions: Include DTT or β-mercaptoethanol if cysteine residues are present

For storage and handling of the recombinant protein, follow the recommendations provided earlier: store at -20°C/-80°C with glycerol as a cryoprotectant, and avoid repeated freeze-thaw cycles .