Recombinant Pyrococcus horikoshii Protein CrcB homolog (crcB)

What is Pyrococcus horikoshii Protein CrcB homolog and what is its significance in research?

Pyrococcus horikoshii Protein CrcB homolog (crcB) is a putative fluoride ion transporter protein encoded by the crcB gene (also designated as PH1502) in the hyperthermophilic archaeon Pyrococcus horikoshii strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3 . This protein belongs to the CrcB family of membrane proteins that are involved in fluoride ion transport across cellular membranes.

The significance of this protein in research stems from several factors: (1) It represents an important component of ion homeostasis in extremophilic archaea; (2) As a membrane protein from a hyperthermophile, it offers insights into protein stability under extreme conditions; (3) Understanding fluoride transport mechanisms has implications for microbial resistance to environmental fluoride toxicity; and (4) Comparative studies with homologous proteins across domains of life can shed light on evolutionary conservation of ion transport mechanisms.

What are the structural characteristics of Pyrococcus horikoshii CrcB homolog?

The Pyrococcus horikoshii CrcB homolog is a full-length protein comprising 123 amino acids with a sequence of: MNAKIIALLIIGGGLGALTRYYISGVLPVYKDFPIGTLIVNSLASFLLGYIYGLLFSGFDISPEWRIFLGTGFCGGLSTFSTFSYETFSLLREGEIWLAFANITTNILVTIFLVFLGFILARR .

Structural analysis indicates that CrcB homolog is a transmembrane protein with characteristic hydrophobic regions consistent with membrane-spanning domains. These hydrophobic stretches are interspersed with charged residues that likely facilitate ion transport. While high-resolution crystal structures of this specific CrcB homolog have not been widely reported, homology modeling suggests a structure with multiple transmembrane helices forming a channel or pore for fluoride ion passage. The protein's adaptation to the hyperthermophilic environment of P. horikoshii (which grows optimally at around 98°C) suggests structural features that contribute to thermostability, such as increased hydrophobic interactions and salt bridges.

How is Recombinant Pyrococcus horikoshii CrcB homolog typically expressed and purified for research applications?

For research applications, Recombinant Pyrococcus horikoshii CrcB homolog is typically expressed using in vitro E. coli expression systems . The expression process generally follows these methodological steps:

• Gene synthesis or cloning of the crcB gene (PH1502) into an appropriate expression vector

• Transformation into an E. coli strain optimized for membrane protein expression

• Induction of protein expression under controlled conditions

• Cell lysis and membrane fraction isolation

• Detergent-based membrane protein solubilization

• Affinity chromatography purification utilizing the N-terminal 10xHis-tag

• Buffer exchange and concentration

For enhanced purification, the protein is often produced with an N-terminal 10xHis-tag that facilitates affinity chromatography purification . Challenges in expression often include proper membrane integration, potential toxicity to host cells, and maintaining proper folding. Alternative expression systems, including cell-free synthesis methods, may be employed for difficult-to-express membrane proteins.

What expression systems are most effective for producing functional CrcB homolog for research?

The most effective expression systems for producing functional CrcB homolog balance protein yield with proper folding and membrane integration. Based on research practices with similar archaeal membrane proteins, several approaches yield favorable results:

• E. coli-based systems: Most commonly used due to their simplicity and cost-effectiveness, with BL21(DE3) and C41/C43(DE3) strains being particularly suitable for membrane proteins . These systems typically employ vectors with inducible promoters (T7, tac) and fusion tags to aid solubility and purification.

• Archaeal host systems: While more challenging to implement, expression in archaeal hosts such as Thermococcus kodakarensis or modified P. horikoshii strains can provide native-like membrane environments.

• Cell-free expression systems: These bypass issues of toxicity and can incorporate detergents or lipids during synthesis to facilitate proper folding of membrane proteins.

• Yeast systems: Pichia pastoris or Saccharomyces cerevisiae can provide eukaryotic-like membrane environments while maintaining relatively high yields.

The expression method should be selected based on downstream application requirements, with considerations for protein authenticity versus yield. For structural studies, archaeal hosts may provide more natively folded protein, while E. coli systems often suffice for initial functional characterization.

What are the recommended storage and handling conditions for maintaining CrcB homolog activity?

Proper storage and handling of Recombinant Pyrococcus horikoshii CrcB homolog are critical for maintaining its structural integrity and functional activity. The recommended conditions include:

The protein can be provided in either liquid form or as a lyophilized powder . For liquid preparations, the protein is typically stored in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 . This formulation helps maintain protein stability during storage and freeze-thaw cycles.

For optimal storage:

• Store at -20°C to -80°C upon receipt

• Aliquot the protein to avoid repeated freeze-thaw cycles, which can lead to protein denaturation

• For liquid formulations, the shelf life is approximately 6 months at -20°C/-80°C

• Lyophilized preparations have extended stability, with a shelf life of approximately 12 months at -20°C/-80°C

When handling the protein for experiments:

• Thaw aliquots on ice

• Maintain cold chain during experimental setup

• Consider the thermostable nature of this archaeal protein when designing assays

• Include appropriate stabilizing agents or detergents in working buffers to maintain solubility of this membrane protein

What experimental approaches are most effective for characterizing the fluoride transport function of CrcB homolog?

Characterizing the fluoride transport function of CrcB homolog requires specialized experimental approaches that account for its membrane protein nature and specific ion transport properties. The most effective methodologies include:

• Fluoride-selective electrode measurements: This direct approach monitors fluoride concentration changes across membranes containing reconstituted CrcB. The setup typically includes:

  • Proteoliposomes with incorporated CrcB homolog

  • Buffer systems with controlled pH and ionic composition

  • Fluoride-selective electrodes for real-time concentration measurement

  • Control measurements with inactive protein or empty liposomes

• Fluorescent indicator assays: Using fluoride-sensitive fluorescent probes to monitor transport activity:

  • PBFI (potassium-binding benzofuran isophthalate) adapted for fluoride sensitivity

  • Fluorescence measurements in real-time during transport assays

  • Calibration with known fluoride concentrations to quantify transport rates

• Isotope flux assays: Using radioactive fluoride isotopes (18F) to track movement across membranes:

  • Inside-out membrane vesicles or proteoliposomes loaded with purified CrcB

  • Time-course measurements of isotope accumulation

  • Scintillation counting to quantify transport rates

• Electrophysiological approaches: Whole-cell patch clamp or reconstituted bilayer recordings:

  • Single-channel conductance measurements

  • Current-voltage relationship determination under varying fluoride concentrations

  • Ion selectivity profiling against other halides

The integration of multiple methodologies provides the most comprehensive characterization of CrcB transport function, with careful attention to experimental controls to distinguish specific transport from nonspecific leakage.

How can structural biology techniques be applied to study the mechanism of CrcB-mediated fluoride transport?

Structural biology techniques provide crucial insights into the mechanism of CrcB-mediated fluoride transport by revealing the protein's three-dimensional architecture and conformational changes during transport cycles. The most valuable approaches include:

• X-ray crystallography: While challenging for membrane proteins, this technique can provide atomic-resolution structures:

  • Requires purification of CrcB in detergent micelles or lipidic cubic phases

  • Crystallization trials with various detergents and precipitation agents

  • Heavy atom derivatives for phase determination

  • Structure determination at different states (apo, fluoride-bound) to understand the transport cycle

• Cryo-electron microscopy (cryo-EM): Increasingly powerful for membrane protein structure determination:

  • Sample preparation in detergent micelles, nanodiscs, or amphipols

  • Single-particle analysis for structure determination

  • Potential for visualizing different conformational states without crystallization

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Solution NMR for dynamics studies of specific domains

  • Solid-state NMR for membrane-embedded structural analysis

  • Chemical shift analysis to identify fluoride binding sites

• Molecular dynamics simulations:

  • Atomistic simulations of CrcB embedded in lipid bilayers

  • Analysis of ion permeation pathways and energy barriers

  • Prediction of key residues involved in transport

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Identification of regions with altered solvent accessibility during transport

  • Mapping conformational changes induced by fluoride binding

These approaches, when combined with functional assays and mutagenesis studies, can elucidate the molecular mechanism of fluoride recognition, binding, and translocation through the CrcB transport pathway.

What site-directed mutagenesis strategies can be employed to identify key functional residues in CrcB homolog?

Site-directed mutagenesis represents a powerful approach for identifying key functional residues in the CrcB homolog. Based on the protein's sequence and putative function as a fluoride transporter, several strategic approaches can be implemented:

• Conserved residue targeting: Identify amino acids conserved across CrcB homologs from different species by multiple sequence alignment. The amino acid sequence (MNAKIIALLIIGGGLGALTRYYISGVLPVYKDFPIGTLIVNSLASFLLGYIYGLLFSGFDISPEWRIFLGTGFCGGLSTFSTFSYETFSLLREGEIWLAFANITTNILVTIFLVFLGFILARR) can be analyzed for conserved motifs across the CrcB family .

• Charge-swap mutations: Target charged residues (D, E, K, R) that might form the ion conduction pathway:

  • Acidic to basic substitutions (D→K, E→R)

  • Basic to acidic substitutions (K→E, R→D)

  • Charge neutralization (D→N, E→Q, K→Q, R→Q)

• Transmembrane domain alterations: Based on hydrophobicity analysis, mutate residues in predicted transmembrane domains that might line the transport pore.

• Polarity-based substitutions: Replace polar residues with nonpolar ones and vice versa to disrupt potential hydrogen bonding networks involved in ion coordination.

• Systematic alanine scanning: Replace consecutive residues with alanine to identify regions critical for function.

The mutants should be functionally characterized using fluoride transport assays, and expression levels should be verified to ensure that altered activity isn't simply due to expression defects. Thermostability assays can also provide insights into whether mutations affect the protein's characteristic stability as an archaeal protein.

How do CrcB homologs from different extremophilic archaea compare in terms of structure and function?

Comparative analysis of CrcB homologs from different extremophilic archaea reveals important adaptations and conserved features that illuminate the evolution and specialized functions of these transporters across diverse environmental niches. This comparison can be approached systematically:

• Sequence conservation analysis:

  • Core functional residues tend to be highly conserved across archaeal CrcB homologs

  • The Pyrococcus horikoshii CrcB homolog sequence can be compared with homologs from related thermophiles (Thermococcus, Archaeoglobus) and other extremophiles (halophiles, acidophiles)

  • Unique substitutions may correlate with specific environmental adaptations

• Structural comparisons:

  • Hyperthermophilic archaea (like P. horikoshii) typically show increased hydrophobic core packing and additional salt bridges

  • Halophilic archaea often exhibit increased negative surface charge to maintain protein solubility

  • Acidophilic archaeal proteins may contain additional acid-stable features

• Functional adaptations:

  • Kinetic parameters of fluoride transport (Km, Vmax) vary based on environmental niches

  • Temperature optima correlate with growth conditions of source organisms

  • pH dependence reflects environmental adaptation

The table below summarizes key comparative features of CrcB homologs from selected archaeal species:

These comparisons provide valuable insights into how evolutionary pressures in extreme environments have shaped the structure-function relationship of CrcB homologs.

What reconstitution methods are most effective for studying CrcB function in artificial membrane systems?

Reconstitution of CrcB homolog into artificial membrane systems is crucial for detailed functional studies of this putative fluoride transporter. Several methodologies have been developed, each with specific advantages for particular experimental objectives:

• Proteoliposome reconstitution:

  • Detergent-mediated reconstitution: Purified CrcB (with N-terminal 10xHis-tag) is mixed with lipids in detergent, followed by controlled detergent removal

  • Direct incorporation during liposome formation: Protein is added during the hydration of dried lipid films

  • Optimized lipid composition: Typically contains archaeal lipids or synthetic lipids with branched chains to accommodate the native membrane environment of P. horikoshii

  • Size control: Extrusion through polycarbonate filters produces uniform vesicle populations

• Planar lipid bilayer systems:

  • Painting method: Lipids dissolved in organic solvent are applied across an aperture separating two aqueous chambers

  • Folding method: Two lipid monolayers are combined to form a bilayer

  • Permits direct electrical measurements of transport activity

  • Allows controlled manipulation of conditions on both sides of the membrane

• Nanodiscs and lipid nanodiscs:

  • Membrane scaffold protein (MSP)-based nanodiscs provide a native-like bilayer environment

  • Polymer-based nanodiscs (SMALP, DIBMA) can extract CrcB directly from membranes

  • Advantages include enhanced stability and accessibility for structural studies

  • Size-tunable to accommodate CrcB oligomeric states

• Lipid cubic phases:

  • Particularly useful for crystallization attempts with membrane proteins

  • Creates a continuous bilayer structure that can maintain protein function

  • Facilitates both functional and structural studies

• Cell-free expression with immediate reconstitution:

  • Co-translational insertion into liposomes or nanodiscs

  • Minimizes aggregation and misfolding by avoiding a solubilized intermediate state

For thermostable proteins like P. horikoshii CrcB, reconstitution protocols must account for temperature stability, with potential benefits from performing procedures at elevated temperatures (40-60°C) that would denature mesophilic proteins but may enhance folding of this archaeal protein.

What controls are essential when designing experiments to study CrcB fluoride transport activity?

Designing rigorous experiments to study CrcB fluoride transport requires comprehensive controls to ensure reliable and interpretable results. The following controls are essential for robust experimental design:

• Negative controls for transport specificity:

  • Empty liposomes/vesicles (without CrcB) to establish baseline leakage rates

  • Heat-inactivated CrcB (denatured at temperatures exceeding its thermostability threshold)

  • CrcB with key mutations in predicted transport pathway residues

  • Transport assays in the presence of known fluoride transport inhibitors

• Positive controls for assay validation:

  • Known fluoride transporters (if available) reconstituted under identical conditions

  • Ionophores that facilitate nonspecific ion movement across membranes

  • Calibration standards for fluoride detection methods

• Specificity controls:

  • Transport assays with other halide ions (Cl−, Br−, I−) to determine selectivity

  • Competition assays with mixed ion compositions

  • pH-dependent measurements to identify potential proton coupling

• Technical controls:

  • Verification of CrcB incorporation into membranes (Western blot, fluorescence if tagged)

  • Liposome/vesicle integrity checks before and after assays

  • Orientation analysis of reconstituted CrcB (inside-out vs. right-side-out)

  • Multiple detection methods to confirm transport measurements

• Expression and purification controls:

  • Empty vector expression products processed identically to CrcB

  • Verification of protein purity by SDS-PAGE and mass spectrometry

  • Confirmation of proper folding through circular dichroism or limited proteolysis

Each experiment should include appropriate combinations of these controls to isolate specific CrcB-mediated transport effects from background artifacts and to establish the physiological relevance of observed activities.

How can researchers optimize experimental conditions for studying thermostable archaeal membrane proteins like CrcB?

Optimizing experimental conditions for thermostable archaeal membrane proteins like Pyrococcus horikoshii CrcB requires specialized approaches that account for their unique properties. The following methodological considerations are crucial:

• Temperature optimization:

  • Purification at elevated temperatures (40-60°C) can increase protein stability

  • Functional assays should include measurements at multiple temperatures (25-95°C)

  • Include temperature gradient studies to determine optimal activity range

  • Consider temperature-resistant buffers and reagents (HEPES, phosphate) that maintain pH at high temperatures

• Buffer and pH considerations:

  • Use buffers with minimal temperature coefficients for pH stability

  • Include stabilizing agents (glycerol, trehalose) in storage buffers

  • Optimize ionic strength based on the native environment of P. horikoshii

  • Evaluate pH range for activity, accounting for different internal pH of thermophilic archaea

• Membrane mimetic environments:

  • Test multiple detergent classes (maltoside, glucoside, fos-choline) for optimal extraction

  • Consider archaeal-like lipids (tetraether lipids, branched-chain phospholipids)

  • Evaluate protein stability in nanodiscs vs. liposomes vs. detergent micelles

  • Adjust lipid composition to match the native membrane environment

• Expression optimization:

  • Consider codon optimization for expression host

  • Test induction at elevated temperatures

  • Evaluate periplasmic vs. cytoplasmic expression strategies

  • Assess fusion partners that enhance stability (MBP, SUMO)

• Purification strategies:

  • Utilize the N-terminal 10xHis-tag for initial affinity purification

  • Consider thermal purification steps (heat treatment)

  • Implement size exclusion chromatography to isolate properly folded protein

  • Verify oligomeric state by native PAGE or analytical ultracentrifugation

• Storage conditions:

  • Evaluate protein stability at -20°C vs. -80°C

  • Test additives (glycerol, sugars, specific ions) for long-term storage

  • Determine whether liquid or lyophilized storage is optimal

By systematically optimizing these conditions, researchers can maintain the native-like structure and function of CrcB while adapting experimental protocols to accommodate its thermostable archaeal nature.

What are the key considerations for designing comparative genomic studies of CrcB homologs across different domains of life?

Designing comparative genomic studies of CrcB homologs across different domains of life requires careful consideration of multiple factors to ensure meaningful evolutionary and functional insights. Key methodological considerations include:

• Sequence identification and curation:

  • Use sensitive profile-based methods (PSI-BLAST, HMMer) to identify distant CrcB homologs

  • Start with well-characterized references including P. horikoshii CrcB homolog (PH1502)

  • Filter out partial sequences and potential pseudogenes

  • Create a representative dataset spanning archaea, bacteria, and eukaryotes

• Alignment methodologies:

  • Apply membrane protein-specific alignment algorithms that account for hydrophobic conservation patterns

  • Consider structure-guided alignments where structural data is available

  • Evaluate alignment quality with objective metrics (CORE index, TCS score)

  • Manually inspect transmembrane domain alignments for biological plausibility

• Phylogenetic analysis approaches:

  • Test multiple tree-building methods (Maximum Likelihood, Bayesian, Neighbor-Joining)

  • Apply appropriate evolutionary models for membrane proteins (CAT+GTR, LG+F+G)

  • Implement bootstrap or posterior probability analysis for branch support

  • Root trees using ancient paralogs or midpoint rooting if outgroups are unclear

• Genomic context analysis:

  • Examine operon structure and gene neighborhood across species

  • Identify co-evolving genes that may indicate functional relationships

  • Map chromosomal locations to identify potential horizontal gene transfer events

• Structural comparison:

  • Map sequence conservation onto structural models to identify functional hotspots

  • Compare predicted transmembrane topologies across evolutionary distance

  • Identify structural elements unique to specific clades or environments

• Functional prediction:

  • Correlate sequence variations with known functional differences

  • Identify potential substrate specificity determinants

  • Predict regulatory elements and expression patterns across lineages

This comprehensive approach allows researchers to reconstruct the evolutionary history of CrcB proteins, identify key adaptation events, and generate testable hypotheses about functional specialization across diverse organisms from all three domains of life.

How should researchers interpret conflicting results in CrcB functional studies?

When encountering conflicting results in CrcB functional studies, researchers should implement a systematic approach to interpretation that considers methodological variations, experimental conditions, and underlying biological complexity. The following framework provides guidance for resolving such conflicts:

• Methodological evaluation:

  • Compare experimental systems (in vivo vs. in vitro, different expression hosts)

  • Assess protein preparation methods (detergents, purification protocols)

  • Examine differences in reconstitution approaches (liposomes, nanodiscs, bilayers)

  • Consider detection method sensitivities and limitations

• Condition-dependent variables:

  • Temperature effects on CrcB activity (especially relevant for thermostable P. horikoshii CrcB)

  • Buffer composition differences (pH, ionic strength, specific ions)

  • Lipid environment variations (headgroups, acyl chains, membrane thickness)

  • Protein concentration and orientation in membranes

• Data integration approaches:

  • Weigh evidence based on methodological rigor and reproducibility

  • Consider orthogonal validation approaches to confirm key findings

  • Apply Bayesian analysis to integrate multiple studies with different confidence levels

  • Develop computational models that accommodate apparently conflicting data

• Biological explanations for discrepancies:

  • Potential allosteric regulation under different conditions

  • Multiple functional states with different activities

  • Post-translational modifications or structural alterations

  • Interactions with unidentified accessory proteins

• Resolution strategies:

  • Design critical experiments specifically targeting the source of conflict

  • Implement collaborative cross-laboratory validation studies

  • Utilize a broader range of techniques to triangulate true function

  • Consider whether conflicting results actually reflect multifunctional properties

When analyzing conflicting data regarding CrcB function, it is particularly important to consider the protein's extremophilic origin and how experimental conditions might diverge from its native environment. Transport rates, substrate specificity, and regulatory mechanisms may all vary significantly depending on temperature, pressure, and ionic composition of the experimental system.

What statistical approaches are recommended for analyzing ion transport data from CrcB studies?

Analyzing ion transport data from CrcB studies requires robust statistical approaches that account for the unique characteristics of transport kinetics and the potential sources of variability in membrane protein experiments. The following statistical methodologies are recommended:

• Kinetic parameter estimation:

  • Nonlinear regression for Michaelis-Menten kinetics (Km, Vmax determination)

  • Global fitting approaches for complex kinetic models

  • Bootstrap resampling to establish confidence intervals for kinetic parameters

  • Comparison of different kinetic models using Akaike Information Criterion (AIC) or Bayesian Information Criterion (BIC)

• Time-series analysis:

  • Calculation of initial rates from early linear phase of transport

  • Exponential fitting for equilibration kinetics

  • Deconvolution analysis for overlapping processes

  • Area under curve (AUC) measurements for total transport

• Hypothesis testing frameworks:

  • ANOVA with post-hoc tests for multi-condition comparisons

  • Mixed-effects models to account for batch-to-batch variability

  • Non-parametric tests when normality assumptions are violated

  • Multiple comparison correction (Bonferroni, Benjamini-Hochberg) to control false discovery rate

• Variability analysis:

  • Propagation of error calculations for derived parameters

  • Repeatability and reproducibility assessment (intra- and inter-assay coefficients of variation)

  • Sensitivity analysis to identify influential variables

  • Monte Carlo simulations to model expected variability

• Specialized approaches for fluoride transport:

  • Correction for background leakage using control measurements

  • Normalization strategies based on protein concentration or vesicle volume

  • Hill equation fitting for cooperative binding/transport

  • Competition analysis for ion selectivity studies

The table below illustrates a typical data analysis workflow for CrcB fluoride transport experiments:

What are the most promising future research directions for CrcB homolog studies?

The study of Pyrococcus horikoshii CrcB homolog presents several promising research directions that could significantly advance our understanding of membrane transport mechanisms, extremophile adaptations, and potential biotechnological applications. Key future directions include:

• Structural determination at atomic resolution: Obtaining high-resolution structures of CrcB in different conformational states would provide unprecedented insights into the transport mechanism. Cryo-EM advances make this increasingly feasible for membrane proteins and could reveal the molecular basis of fluoride selectivity .

• Systems biology integration: Exploring how CrcB functions within the broader context of P. horikoshii physiology, including potential interactions with other membrane proteins and metabolic networks, would provide a more comprehensive understanding of its biological role.

• Comparative functional genomics: Expanding functional characterization across diverse CrcB homologs from different extremophilic environments could reveal evolutionary adaptations in ion transport mechanisms and potentially uncover novel functions beyond fluoride transport.

• Synthetic biology applications: Engineered CrcB variants could be developed for applications in bioremediation of fluoride-contaminated environments, biosensors for fluoride detection, or as components in artificial cell systems designed to function in extreme conditions.

• Drug discovery applications: The uniqueness of archaeal transporters like CrcB offers potential targets for developing antimicrobials with novel mechanisms of action, particularly against pathogenic archaea or bacteria with essential CrcB homologs.