Recombinant Psychrobacter cryohalolentis Protein CrcB homolog (crcB)

What is the CrcB homolog protein from Psychrobacter cryohalolentis?

The CrcB homolog protein from Psychrobacter cryohalolentis (strain K5) is a membrane protein encoded by the crcB gene (locus tag: Pcryo_0235). This protein consists of 123 amino acids and functions as a putative fluoride ion transporter. The protein has a molecular structure characterized by multiple transmembrane domains, which is typical of membrane transport proteins . The full amino acid sequence is: MQWLAIGLGAAIGACLRGWLARFNPMHHWIPLGTLGANVLGGLLIGLALVWFERVGSGLSPNIRLFVITGFLGGLTTFSTFSVEVFTFIHNGKLLAGLGLIGLHVGLTLLATALGFYFFKLVL .

What organism is Psychrobacter cryohalolentis and what are its unique properties?

Psychrobacter cryohalolentis K5 is a psychrotolerant (cold-tolerant) microorganism originally isolated from a Siberian permafrost cryopeg . This extremophile has adapted to survive in environments characterized by low temperatures, high salinity, and desiccation conditions. The bacterium has gained research interest due to its remarkable ability to maintain metabolic activity at temperatures as low as -12.5°C and its resistance to multiple environmental stressors . The SDWF2-4 strain has been noted for producing bacteriocins with obvious antibacterial activity, wide antibacterial spectrum, and good thermal stability, making it valuable for applications in food preservation .

How is the CrcB homolog classified in protein databases?

The CrcB homolog from Psychrobacter cryohalolentis is classified under UniProt accession number Q1QE84 . It belongs to the CrcB protein family, which includes membrane proteins associated with fluoride ion transport across cellular membranes. These proteins typically contain multiple transmembrane domains and are conserved across various bacterial species. The protein is functionally classified as a transport protein, specifically involved in ion homeostasis. Structurally, it features multiple alpha-helical transmembrane segments characteristic of integral membrane proteins involved in solute transport.

What are the optimal conditions for expressing recombinant Psychrobacter cryohalolentis CrcB protein in E. coli?

For optimal expression of recombinant P. cryohalolentis CrcB protein in E. coli, researchers should consider the following methodology:

• Expression vector selection: pET-based vectors with T7 promoter systems have shown high efficiency for membrane proteins like CrcB.

• Host strain: BL21(DE3) or C41(DE3) strains are preferred for membrane protein expression.

• Temperature: Induction at lower temperatures (16-20°C) rather than 37°C improves proper folding.

• Induction conditions: 0.1-0.5 mM IPTG for 16-20 hours at 18°C.

• Media composition: Use of TB (Terrific Broth) supplemented with 1% glucose during growth phase and switching to lactose during induction.

This approach minimizes the formation of inclusion bodies and increases the yield of correctly folded protein. For psychrophilic proteins like those from P. cryohalolentis, lower expression temperatures are particularly important to maintain proper folding and activity .

What purification strategy is most effective for obtaining high-purity CrcB homolog protein?

The most effective purification strategy for CrcB homolog protein involves multiple chromatographic steps:

• Initial solubilization: Membrane fractions should be solubilized using mild detergents such as n-dodecyl-β-D-maltopyranoside (DDM) at 1% concentration.

• Affinity chromatography: Using Ni-NTA resin for His-tagged proteins with an optimized buffer system:

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% DDM, 10% glycerol

  • Wash buffer: Same as binding buffer with 20-40 mM imidazole

  • Elution buffer: Same as binding buffer with 250-300 mM imidazole

• Size exclusion chromatography: As a polishing step using Superdex 200 with buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.03% DDM, 5% glycerol.

This protocol typically yields protein with >90% purity as determined by SDS-PAGE . Storage in 50% glycerol at -20°C/-80°C maintains protein stability for extended periods, though working aliquots should be kept at 4°C for no more than one week to prevent repeated freeze-thaw cycles .

How can researchers verify the functional activity of purified recombinant CrcB homolog?

To verify the functional activity of purified recombinant CrcB homolog, researchers should employ multiple complementary approaches:

• Fluoride ion transport assay:

  • Reconstitute purified protein in liposomes loaded with a pH-sensitive fluorescent dye

  • Monitor fluorescence changes upon addition of NaF to measure fluoride transport activity

  • Compare with control liposomes without protein incorporation

• Complementation assay:

  • Transform crcB knockout E. coli strains with plasmids expressing the recombinant CrcB

  • Test growth recovery in media containing fluoride at inhibitory concentrations

  • Quantify growth rates compared to wild-type and negative controls

• Binding assays:

  • Isothermal titration calorimetry (ITC) to measure direct binding of fluoride ions

  • Fluorescent probe displacement assays to determine binding affinity

A functional CrcB homolog should demonstrate fluoride transport activity in vitro and the ability to rescue fluoride sensitivity in crcB-deficient bacteria in vivo . Results should be analyzed for both kinetic parameters and substrate specificity to confirm proper protein folding and activity.

What is the predicted membrane topology of the CrcB homolog protein?

Based on bioinformatic analyses and comparative studies with related proteins, the CrcB homolog from P. cryohalolentis is predicted to have the following membrane topology:

The protein contains four transmembrane α-helical domains with both N and C termini likely oriented toward the cytoplasm . The transmembrane regions are predominantly hydrophobic, containing residues like leucine, isoleucine, and valine, while the connecting loops contain more polar and charged residues. This topology is consistent with its function as an ion channel or transporter protein.

How does the CrcB homolog from P. cryohalolentis differ structurally from homologs in mesophilic bacteria?

The CrcB homolog from P. cryohalolentis exhibits several structural adaptations compared to its mesophilic counterparts:

• Amino acid composition differences:

  • Increased proportion of glycine residues (higher flexibility)

  • Decreased proline content (lower rigidity)

  • Higher ratio of hydrophobic residues with small side chains

• Secondary structure elements:

  • Slightly shorter α-helical segments in transmembrane domains

  • More flexible loop regions connecting transmembrane segments

• Stability-flexibility balance:

  • Reduced number of rigid structural elements

  • Increased number of weak interactions rather than strong hydrogen bonds

What is the proposed mechanism for fluoride transport by the CrcB homolog?

The proposed mechanism for fluoride transport by the CrcB homolog involves several coordinated steps:

• Recognition and binding: Fluoride ions are recognized by positively charged residues (likely arginine or lysine) in the channel entrance.

• Channel formation: The four transmembrane helices assemble to form a narrow, hydrophilic pore through which fluoride ions can pass.

• Selectivity filter: A constriction region within the pore contains residues that selectively coordinate fluoride ions while excluding larger anions and restricting passage of water molecules.

• Transport facilitation: Protonation/deprotonation events of key residues likely drive conformational changes that facilitate ion movement through the channel.

• Release: The fluoride ion exits the channel on the opposite side of the membrane, completing the transport cycle.

This transport mechanism is likely energy-independent and operates through a channel-like process rather than an active transport mechanism. The protein's adaptation to cold environments may involve modifications to maintain flexibility of these key transport elements at low temperatures where molecular motion is reduced .

How can the CrcB homolog from Psychrobacter cryohalolentis be utilized in cold-adaptation studies?

The CrcB homolog from P. cryohalolentis serves as an excellent model system for studying protein cold-adaptation strategies through several research approaches:

• Comparative structural analysis:

  • Detailed comparison with mesophilic homologs to identify cold-adaptive modifications

  • Structure-function relationship studies at various temperatures (4°C to 30°C)

  • Identification of specific amino acid substitutions that confer cold activity

• Protein engineering applications:

  • Use as a scaffold for designing cold-active variants of industrial enzymes

  • Creation of chimeric proteins combining cold-adaptive domains with functional domains from mesophilic proteins

  • Site-directed mutagenesis to test specific cold-adaptation hypotheses

• Extremophile biology insights:

  • Understanding how psychrophilic organisms maintain membrane integrity and ion homeostasis at low temperatures

  • Elucidation of specific adaptations in membrane proteins that allow function in cold environments

This protein offers valuable insights into the molecular basis of cold adaptation in membrane proteins, which differ significantly from soluble proteins in their adaptation strategies. Its study can inform biotechnological applications requiring protein function at low temperatures, such as bioremediation in cold environments and food processing technologies .

What role does CrcB play in fluoride resistance and how can it be studied experimentally?

CrcB plays a critical role in fluoride resistance by exporting toxic fluoride ions from the cytoplasm, thereby maintaining cellular viability in fluoride-rich environments. This function can be studied through several experimental approaches:

• Growth inhibition assays:

  • Comparing growth of wild-type, crcB knockout, and crcB-overexpressing strains in media with varying fluoride concentrations

  • Measuring minimum inhibitory concentration (MIC) for fluoride

  • Time-kill kinetics in the presence of fluoride

• Fluoride accumulation measurements:

  • Using fluoride-specific electrodes to measure intracellular vs. extracellular fluoride concentrations

  • Fluorescent probes to visualize fluoride distribution in cells

  • Radioactive fluoride (18F) uptake and efflux studies

• Resistance mechanism characterization:

  • Transcriptomic analysis of fluoride stress response

  • Proteomic changes in response to fluoride exposure

  • Metabolomic assessment of fluoride toxicity mechanisms

The table below summarizes typical experimental results comparing fluoride sensitivity:

These studies provide insights into both the physiological role of CrcB and the potential applications for fluoride bioremediation or as targets for antimicrobial development .

What insights can be gained from studying psychrophilic membrane proteins like CrcB for astrobiology?

Studying psychrophilic membrane proteins like CrcB from P. cryohalolentis offers valuable insights for astrobiology, particularly regarding the potential for life in extremely cold extraterrestrial environments:

• Mars habitability models:

  • P. cryohalolentis has been directly studied under simulated Martian conditions

  • Understanding how membrane proteins maintain functionality under extreme conditions informs predictions about potential extraterrestrial life

  • Data shows P. cryohalolentis cells embedded in a medium/salt matrix (MSM) showed 1-3 orders of magnitude better survival under desiccating conditions

• Cellular adaptation to multiple extreme stressors:

  • P. cryohalolentis demonstrates survival mechanisms against combined stressors (cold, radiation, desiccation, low pressure)

  • Membrane protein stability is crucial for maintaining cellular viability under these conditions

  • Research shows survivability increases with UVC attenuation, though populations still decline several orders of magnitude over 8-hour exposure periods

• Biocontainment considerations:

  • Understanding psychrophilic organisms' adaptability informs planetary protection protocols

  • Research on P. cryohalolentis demonstrates that terrestrial microorganisms surviving interplanetary transit might compromise extraterrestrial life detection

These studies provide experimental data on how life might adapt to extreme environments beyond Earth, particularly ice-covered planets and moons like Europa or Enceladus. The protein-level adaptations observed in CrcB and other membrane proteins from P. cryohalolentis provide molecular markers that could be sought in extraterrestrial samples .

What are common challenges in expressing and purifying CrcB homolog and how can they be addressed?

Researchers frequently encounter several challenges when working with the CrcB homolog protein. Here are the most common issues and recommended solutions:

• Low expression yields:

  • Problem: Membrane proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for E. coli, use specialized expression strains (C41/C43), and reduce expression temperature to 16-18°C

  • Advanced approach: Consider cell-free expression systems specifically optimized for membrane proteins

• Protein aggregation and inclusion body formation:

  • Problem: Improper folding leading to non-functional protein

  • Solution: Add solubilizing agents (0.5-1% glycerol) during expression, use fusion partners (MBP, SUMO)

  • Advanced approach: Express protein with specific chaperones like GroEL/GroES

• Inefficient membrane extraction:

  • Problem: Insufficient solubilization of target protein

  • Solution: Screen different detergents (DDM, LDAO, FC-12) at various concentrations

  • Optimization strategy: Use a two-step extraction with increasing detergent concentrations

• Protein instability during purification:

  • Problem: Rapid activity loss after extraction

  • Solution: Include stabilizing agents (glycerol 10%, specific lipids) in all buffers

  • Advanced approach: Reconstitute into nanodiscs or liposomes immediately after initial purification

Implementing these strategies significantly improves the quality and yield of functional CrcB homolog protein. Most researchers report 2-5 fold increases in functional protein yield when optimizing these parameters specifically for psychrophilic membrane proteins .

How can researchers optimize functional assays to accurately measure CrcB activity at low temperatures?

Optimizing functional assays for CrcB activity at low temperatures requires specific modifications to standard protocols:

• Temperature equilibration considerations:

  • Pre-equilibrate all buffers and reaction components at the target temperature for at least 30 minutes

  • Use temperature-controlled microplate readers with bottom reading capability

  • Implement temperature calibration controls within each assay plate

• Fluoride transport assay optimization:

  • Modify standard liposome-based assays using temperature-sensitive fluorescent dyes (e.g., BCECF instead of HPTS)

  • Increase lipid fluidity by incorporating unsaturated phospholipids (18:1 PC/PE) in liposome preparation

  • Extend measurement times to account for slower kinetics at low temperatures

• Control experiments essential for cold-temperature assays:

  • Include positive controls with known cold-active transporters

  • Perform parallel experiments at multiple temperatures (4°C, 15°C, 25°C) to establish temperature dependency

  • Use heat-inactivated protein as negative control rather than empty liposomes alone

• Data analysis adjustments:

  • Apply temperature-correction factors to kinetic parameters

  • Use initial velocity measurements rather than endpoint readings

  • Implement non-linear regression models that account for temperature effects on protein dynamics

This methodological framework ensures accurate measurement of CrcB activity at temperatures relevant to its native functioning (4-15°C). When correctly optimized, these assays can detect as little as 20-30% changes in transport activity, which is essential for structure-function studies of cold-adapted membrane proteins .

What quality control measures should be employed to ensure the recombinant protein retains native-like structure and function?

Comprehensive quality control measures for the recombinant CrcB homolog protein should include:

• Purity and homogeneity assessment:

  • SDS-PAGE analysis confirming >90% purity

  • Size exclusion chromatography to verify monodispersity in solution

  • Dynamic light scattering to detect aggregation

  • Mass spectrometry to confirm accurate molecular weight and sequence integrity

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to assess secondary structure composition

  • Thermal shift assays to determine stability at different temperatures

  • Limited proteolysis to confirm proper folding (properly folded membrane proteins show characteristic digestion patterns)

• Functional validation:

  • Fluoride binding assays using isothermal titration calorimetry

  • Transport activity in reconstituted systems (proteoliposomes)

  • Complementation of crcB-deficient bacterial strains

• Critical quality attributes to monitor:

  • Thermal stability (Tm) should be consistent between batches (±2°C)

  • α-helical content from CD should match theoretical predictions (±5%)

  • Specific activity should be within 80-120% of reference standards

These quality control measures should be applied systematically to each protein preparation. For cold-adapted proteins like CrcB from P. cryohalolentis, particular attention should be paid to testing functional activity at both the organism's optimal growth temperature (~4-15°C) and standard laboratory temperatures to ensure the recombinant protein exhibits the expected cold adaptation properties .

How might genetic engineering approaches be used to study structure-function relationships in CrcB homolog?

Advanced genetic engineering approaches offer powerful tools for elucidating structure-function relationships in the CrcB homolog protein:

• Site-directed mutagenesis strategies:

  • Alanine-scanning mutagenesis of putative channel-forming residues

  • Conservative vs. non-conservative substitutions at key positions to identify critical amino acids

  • Introduction of reporter groups (e.g., cysteine residues for fluorescent labeling) at specific locations

• Domain swapping experiments:

  • Creating chimeric proteins between psychrophilic and mesophilic CrcB homologs

  • Swapping transmembrane domains to identify cold-adaptation determinants

  • Reassigning loop regions to determine their role in temperature adaptation

• Recombineering approaches:

  • Using homologous recombination techniques to introduce mutations directly into the P. cryohalolentis chromosome

  • Creating genomic libraries with random mutations in the crcB gene

  • Applying bacterial recombineering systems as described by Thomason et al. for precise genetic modifications

• Advanced protein engineering:

  • Directed evolution experiments under selective pressure (fluoride presence and low temperature)

  • Creation of protein fusions with split fluorescent proteins to study oligomerization

  • Introduction of unnatural amino acids at specific positions to probe function

These approaches can be combined with computational modeling to formulate and test hypotheses about the molecular basis of CrcB function, particularly focusing on the adaptations that allow activity at low temperatures. The resulting structure-function insights may guide the rational design of cold-active membrane proteins for biotechnological applications .

What comparative genomic and proteomic approaches could reveal about CrcB evolution in psychrophilic bacteria?

Comprehensive comparative genomic and proteomic approaches can provide significant insights into CrcB evolution in psychrophilic bacteria:

• Phylogenomic analysis framework:

  • Construction of phylogenetic trees based on CrcB sequences from psychrophilic, mesophilic, and thermophilic organisms

  • Calculation of dN/dS ratios to identify positions under positive selection in cold-adapted lineages

  • Ancestral sequence reconstruction to trace the evolutionary trajectory of cold adaptation

• Genomic context analysis:

  • Comparison of operonic structure and gene neighborhoods across bacterial lineages

  • Identification of co-evolving genes that may functionally interact with CrcB

  • Assessment of horizontal gene transfer events in the acquisition of fluoride resistance mechanisms

• Structural proteomics approaches:

  • Homology modeling based on available CrcB crystal structures

  • Molecular dynamics simulations at different temperatures to identify adaptations in protein dynamics

  • Comparison of predicted protein flexibility profiles between psychrophilic and mesophilic homologs

• Systematic analysis of amino acid substitution patterns:

  • Identification of consistent substitution patterns across independently evolved psychrophilic lineages

  • Quantification of specific physiochemical property changes (charge, hydrophobicity, etc.)

  • Statistical analysis of amino acid frequencies at equivalent positions in the protein

These approaches can reveal whether cold adaptation in CrcB evolved multiple times independently (convergent evolution) or was inherited from a common cold-adapted ancestor (divergent evolution). The patterns identified can inform broader questions about molecular evolution in extreme environments and potentially identify universal principles of protein cold adaptation .

What are the potential implications of studying CrcB function for developing novel antimicrobial strategies?

The study of CrcB function has significant implications for developing novel antimicrobial strategies through several potential mechanisms:

• CrcB as a direct antimicrobial target:

  • CrcB inhibitors could potentiate the toxicity of fluoride ions against pathogenic bacteria

  • Structural insights from the P. cryohalolentis CrcB could guide rational design of inhibitors

  • Combination therapy approaches coupling CrcB inhibitors with fluoride-containing compounds

• Psychrobacter-derived antimicrobial compounds:

  • Research shows that P. cryohalolentis SDWF2-4 produces bacteriocins with broad-spectrum antibacterial activity

  • These bacteriocins demonstrate good thermal stability and activity across varied conditions

  • The molecular basis of their activity could be characterized and optimized for therapeutic applications

• Novel antibiotic discovery strategies:

  • Understanding bacterial ion homeostasis mechanisms can reveal new vulnerability points

  • Comparative analysis between pathogenic and non-pathogenic species can identify selective targets

  • Cold-active antimicrobial compounds may have advantages for treating infections in relatively cooler body sites

• Resistance mechanism insights:

  • Study of CrcB and related transporters may reveal resistance mechanisms to fluoride-containing drugs

  • Pre-emptive engineering of antimicrobials to avoid such resistance mechanisms

  • Development of adjuvants that specifically target transport-based resistance

The detailed understanding of membrane transport proteins like CrcB can guide the development of novel antimicrobial compounds with mechanisms distinct from conventional antibiotics, potentially addressing the growing challenge of antimicrobial resistance. Additionally, the bacteriocins produced by P. cryohalolentis represent a promising source of new antimicrobial agents with activity preserved across a wide temperature range .