Recombinant Shewanella amazonensis Protein CrcB homolog (crcB)

What is Shewanella amazonensis Protein CrcB homolog and what is its basic function?

Shewanella amazonensis Protein CrcB homolog (crcB) is a 124-amino acid protein (UniProt ID: A1S6H4) that functions as a putative fluoride ion transporter . The protein is encoded by the crcB gene (also known as Sama_1775) in S. amazonensis SB2B, a bacterium originally isolated from the Amazon River delta . CrcB belongs to a family of membrane proteins involved in ion homeostasis, specifically fluoride ion efflux, which is critical for bacterial survival in environments containing fluoride. The protein's full amino acid sequence is:

MNNVLYIAAGGAIGAVLRYSISILALQLFGTGFPFGTLIVNVAGSFLMGCIYALAELSHI GPEWKALIGVGLLGALTTFSTFSNETLLLLQQGELVKASLNVLLNLILCLTVVYLGQQLI YSRV

How is recombinant Shewanella amazonensis CrcB produced for research purposes?

The production of recombinant Shewanella amazonensis CrcB typically involves heterologous expression in E. coli expression systems. The full-length protein (amino acids 1-124) is commonly fused with an N-terminal His-tag to facilitate purification . The methodology involves:

• Cloning the crcB gene from S. amazonensis into an appropriate expression vector

• Transforming E. coli with the recombinant vector

• Inducing protein expression under optimized conditions

• Extracting and purifying the protein using affinity chromatography

• Processing into lyophilized powder form for long-term storage

What are the recommended storage and handling conditions for recombinant CrcB protein?

For optimal research results when working with recombinant CrcB protein, the following storage and handling protocols are recommended:

What techniques are most effective for studying CrcB protein function and structure?

Investigating CrcB protein function and structure requires a multi-faceted experimental approach:

• Membrane protein structural analysis:

  • Cryo-electron microscopy for native structure determination

  • X-ray crystallography of purified protein (challenging due to membrane protein properties)

  • Circular dichroism spectroscopy for secondary structure assessment

• Functional characterization:

  • Fluoride ion transport assays using fluoride-selective electrodes

  • Vesicle-based transport assays with fluorescent ion indicators

  • Electrophysiological measurements in reconstituted membrane systems

• Localization studies:

  • Fluorescent protein tagging for cellular localization

  • Immunolocalization with specific antibodies

  • Membrane fractionation followed by Western blotting

• Interactome analysis:

  • Pull-down assays using the His-tagged recombinant protein

  • Bacterial two-hybrid systems for protein-protein interaction detection

  • Cross-linking mass spectrometry for identifying interacting partners

How can site-directed mutagenesis be applied to investigate functional domains of Shewanella amazonensis CrcB?

Site-directed mutagenesis represents a powerful approach to dissect the structure-function relationship of the CrcB protein. Based on amino acid sequence analysis and predicted transmembrane topology, researchers should:

• Generate a predictive structural model using tools like AlphaFold or similar protein prediction algorithms

• Identify conserved residues likely involved in the fluoride ion channel formation

• Create a systematic mutation library focusing on:

  • Charged residues potentially involved in ion coordination

  • Highly conserved residues across CrcB homologs

  • Residues in predicted transmembrane regions

The experimental workflow should include:

• PCR-based site-directed mutagenesis of the crcB gene

• Expression and purification of mutant proteins following the same protocol as the wild-type

• Comparative functional assays measuring fluoride transport efficiency

• Structural integrity assessment using circular dichroism

• Fluoride resistance complementation assays in CrcB-deficient bacterial strains

What is known about the ecological significance of CrcB in Shewanella amazonensis?

Shewanella amazonensis SB2B was isolated from the Amazon River delta and has demonstrated remarkable metabolic versatility compared to other Shewanella species . Research has shown that:

• S. amazonensis SB2B can utilize 60 different carbon compounds, significantly more than other Shewanella strains from different environments (e.g., S. sp. strain W3-18-1 from deep marine sediment can only utilize 25)

• The bacterium shows particular proficiency in utilizing glucose multimers including α-, β-, and γ-cyclodextrin, dextrin, maltose, maltotriose, and sucrose

The CrcB protein likely contributes to the ecological fitness of S. amazonensis by:

• Providing protection against naturally occurring fluoride in the Amazon River delta environment

• Potentially participating in broader ion homeostasis mechanisms

• Contributing to the bacterium's adaptation to its specific ecological niche

Further investigation of CrcB expression patterns under different environmental conditions would provide valuable insights into its ecological role.

How does Shewanella amazonensis CrcB compare to homologs in other bacterial species?

Comparative genomic analysis of CrcB homologs across bacterial species reveals:

• CrcB proteins are widely distributed across bacterial phyla, indicating their fundamental importance

• The protein typically consists of approximately 120-130 amino acids with multiple transmembrane domains

• Functional conservation exists despite sequence variation, suggesting structural constraints on the fluoride channel function

Researchers investigating comparative aspects should:

• Perform multiple sequence alignments to identify conserved motifs

• Analyze the genomic context of crcB genes across species to identify potential functional associations

• Compare expression patterns under varying fluoride concentrations

• Consider the co-evolution of CrcB with other fluoride resistance mechanisms

What expression systems can be utilized to optimize recombinant CrcB protein yield and activity?

While E. coli is commonly used for CrcB expression , researchers seeking to optimize yield and activity should consider:

• Alternative expression hosts:

  • Bacillus subtilis for gram-positive expression

  • Pichia pastoris for eukaryotic expression with proper membrane protein folding

  • Cell-free expression systems for toxic membrane proteins

• Expression optimization strategies:

  • Codon optimization for the selected expression host

  • Testing different fusion tags beyond His-tag (e.g., MBP, GST, SUMO)

  • Screening various induction conditions (temperature, inducer concentration, time)

  • Membrane-targeted expression with appropriate signal sequences

• Purification refinement:

  • Detergent screening for optimal solubilization

  • Lipid nanodisc incorporation for native-like environment

  • Size exclusion chromatography to ensure homogeneity

How can computational approaches enhance understanding of CrcB structure and function?

Computational methods offer powerful tools for CrcB research when combined with experimental data:

• Structural prediction and analysis:

  • Homology modeling based on related ion channel structures

  • Molecular dynamics simulations to study conformational changes

  • Prediction of ion coordination sites and gating mechanisms

• Evolutionary analysis:

  • Phylogenetic analysis of CrcB across bacterial species

  • Detection of positive selection signals in specific lineages

  • Co-evolution analysis with interacting partners

• Systems biology integration:

  • Contextualizing CrcB within the broader ion homeostasis network

  • Predicting regulatory elements controlling crcB expression

  • Modeling the impact of CrcB function on cellular physiology

What are common challenges in working with recombinant CrcB and how can they be addressed?

Researchers working with CrcB often encounter several technical challenges:

• Low expression yields:

  • Solution: Optimize codon usage, reduce expression temperature, or use specialized strains designed for membrane protein expression

  • Alternative: Consider fusion partners known to enhance solubility

• Protein aggregation:

  • Solution: Screen different detergents for solubilization

  • Alternative: Express truncated constructs based on domain prediction

• Loss of activity during purification:

  • Solution: Include stabilizing agents like glycerol in buffers

  • Alternative: Reconstitute in lipid environments mimicking native membranes

• Reproducibility issues in functional assays:

  • Solution: Standardize protein:lipid ratios in reconstitution experiments

  • Alternative: Develop robust in vivo functional complementation assays

How can researchers verify the functional integrity of purified recombinant CrcB?

Ensuring that purified recombinant CrcB maintains its native functional properties is crucial for meaningful research. Validation approaches include:

• Structural integrity assessment:

  • Circular dichroism to confirm secondary structure content

  • Size exclusion chromatography to verify oligomeric state

  • Thermal shift assays to measure protein stability

• Functional verification:

  • Fluoride binding assays using isothermal titration calorimetry

  • Reconstitution into proteoliposomes followed by ion flux measurements

  • Complementation of CrcB-deficient bacterial strains

• Quality control metrics:

  • SDS-PAGE with greater than 90% purity

  • Mass spectrometry to confirm protein identity

  • Dynamic light scattering to assess homogeneity