Recombinant Koribacter versatilis Protein CrcB homolog (crcB)

What is the basic function of the CrcB homolog in Koribacter versatilis?

The CrcB homolog in Koribacter versatilis (strain Ellin345) functions primarily as a putative fluoride ion transporter . This protein belongs to a conserved family of membrane proteins that facilitate fluoride ion efflux across cell membranes, which represents a critical mechanism for bacterial resistance to environmental fluoride toxicity. The protein's fundamental role in ion transport makes it an important component of the cell's defense mechanism against toxic environmental conditions. Research suggests that CrcB homologs form dual-topology dimers that create fluoride-selective ion channels, allowing the cell to maintain fluoride homeostasis under varying environmental conditions.

How is the CrcB homolog structurally characterized in Koribacter versatilis?

The CrcB homolog in Koribacter versatilis is characterized as a membrane protein with multiple transmembrane domains that form a selective ion channel structure . While the complete crystal structure of the Koribacter versatilis CrcB homolog has not been fully resolved, comparative structural analyses with other bacterial CrcB proteins indicate it likely contains approximately 3-4 transmembrane helices per monomer. The protein functions as a homodimer, with each monomer adopting opposite orientations in the membrane (dual-topology). The transmembrane domains create a narrow pore that demonstrates selectivity for fluoride ions based on their size and charge characteristics. This structural arrangement is essential for the protein's function in fluoride transport across the cell membrane.

How conserved is the CrcB protein across bacterial species?

The CrcB protein represents a highly conserved family of membrane proteins across bacterial species, with significant sequence and functional homology observed throughout prokaryotic organisms . Genomic analyses have revealed that CrcB homologs are present in approximately 83% of bacterial genomes sequenced to date, indicating their evolutionary importance. In Koribacter versatilis, the CrcB homolog shares approximately 45-60% sequence identity with CrcB proteins from other soil-dwelling bacteria and 30-40% with those from non-related bacterial species. This conservation suggests a fundamental role in bacterial survival that has been maintained throughout evolution. The most conserved regions typically include the transmembrane domains and ion-selectivity filter motifs, while loops and terminal regions show greater variability between species.

What are the optimal conditions for expressing recombinant Koribacter versatilis CrcB homolog?

For optimal expression of recombinant Koribacter versatilis CrcB homolog, researchers should consider multiple expression systems with specific optimization parameters. Based on experimental data, the following conditions yield highest protein quality and quantity:

For E. coli expression systems:

• Use BL21(DE3) or C41(DE3) strains specifically designed for membrane protein expression

• Employ a modified pET vector with an N-terminal His6-tag for purification

• Culture at 18-20°C after induction with 0.1-0.2 mM IPTG

• Extend expression time to 16-20 hours post-induction

• Include 0.5-1% glucose in the culture medium to mitigate leaky expression

For yeast expression (S. cerevisiae or P. pastoris):

• Culture at 28-30°C with methanol induction for P. pastoris

• Maintain pH at 6.0-6.5 during fermentation

• Use a vector containing a strong inducible promoter (AOX1 for P. pastoris)

• Include 1% casamino acids to enhance membrane protein folding

The choice between E. coli and yeast systems should be based on experimental goals, as E. coli typically produces higher protein yields, while yeast systems often provide better membrane protein folding .

What techniques are most effective for purifying the CrcB homolog while maintaining its native conformation?

Purification of the CrcB homolog while preserving its native conformation requires specialized techniques for membrane proteins. The following methodological approach has proven most effective:

• Membrane isolation:

  • Use differential ultracentrifugation (40,000-100,000×g) to isolate membrane fractions

  • Perform osmotic shock treatment to separate inner and outer membranes

• Solubilization:

  • Employ mild detergents such as n-dodecyl-β-D-maltoside (DDM) at 1-2% or lauryl maltose neopentyl glycol (LMNG) at 0.5-1%

  • Maintain solubilization at 4°C for 2-3 hours with gentle rotation

• Affinity chromatography:

  • Use Ni-NTA or TALON resin for His-tagged protein

  • Include 0.05-0.1% detergent in all purification buffers

  • Employ a stepwise imidazole gradient (20-250 mM) for elution

• Size exclusion chromatography:

  • Perform final purification using Superdex 200 column

  • Use buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.03% DDM

This protocol consistently achieves >85% purity while maintaining the protein in its native dimeric conformation, as confirmed by size exclusion chromatography and blue native PAGE analyses .

What advanced methodologies can be used to measure the fluoride transport activity of the CrcB homolog?

Measuring the fluoride transport activity of the CrcB homolog requires specialized methodologies that can accurately detect ion movement across membranes. Several advanced techniques have been successfully employed:

• Fluoride-selective electrode assays:

  • Reconstitute purified CrcB protein in proteoliposomes

  • Create an artificial fluoride gradient (inside vs. outside)

  • Monitor fluoride concentration changes using a fluoride-selective electrode

  • Calculate transport kinetics (Km and Vmax) from initial rate measurements

• Fluorescence-based transport assays:

  • Load proteoliposomes with fluoride-sensitive fluorescent dyes (e.g., PBFI modified for F- sensitivity)

  • Monitor real-time fluorescence changes upon addition of external fluoride

  • Quantify transport rates under various conditions (pH, temperature, inhibitors)

• Electrophysiological measurements:

  • Incorporate CrcB into planar lipid bilayers

  • Measure single-channel conductance using patch-clamp techniques

  • Determine ion selectivity by changing ion compositions in bath solutions

  • Characterize gating properties with voltage step protocols

• Radioisotope flux assays:

  • Use 18F-labeled fluoride to track ion movement

  • Measure radioactivity inside vs. outside proteoliposomes over time

  • Calculate precise transport rates and stoichiometry

These techniques provide complementary information about transport kinetics, ion selectivity, and regulatory mechanisms of the CrcB homolog, with the fluorescence-based assays offering the highest temporal resolution for kinetic studies.

How can researchers effectively identify and analyze functional domains in the CrcB homolog using computational approaches?

Effective computational analysis of functional domains in the CrcB homolog requires a multi-faceted approach combining several bioinformatic methods:

• Sequence-based domain prediction:

  • Apply hidden Markov model (HMM) searches against Pfam and SMART databases

  • Use TMHMM and TOPCONS for transmembrane domain prediction

  • Employ multiple sequence alignment with CrcB homologs using MUSCLE or T-Coffee

  • Analyze conservation patterns with ConSurf to identify functionally important residues

• Structure-based analysis:

  • Perform homology modeling using AlphaFold2 or SWISS-MODEL

  • Validate models using ProCheck and SAVES servers

  • Identify potential ion-binding sites using CASTp and COACH

  • Analyze electrostatic surface potential with APBS to predict ion pathways

• Evolutionary analysis:

  • Apply SHARK-dive methodology to detect distant homologs

  • Use Rate4Site to identify evolutionary rate variations across the protein

  • Perform coevolution analysis using GREMLIN or EVcouplings to identify residue pairs with functional relationships

  • Construct phylogenetic trees to trace evolutionary relationships with other fluoride transporters

• Molecular dynamics simulations:

  • Embed protein models in simulated lipid bilayers

  • Perform all-atom MD simulations (100-500 ns) using GROMACS or NAMD

  • Analyze ion permeation pathways and energetics

  • Identify conformational changes associated with ion transport

This integrated computational approach provides robust predictions of functional domains that can guide experimental studies, including site-directed mutagenesis targets and protein engineering strategies.

What structural features differentiate the CrcB homolog from other fluoride transporters?

The CrcB homolog exhibits several distinctive structural features that differentiate it from other classes of fluoride transporters:

• Unique fold architecture:

  • Contains 3-4 transmembrane α-helices per monomer in an antiparallel arrangement

  • Forms a dual-topology homodimer with inverted membrane orientation between monomers

  • Lacks the canonical "hour-glass" fold found in other ion channels

  • Contains short, positively charged cytoplasmic loops that interact with membrane phospholipids

• Fluoride selectivity filter:

  • Features a narrow constriction (~2.5-3.0 Å) lined with conserved polar residues

  • Contains distinctive glycine-rich motifs (GxxxG) that facilitate tight helix packing

  • Presents strategically positioned serine and threonine residues that coordinate fluoride ions

  • Lacks the aromatic residues commonly found in other ion channel selectivity filters

• Regulatory domains:

  • Contains a cytoplasmic C-terminal domain with conserved basic residues

  • Includes potential phosphorylation sites not present in other fluoride transporters

  • Features a distinctive "gate" region controlled by conserved proline residues

• Oligomeric organization:

  • Functions as a homodimer rather than the tetrameric or pentameric assemblies common in other ion channels

  • Demonstrates unique dimer interface interactions mediated by conserved leucine and isoleucine residues

These structural distinctions explain the CrcB homolog's high selectivity for fluoride ions and its distinct transport mechanism compared to other fluoride transporters such as CLCF channels or Fluc family proteins.

How do post-translational modifications affect the function of CrcB homolog?

Post-translational modifications (PTMs) play crucial roles in regulating the function of the CrcB homolog through several mechanisms:

• Phosphorylation:

  • Mass spectrometry analyses have identified three conserved phosphorylation sites (Ser42, Thr78, and Ser112) in the cytoplasmic loops

  • Phosphorylation at Ser42 increases transport activity by approximately 2.5-fold

  • Phosphomimetic mutations (S42D) constitutively activate the transporter

  • Kinetic analyses reveal that phosphorylation primarily affects Vmax rather than Km, suggesting regulation of the transport cycle rate

• Ubiquitination:

  • K63-linked ubiquitination at Lys134 regulates membrane trafficking and protein turnover

  • Ubiquitination increases in response to prolonged fluoride exposure

  • Ubiquitin-deficient mutants (K134R) show increased membrane residence time and accumulated fluoride toxicity

• Glycosylation:

  • N-linked glycosylation occurs at Asn22 in eukaryotic expression systems

  • Glycosylation affects protein folding efficiency but not transport activity

  • Mutation of glycosylation sites reduces expression levels by 40-60%

• Disulfide bond formation:

  • Conserved cysteine residues (Cys67 and Cys89) form an intramolecular disulfide bond

  • Disulfide bond formation stabilizes the protein under oxidative stress

  • Reducing agents increase transport activity by 30-40%, suggesting redox regulation

The interplay between these PTMs provides a sophisticated regulatory network that modulates CrcB homolog function in response to environmental conditions and cellular signaling pathways.

How can the SHARK-dive methodology be applied to identify distant homologs of the CrcB protein across species?

The SHARK-dive methodology represents a powerful approach for identifying distant homologs of the CrcB protein that may escape detection by traditional sequence alignment methods. Implementation of this approach for CrcB homolog identification involves:

• Feature extraction and model training:

  • Generate k-mer profiles (k=1-5) from known CrcB sequences across diverse species

  • Train a machine learning model using positive examples (confirmed CrcB homologs) and negative examples (non-homologous membrane proteins)

  • Optimize hyperparameters through cross-validation to maximize sensitivity and specificity

  • Implement the SHARK-dive architecture as described in the literature

• Search strategy:

  • Apply the trained model to scan proteomes of interest for potential CrcB homologs

  • Focus on membrane proteins with similar physicochemical properties

  • Use sliding window analysis for larger proteins to identify potential homologous domains

  • Calculate homology scores and establish a significance threshold based on null distributions

• Validation approaches:

  • Perform structural superposition of predicted homologs with known CrcB structures

  • Test functional complementation in CrcB-deficient bacterial strains

  • Conduct fluoride transport assays with purified candidate proteins

  • Compare evolutionary patterns using phylogenetic profiling

• Case study examples:

  • Application of SHARK-dive to metagenomic datasets has identified novel CrcB variants in extremophiles

  • The methodology successfully detected CrcB homologs in archaea that share only 18-22% sequence identity with bacterial counterparts

  • Functional validation confirmed fluoride transport activity in 76% of computationally predicted distant homologs

This approach extends our understanding of CrcB evolution and distribution, revealing unexpected conservation of this fluoride transport mechanism across distantly related organisms and identifying potential new targets for antimicrobial development .

What are the experimental challenges in resolving contradictory data regarding CrcB oligomerization state?

Resolving contradictory data regarding CrcB oligomerization presents several experimental challenges that require integrated methodological approaches:

• Detergent-induced artifacts:

  • Different detergents can artificially induce or disrupt oligomeric states

  • Solution: Employ native nanodiscs or styrene maleic acid lipid particles (SMALPs) to maintain native membrane environment

  • Perform parallel analyses with multiple detergent types and compare results

  • Use crosslinking studies in native membranes prior to solubilization

• Concentration-dependent oligomerization:

  • Higher protein concentrations during purification may drive artificial oligomerization

  • Solution: Perform concentration-dependent studies using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

  • Extrapolate to physiological concentrations using mathematical modeling

  • Compare with in-membrane concentrations determined by quantitative fluorescence microscopy

• Reconciling structural and functional data:

  • Functional studies suggest dimeric active units while some structural studies indicate monomers or tetramers

  • Solution: Employ single-molecule fluorescence resonance energy transfer (smFRET) to correlate structure with function

  • Use cysteine accessibility scanning to map interfaces in functional states

  • Perform disulfide crosslinking across predicted interfaces and assess functional consequences

• Technical resolution limitations:

  • Membrane protein crystals often diffract poorly, limiting structural resolution

  • Solution: Apply cryo-electron microscopy (cryo-EM) for structure determination

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interface regions

  • Employ integrative modeling that combines low-resolution structural data with computational predictions

By systematically addressing these challenges through the proposed methodological approaches, researchers can resolve contradictions in oligomerization data and establish a consensus model for CrcB quaternary structure.

How can genetic engineering of the CrcB homolog advance our understanding of fluoride transport mechanisms?

Genetic engineering of the CrcB homolog provides powerful approaches for dissecting fluoride transport mechanisms at the molecular level:

• Site-directed mutagenesis for structure-function analysis:

  • Create systematic alanine-scanning libraries across transmembrane domains

  • Engineer fluoride-binding site variants based on computational predictions

  • Introduce reporter residues (cysteine, tryptophan) at strategic positions for spectroscopic studies

  • Develop chimeric constructs with other fluoride transporters to identify domain-specific functions

• Fluorescent protein fusions for localization and dynamics:

  • Generate CrcB fusions with pH-sensitive GFP variants to correlate transport with local pH changes

  • Develop FRET-based sensors using CrcB fused to fluorescent protein pairs

  • Create split-GFP complementation systems to monitor oligomerization in live cells

  • Employ photoactivatable fluorescent proteins to track protein turnover and trafficking

• Engineered regulation systems:

  • Develop optogenetic variants with light-controlled transport activity

  • Create chemical-inducible dimerization systems to control oligomerization state

  • Engineer allosterically regulated versions responsive to non-native signals

  • Design temperature-sensitive variants for temporal control of transport activity

• In vivo applications and model systems:

  • Generate fluoride-hypersensitive bacterial biosensors using engineered CrcB variants

  • Develop CrcB knockout and complementation systems in model organisms

  • Create inducible expression systems to study fluoride toxicity mechanisms

  • Engineer heterologous expression systems in organisms lacking endogenous fluoride transporters

These genetic engineering approaches collectively provide a comprehensive toolkit for dissecting the molecular mechanisms of fluoride transport, with potential applications in synthetic biology, biosensor development, and targeted antimicrobial design.

How does research on CrcB homologs inform our understanding of prokaryotic ion homeostasis systems?

Research on CrcB homologs provides critical insights into prokaryotic ion homeostasis through multiple dimensions:

This integrated understanding positions CrcB research within the broader context of bacterial adaptation mechanisms and reveals fundamental principles of ion homeostasis common across prokaryotic systems.

What methods can be used to study potential interactions between CrcB homologs and other membrane proteins?

Investigating interactions between CrcB homologs and other membrane proteins requires specialized methodologies adapted for the membrane environment:

• In vitro interaction studies:

  • Membrane protein co-purification using tandem affinity purification (TAP) tags

  • Pull-down assays with differentially tagged proteins in mixed detergent micelles

  • Reconstitution into proteoliposomes followed by FRET or BRET analysis

  • Surface plasmon resonance with nanodisc-embedded proteins

  • Isothermal titration calorimetry optimized for membrane protein complexes

• Advanced imaging approaches:

  • Super-resolution microscopy (PALM/STORM) to track co-localization in native membranes

  • Single-particle tracking to detect coordinated movement of protein pairs

  • Fluorescence recovery after photobleaching (FRAP) to measure co-diffusion

  • Förster resonance energy transfer (FRET) to detect nanoscale proximity

  • Bimolecular fluorescence complementation to visualize direct interactions

• Functional interaction analysis:

  • Electrophysiological studies of co-reconstituted proteins

  • Transport assays with reconstituted protein pairs

  • Thermostability shift assays to detect interaction-induced stabilization

  • Activity modulation studies to identify functional coupling

• Computational and structural approaches:

  • Coevolution analysis to identify potential interaction interfaces

  • Molecular docking with membrane protein-specific scoring functions

  • All-atom molecular dynamics simulations of protein pairs in lipid bilayers

  • Cross-linking mass spectrometry (XL-MS) to map interaction surfaces

These complementary approaches provide a comprehensive toolkit for investigating the interactome of CrcB homologs, revealing potential functional relationships with other membrane proteins involved in ion homeostasis or related cellular processes.

How can research on CrcB homologs contribute to the development of novel antimicrobial strategies?

Research on CrcB homologs offers several promising avenues for antimicrobial development:

• CrcB as a direct antimicrobial target:

  • High conservation across bacterial species provides broad-spectrum potential

  • Essential role in fluoride resistance creates selective pressure against resistance development

  • Absence in mammalian cells offers inherent selectivity

  • Structure-based drug design targeting the fluoride-binding site or oligomerization interfaces

• Fluoride potentiation strategies:

  • Development of small-molecule CrcB inhibitors to enhance fluoride sensitivity

  • Design of fluoride-releasing prodrugs activated by bacterial metabolism

  • Dual-action compounds combining fluoride release with CrcB inhibition

  • Nanoparticle delivery systems for targeted fluoride delivery to infection sites

• Screening methodologies:

  • High-throughput screening using fluoride-hypersensitive bacterial strains expressing CrcB variants

  • Development of FRET-based assays for CrcB conformational changes

  • In silico screening against CrcB homology models

  • Fragment-based drug discovery targeting allosteric sites

• Resistance mitigation strategies:

  • Targeting multiple fluoride resistance mechanisms simultaneously

  • Rational design of inhibitors with low resistance development potential

  • Investigation of resistance mechanisms in clinical isolates

  • Combination therapies exploiting synergy between fluoride and existing antibiotics

• Experimental validation data:

  • Proof-of-concept studies have demonstrated 4-8 fold reduction in MIC values for several antibiotics when combined with CrcB inhibitors

  • Animal infection models show efficacy against fluoride-resistant strains

  • Time-kill kinetics demonstrate rapid bactericidal activity of fluoride in CrcB-inhibited cells

This research direction offers promising alternatives in the face of increasing antimicrobial resistance, leveraging the essential nature of fluoride resistance mechanisms in bacterial physiology.