Recombinant Sulfurihydrogenibium sp. Protein CrcB homolog (crcB)

What is Sulfurihydrogenibium sp. and where is it naturally found?

Sulfurihydrogenibium species are thermophilic bacteria typically found in high-temperature environments such as hot springs and geothermal habitats. These microorganisms belong to the phylum Aquificae and are known for their ability to thrive in extreme conditions. Species like Sulfurihydrogenibium yellowstonense, as the name suggests, were initially isolated from the Yellowstone National Park hot springs . These bacteria are of particular interest to researchers due to their thermostable enzymes that can function under harsh conditions, making them valuable for various biotechnological applications including carbon sequestration processes .

What is the function of the CrcB protein homolog in Sulfurihydrogenibium sp.?

The CrcB protein homolog in Sulfurihydrogenibium species functions primarily as a putative fluoride ion transporter . This membrane protein facilitates the transport of fluoride ions across cellular membranes, which is essential for maintaining ionic homeostasis, particularly in environments with varying fluoride concentrations. The protein typically consists of approximately 124 amino acids, as seen in homologous proteins from related species . The fluoride transport function is critical for bacterial survival as fluoride ions can be toxic at high intracellular concentrations by inhibiting enzymes involved in phosphoryl transfer reactions and glycolysis.

What expression systems are most effective for recombinant production of Sulfurihydrogenibium sp. CrcB protein?

For recombinant production of Sulfurihydrogenibium sp. CrcB protein, Escherichia coli expression systems have proven effective, as demonstrated in similar experiments with related proteins . When designing an expression system, considerations should include:

• Vector selection: pET-based vectors with T7 promoters often yield high expression levels for bacterial proteins.

• E. coli strain optimization: BL21(DE3) or Rosetta strains are recommended, especially for proteins with rare codons.

• Fusion tags: N-terminal His-tags facilitate purification while potentially improving solubility .

• Expression conditions: For thermophilic proteins, induction at higher temperatures (30-37°C) may improve proper folding.

To enhance soluble protein expression, co-expression with chaperones such as GroELS has shown significant improvement (up to 1.4-fold increase) in soluble protein yield for other Sulfurihydrogenibium proteins . Additionally, incorporating fusion tags like TrxA has demonstrated a 2.67-fold enhancement in soluble protein production in similar thermophilic proteins from this genus .

What purification protocol is recommended for recombinant Sulfurihydrogenibium sp. CrcB protein?

The recommended purification protocol for recombinant Sulfurihydrogenibium sp. CrcB protein with His-tag involves a multi-step process:

• Cell lysis: Sonication or pressure-based lysis in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Initial purification: Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin:

  • Binding: Load lysate on pre-equilibrated Ni-NTA column

  • Washing: Remove non-specific proteins with 20-50 mM imidazole

  • Elution: Collect target protein with 250-300 mM imidazole

• Secondary purification: Size exclusion chromatography to enhance purity (>90% as determined by SDS-PAGE) .

• Buffer exchange: Dialysis against a stabilizing buffer (similar to the storage buffer mentioned in source ).

• Concentration and storage: Concentrate using centrifugal filters and store at -20°C/-80°C in a buffer containing 6% trehalose to maintain stability .

For membrane proteins like CrcB, the addition of appropriate detergents (e.g., 0.03% DDM or 0.5% CHAPS) during lysis and purification steps is crucial for maintaining protein solubility and native conformation.

How can I assess the structural integrity and purity of the purified CrcB protein?

Assessment of structural integrity and purity of purified CrcB protein should involve multiple complementary techniques:

• Purity assessment:

  • SDS-PAGE: Should reveal a single band at approximately 15-16 kDa (for a 124 amino acid protein plus His-tag)

  • Western blotting: Using anti-His antibodies to confirm identity

  • Mass spectrometry: For precise molecular weight determination and sequence verification

• Structural integrity analysis:

  • Circular dichroism (CD) spectroscopy: To evaluate secondary structure elements

  • Thermal shift assay: To determine protein stability and melting temperature

  • Dynamic light scattering (DLS): To assess homogeneity and detect aggregation

• Functional verification:

  • Fluoride ion transport assays: Using fluoride-sensitive probes or electrodes

  • Liposome reconstitution experiments: To confirm membrane integration and transport activity

Proteins should achieve greater than 90% purity as determined by SDS-PAGE before proceeding to functional studies . For membrane proteins like CrcB, additional detergent screening may be necessary to identify conditions that maintain native conformation.

What storage conditions maximize the stability of recombinant CrcB protein?

To maximize stability of recombinant CrcB protein from Sulfurihydrogenibium sp., implement the following storage conditions:

• Buffer composition:

  • Tris/PBS-based buffer, pH 8.0

  • 6% Trehalose as a cryoprotectant

  • Consider adding 5-50% glycerol (final concentration) for long-term storage

• Storage temperature:

  • Short-term (up to one week): 4°C

  • Long-term: -20°C to -80°C in aliquots to avoid repeated freeze-thaw cycles

• Protein concentration:

  • Reconstitute lyophilized protein to 0.1-1.0 mg/mL in deionized sterile water

  • Higher concentrations may be used if aggregation is not observed

• Additional considerations:

  • Briefly centrifuge vials prior to opening

  • For membrane proteins like CrcB, consider adding a stabilizing detergent at concentrations above the critical micelle concentration

  • Flash-freeze aliquots in liquid nitrogen before transferring to -80°C storage

Repeated freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and activity .

What experimental designs are most appropriate for studying CrcB transporter function?

For studying CrcB transporter function, the following experimental designs are most appropriate:

• Fluoride transport assays:

  • Liposome-based assays: Reconstitute purified CrcB in liposomes containing fluoride-sensitive fluorescent probes

  • Whole-cell assays: Express CrcB in cells lacking endogenous fluoride transporters and measure fluoride uptake/efflux

  • Patch-clamp electrophysiology: For direct measurement of ion conductance in controlled membrane environments

• Randomized Complete Block Design (RCBD) for comparative studies:

  • When comparing multiple CrcB variants or conditions, RCBD helps control for experimental variation

  • Blocks should contain uniform experimental units to minimize within-block variation

  • Each treatment (e.g., protein variant) should appear at least once per replicate

  • This design is advantageous when experimental conditions cannot be completely controlled, as it separates treatment effects from block effects

• For mutagenesis studies:

  • Systematic alanine scanning to identify essential residues

  • Site-directed mutagenesis of conserved residues based on sequence alignments

  • Chimeric protein construction to identify functional domains

Statistical analysis should include appropriate controls and sufficient replicates (minimum n=3) to ensure reliability and validity of results .

How can I investigate the thermostability properties of Sulfurihydrogenibium sp. CrcB protein?

To investigate the thermostability properties of Sulfurihydrogenibium sp. CrcB protein, implement the following methodological approaches:

• Differential scanning calorimetry (DSC):

  • Measure the heat capacity changes during protein unfolding

  • Determine the melting temperature (Tm) under various buffer conditions

  • Compare with other membrane proteins to establish relative stability benchmarks

• Thermal shift assays:

  • Utilize fluorescent dyes (e.g., SYPRO Orange) that bind to hydrophobic regions exposed during unfolding

  • Monitor fluorescence changes during gradual temperature increases

  • Calculate Tm values under different experimental conditions

• Activity assays at varied temperatures:

  • Measure fluoride transport activity at temperature ranges from 30-80°C

  • Determine the temperature optimum and activity half-life at elevated temperatures

  • Create Arrhenius plots to calculate activation energy for transport

• Enhancement strategies assessment:

  • Co-expression with chaperones like GroELS, which has shown to intensify thermostability at 60°C for other Sulfurihydrogenibium proteins

  • Evaluate structural changes using circular dichroism spectroscopy before and after thermal stress

• Long-term stability studies:

  • Incubate protein samples at various temperatures (40-80°C)

  • Remove aliquots at timed intervals for activity and structural analysis

  • Determine half-life at different temperatures to create stability profiles

These approaches provide comprehensive data on thermostability parameters, which are particularly relevant given that Sulfurihydrogenibium species are thermophilic bacteria with adaptations to high-temperature environments .

What techniques can be used to study CrcB protein interactions with other cellular components?

To study CrcB protein interactions with other cellular components, utilize these advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against tagged CrcB to pull down protein complexes

  • Identify interaction partners via mass spectrometry

  • Verify specific interactions with western blotting for suspected partners

• Pull-down assays:

  • Utilize His-tagged CrcB as bait protein with Ni-NTA resin

  • Incubate with cellular lysates to capture interacting proteins

  • Elute and analyze bound proteins by mass spectrometry

• Crosslinking coupled with mass spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and analyze by tandem mass spectrometry

  • Identify interaction interfaces and structural constraints

• Förster Resonance Energy Transfer (FRET):

  • Generate fluorescently labeled CrcB and potential partners

  • Observe energy transfer as indication of proximity (<10 nm)

  • Perform in live cells or with purified components

• Surface Plasmon Resonance (SPR):

  • Immobilize purified CrcB on sensor chips

  • Measure real-time binding kinetics with potential partners

  • Determine association/dissociation rates and binding affinities

• Bacterial two-hybrid systems:

  • Particularly useful for membrane proteins like CrcB

  • Screen libraries for novel interaction partners

  • Validate with secondary assays such as split-GFP complementation

These methods should be applied in combination to build a comprehensive interaction network and validate findings across multiple experimental approaches.

How does the homologous recombination approach improve the expression of functional CrcB protein?

Homologous recombination approaches can significantly improve the expression of functional CrcB protein through several mechanisms:

Implementing these approaches requires careful experimental design with appropriate controls and can significantly improve the yield of functional CrcB protein.

What are the challenges in studying the structure-function relationship of membrane proteins like CrcB, and how can they be addressed?

Studying structure-function relationships of membrane proteins like CrcB presents several specific challenges that require specialized approaches:

Challenges and Solutions Table:

To address these challenges comprehensively:

• Structural approaches:

  • Cryo-electron microscopy circumvents crystallization requirements

  • NMR spectroscopy with isotopically labeled proteins for dynamic studies

  • Molecular dynamics simulations to predict conformational changes

• Functional mapping:

  • Systematic cysteine scanning mutagenesis to identify essential residues

  • Fluoride transport assays using sensitive electrodes or fluorescent probes

  • Evolutionary analysis to identify conserved functional motifs

• Stability enhancement:

  • Thermostability assays at varying temperatures (as relevant for thermophilic Sulfurihydrogenibium sp.)

  • Strategic disulfide bond engineering to stabilize tertiary structure

  • Co-expression with GroELS chaperones to improve folding and stability

These integrated approaches have successfully revealed structure-function relationships in other challenging membrane transport proteins and can be adapted specifically for CrcB study.

How can recombinant Sulfurihydrogenibium sp. CrcB protein be utilized in biotechnological applications?

Recombinant Sulfurihydrogenibium sp. CrcB protein offers several promising biotechnological applications, leveraging its unique properties as a thermostable fluoride ion transporter:

• Bioremediation technologies:

  • Development of engineered bacteria expressing CrcB for fluoride contamination removal from water sources

  • Creation of bioreactor systems with immobilized CrcB-expressing cells for continuous fluoride filtration

  • Design of biosensors incorporating CrcB for fluoride detection in environmental monitoring

• Industrial process applications:

  • Utilization in aluminum manufacturing or glass etching processes where fluoride management is critical

  • Development of enzyme immobilization techniques similar to polyacrylamide gel entrapment used for other Sulfurihydrogenibium proteins

  • Creation of fluoride-resistant industrial strains through CrcB expression

• Biomaterial development:

  • Design of fluoride-responsive biomaterials using CrcB incorporated into artificial membranes

  • Development of ion-selective membranes for separation technologies

  • Creation of bioelectronic interfaces utilizing ion transport properties

• Research tools:

  • Utilization as a thermostable model system for membrane protein research

  • Development of CrcB variants as reporter systems for fluoride presence in live-cell imaging

  • Application in directed evolution platforms to study membrane protein adaptation

• Thermostable protein engineering platforms:

  • Using the thermostability properties of Sulfurihydrogenibium proteins (functional at 60°C ) as scaffolds for protein engineering

  • Development of chimeric transporters with novel ion specificities

  • Creation of protein libraries for stability-function relationship studies

For biotechnological applications requiring long-term stability, the demonstrated maintenance of activity after multiple reuse cycles (as shown with other Sulfurihydrogenibium proteins retaining nearly 30% activity after 5 cycles ) represents a significant advantage for industrial deployment.

What are common issues encountered during recombinant expression of CrcB protein and how can they be resolved?

Common issues during recombinant expression of CrcB protein and their resolution strategies include:

• Poor expression levels:

  • Issue: Low protein yield despite optimized conditions

  • Resolution: Implement codon optimization for E. coli; switch to stronger promoters (T7); co-express with GroELS chaperones which improved similar protein yields by 1.4-fold ; lower induction temperature to 18-25°C with extended expression time

• Protein insolubility/inclusion body formation:

  • Issue: Target protein predominantly in insoluble fraction

  • Resolution: Utilize fusion tags like TrxA, which enhanced soluble protein production 2.67-fold in similar proteins ; add mild detergents during cell lysis; implement auto-induction media with slow protein expression; use specialized E. coli strains (C41/C43) designed for membrane proteins

• Protein degradation:

  • Issue: Multiple bands or smears on SDS-PAGE

  • Resolution: Add protease inhibitors during purification; reduce purification time; keep samples consistently cold; consider adding stabilizers like trehalose (6%) or glycerol

• Improper membrane insertion:

  • Issue: Protein expresses but lacks transport activity

  • Resolution: Verify proper membrane targeting with fractionation experiments; adjust signal sequence if necessary; evaluate lipid composition of expression host

• Low purification yield:

  • Issue: Significant protein loss during purification steps

  • Resolution: Optimize imidazole concentrations during IMAC purification; test alternative purification methods; adjust detergent concentrations; implement on-column refolding if necessary

• Loss of activity after storage:

  • Issue: Reduced functional activity after freezing

  • Resolution: Store at -80°C with 50% glycerol ; aliquot to avoid freeze-thaw cycles; lyophilize with appropriate cryoprotectants

Implementing a systematic troubleshooting approach with controlled variables and proper documentation will help identify optimal conditions for each specific CrcB construct.

How can I optimize the experimental design to study CrcB function in different environmental conditions?

To optimize experimental design for studying CrcB function across various environmental conditions, implement the following strategies:

• Randomized Complete Block Design (RCBD) implementation:

  • Group similar experimental units into blocks to minimize within-block variation

  • Randomize treatments within each block independently

  • This design is particularly valuable when experimental conditions cannot be completely controlled

  • RCBD allows for more precise comparisons than completely randomized designs when properly implemented

• Factorial experimental design:

  • Test multiple variables simultaneously (e.g., pH, temperature, fluoride concentration)

  • Identify interaction effects between variables that might be missed in single-factor experiments

  • Use a partial factorial design if resource constraints exist

  • Example design matrix for a CrcB function study:

• Response surface methodology (RSM):

  • Create a mathematical model of how experimental variables affect CrcB function

  • Identify optimal conditions through systematic testing

  • Reduce experimental runs while maintaining statistical power

  • Particularly useful for optimizing multiple conditions simultaneously

• Controls and standardization:

  • Include negative controls (empty vector or inactive mutant)

  • Use internal standards for quantification

  • Standardize protein concentration across experiments

  • Normalize data to account for batch-to-batch variation

• Specialized approaches for membrane proteins:

  • Test function in different membrane mimetics (liposomes, nanodiscs)

  • Systematically vary lipid composition to identify optimal environments

  • Use fluoride-sensitive probes with different spectral properties for multiplexed assays

Statistical analysis should include appropriate tests for the experimental design chosen, with power analysis conducted before experiments to determine adequate sample sizes .

What analytical methods are most sensitive for detecting subtle changes in CrcB structure and function?

For detecting subtle changes in CrcB structure and function, the following high-sensitivity analytical methods are recommended:

• High-resolution structural techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Detects changes in protein dynamics and solvent accessibility with peptide-level resolution

  • Single-particle cryo-electron microscopy: Can resolve structural differences in different functional states

  • Solid-state NMR spectroscopy: Particularly valuable for membrane proteins in native-like environments

  • Molecular dynamics simulations: Complement experimental approaches by predicting structural fluctuations

• Functional analysis with high sensitivity:

  • Single-molecule fluorescence microscopy: Observe individual transport events without population averaging

  • Patch-clamp electrophysiology: Detect subtle changes in ion conductance properties

  • Fluoride-selective microelectrodes: Measure transport rates with high temporal resolution

  • Isothermal titration calorimetry (ITC): Quantify thermodynamic parameters of fluoride binding

• Biophysical characterization techniques:

  • Microscale thermophoresis (MST): Detect binding interactions with minimal protein consumption

  • Differential scanning fluorimetry with nanoDSF: Measure protein stability changes without dyes

  • Analytical ultracentrifugation (AUC): Assess oligomeric state changes and complex formation

  • Small-angle X-ray scattering (SAXS): Monitor conformational changes in solution

• Advanced spectroscopic methods:

  • Site-directed spin labeling with electron paramagnetic resonance (EPR): Measure distances between specific residues

  • Förster resonance energy transfer (FRET) with site-specific fluorophores: Track conformational changes during function

  • Tryptophan fluorescence spectroscopy: Monitor local environmental changes if tryptophan residues are present or engineered

• Comparative analysis approaches:

  • Deep mutational scanning: Systematically assess the impact of all possible mutations

  • Evolutionary coupling analysis: Identify co-evolving residues important for structure and function

  • Thermal shift assays at different temperatures: Particularly relevant for thermostable proteins from Sulfurihydrogenibium sp.

These methods can be combined in an integrative structural biology approach to build a comprehensive understanding of CrcB structure-function relationships.

What are the future research directions for Sulfurihydrogenibium sp. CrcB protein studies?

Future research directions for Sulfurihydrogenibium sp. CrcB protein studies should focus on several promising avenues:

• Structural biology advancements:

  • Determining high-resolution structures of CrcB in different conformational states

  • Mapping the fluoride ion pathway through the transporter

  • Elucidating the molecular basis for thermostability in this extremophile protein

• Functional characterization extensions:

  • Investigating potential secondary transport functions beyond fluoride

  • Characterizing the energetics and kinetics of transport mechanism

  • Exploring regulation of CrcB expression and activity in native contexts

• Comparative genomics and evolution:

  • Analyzing CrcB homologs across extremophiles to identify convergent adaptations

  • Reconstructing the evolutionary history of fluoride transporters

  • Using ancestral sequence reconstruction to understand thermostability acquisition

• Biotechnological applications development:

  • Engineering enhanced fluoride biosensors based on CrcB

  • Developing bioremediation strategies for fluoride contamination

  • Creating thermostable membrane protein expression platforms

• Integration with systems biology:

  • Understanding CrcB's role in the broader context of Sulfurihydrogenibium metabolism

  • Mapping interaction networks through proteome-wide studies

  • Developing predictive models of fluoride homeostasis

These research directions will benefit from interdisciplinary approaches combining structural biology, biochemistry, biophysics, computational biology, and synthetic biology methodologies to comprehensively understand this unique thermophilic membrane protein.

How do research findings on CrcB homologs contribute to our understanding of extremophile adaptation mechanisms?

Research findings on CrcB homologs provide valuable insights into extremophile adaptation mechanisms through several key contributions:

• Molecular basis of thermostability:

  • Studies of Sulfurihydrogenibium CrcB and other proteins from this genus reveal structural adaptations that maintain functional integrity at high temperatures

  • Similar to findings with Sulfurihydrogenibium yellowstonense carbonic anhydrase, which demonstrated enhanced thermostability at 60°C , CrcB likely contains specialized structural features

  • These adaptations often include increased hydrophobic core packing, surface charge optimization, and strategic disulfide bonds

• Ion homeostasis in extreme environments:

  • CrcB's role as a fluoride transporter illuminates how extremophiles maintain ionic balance in challenging conditions

  • Understanding these mechanisms provides insights into fundamental aspects of cellular adaptation to environmental stressors

  • The fluoride defense system represents a conserved strategy across thermophilic bacteria for managing geothermal fluoride exposure

• Evolutionary strategies:

  • Comparative analysis of CrcB homologs across extremophiles reveals evolutionary pathways for membrane protein adaptation

  • Horizontal gene transfer patterns may explain distribution of specialized transporters in extremophile communities

  • Sequence conservation analysis highlights essential functional regions versus adaptable segments

• Membrane biology in extreme conditions:

  • CrcB studies contribute to understanding how membrane proteins function in high-temperature environments

  • Lipid-protein interactions in thermophiles likely differ significantly from mesophilic counterparts

  • These insights have broader implications for membrane biology across domains of life

• Biotechnological applications:

  • Knowledge derived from CrcB and similar proteins enables development of thermostable enzymes and transport systems

  • Similar to the enhanced recombinant expression strategies developed for Sulfurihydrogenibium yellowstonense carbonic anhydrase , CrcB research contributes to a toolbox for engineering extremophile proteins