Recombinant Pelobacter carbinolicus Protein CrcB homolog (crcB)

What is Pelobacter carbinolicus CrcB homolog protein and what is its functional classification?

Pelobacter carbinolicus CrcB homolog is a membrane protein encoded by the crcB gene (locus Pcar_0981) in P. carbinolicus strain DSM 2380 / Gra Bd 1. The protein consists of 123 amino acids with the sequence: MHLVYIAIFGALGCLSRFMVSGWVYALIGRALPYGTLAVNVIGSLLLGLLMEGGLRSAALPADIRMGITTGFMGGFTTFSTFSYETVRLLEDGSMVAAGANILLNVTVSVVFAGLGIFLARQL .

Functionally, CrcB homologs are generally classified as membrane proteins involved in ion transport, particularly fluoride ion channels. In bacterial systems, CrcB proteins contribute to fluoride resistance by exporting toxic fluoride ions from the cytoplasm. The protein contains multiple transmembrane domains, as evidenced by its hydrophobic amino acid sequence rich in glycine and alanine residues, which are typical of membrane-spanning segments .

How does Pelobacter carbinolicus fit into microbial metabolic networks?

Pelobacter carbinolicus functions within specialized anaerobic metabolic networks, particularly in syntrophic relationships with methanogenic archaea. This bacterium oxidizes ethanol in cooperation with methanogens such as Methanospirillum hungatei, serving as a model system for syntrophic ethanol oxidation .

The metabolic pathway involves:

• Ethanol oxidation via NAD+-dependent alcohol dehydrogenases

• Acetaldehyde conversion through two distinct enzymatic routes:

  • Benzyl viologen-reducing enzymes that form acetate

  • NAD+-reducing enzymes that form acetyl-CoA

• ATP synthesis from acetyl-CoA via acetyl phosphate

P. carbinolicus possesses specialized gene clusters for comproportionating hydrogenases (gene loci Pcar_1602-Pcar_1605 and Pcar_1633-Pcar_1936) similar to those in Thermotoga maritima, enabling it to participate in electron transfer chains critical for anaerobic metabolism .

What expression systems are recommended for producing recombinant Pelobacter carbinolicus CrcB protein?

For optimal expression of recombinant P. carbinolicus CrcB protein, consider the following methodological approaches:

• Expression System Selection:

  • E. coli-based systems (BL21(DE3), C41(DE3), or C43(DE3)) are recommended for membrane proteins

  • For proper folding, consider lower induction temperatures (16-25°C)

  • IPTG concentrations should be optimized (0.1-1.0 mM range)

• Vector Design:

  • Include purification tags (His6 or Strep) at either N- or C-terminus

  • Incorporate cleavage sites for tag removal (TEV or PreScission protease)

  • Consider fusion partners (MBP or SUMO) to enhance solubility

• Buffer Optimization:

  • Use detergents compatible with membrane proteins (DDM, LDAO, or Fos-choline)

  • Include glycerol (10-20%) for stability

  • Incorporate small amounts of reducing agents (1-5 mM DTT or 2-ME)

The recombinant protein is typically stored in Tris-based buffer with 50% glycerol for optimal stability during storage at -20°C or -80°C .

What are the optimal storage and handling conditions for recombinant P. carbinolicus CrcB protein?

For maximum stability and activity retention of recombinant P. carbinolicus CrcB protein, implement the following evidence-based storage and handling protocol:

• Short-term Storage (1-7 days):

  • Store working aliquots at 4°C

  • Maintain in Tris-based buffer with 50% glycerol

  • Avoid repeated freeze-thaw cycles

• Long-term Storage:

  • Store at -20°C for routine storage

  • For extended preservation, maintain at -80°C

  • Prepare single-use aliquots (10-25 μL) to prevent freeze-thaw damage

• Handling Precautions:

  • Thaw frozen samples slowly on ice

  • Centrifuge briefly after thawing to collect contents

  • Work with the protein under temperature-controlled conditions (4°C when possible)

  • Handle with low-binding pipette tips to minimize protein loss

For experimental work, avoid repeated freeze-thaw cycles as these significantly reduce protein activity and integrity. When working with the reconstituted protein, monitor buffer pH stability as membrane proteins can be particularly sensitive to pH fluctuations.

How can researchers validate the structural integrity of recombinant CrcB protein preparations?

Validation of structural integrity for recombinant CrcB protein requires a multi-method approach:

• Biophysical Characterization Methods:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Thermal shift assays to assess folding stability

  • Dynamic light scattering (DLS) to evaluate homogeneity and aggregation state

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) for molecular weight determination

• Functional Validation:

  • Fluoride ion transport assays using fluoride-sensitive electrodes

  • Liposome reconstitution experiments to assess membrane integration

  • Binding assays with known interaction partners

• Structural Analysis:

  • Limited proteolysis to confirm proper folding

  • Fourier-transform infrared spectroscopy (FTIR) for membrane protein secondary structure

  • Negative stain electron microscopy to visualize protein particles

Researchers should compare results to predicted structural features based on the amino acid sequence (MHLVYIAIFGALGCLSRFMVSGWVYALIGRALPYGTLAVNVIGSLLLGLLMEGGLRSAALPADIRMGITTGFMGGFTTFSTFSYETVRLLEDGSMVAAGANILLNVTVSVVFAGLGIFLARQL) which suggests multiple transmembrane domains characteristic of membrane channel proteins .

What analytical methods are appropriate for studying protein-protein interactions involving CrcB homolog?

To effectively study protein-protein interactions involving CrcB homolog, implement the following analytical workflow:

• In vitro Interaction Analysis:

  • Pull-down assays: Use tagged recombinant CrcB as bait to capture interaction partners

  • Surface plasmon resonance (SPR): Quantify binding kinetics and affinity constants

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

  • Microscale thermophoresis (MST): Measure interactions in solution with minimal sample consumption

• Cellular Interaction Studies:

  • Co-immunoprecipitation (Co-IP): Isolate native protein complexes from P. carbinolicus

  • Proximity labeling (BioID or APEX): Identify transient interaction partners

  • Fluorescence resonance energy transfer (FRET): Visualize interactions in reconstituted membranes

• Computational Methods:

  • Molecular docking: Predict binding interfaces

  • Coevolution analysis: Identify correlated mutations suggesting interaction surfaces

  • Protein-protein interaction network analysis: Place CrcB within broader metabolic contexts

For membrane proteins like CrcB, special considerations include using mild detergents during extraction and purification, employing nanodiscs or liposomes for maintaining native-like membrane environments, and integrating in silico predictions with experimental validation to overcome technical challenges inherent to membrane protein interaction studies.

How does the CrcB homolog in P. carbinolicus compare structurally and functionally to CrcB proteins in other bacterial species?

Comparative analysis of P. carbinolicus CrcB with homologs in other bacterial species reveals important structural and functional relationships:

In terms of structure, P. carbinolicus CrcB likely adopts the characteristic "dual-topology" arrangement seen in other CrcB proteins, forming dimers with opposite orientations in the membrane. The sequence "MHLVYIAIFGALGCLSRFMVSGWVYALIGRAL" corresponds to the first transmembrane domain, with subsequent hydrophobic segments forming additional membrane-spanning regions .

Functionally, while most CrcB homologs serve as fluoride channels providing resistance to environmental fluoride, the exact role in P. carbinolicus remains to be fully characterized, particularly in the context of its unique syntrophic lifestyle and potential integration with comproportionating hydrogenase systems identified in its genome .

What is the relationship between P. carbinolicus CrcB and the organism's metabolic activities during syntrophic growth?

The relationship between P. carbinolicus CrcB and syntrophic metabolism presents a complex interplay that may involve:

• Ion Homeostasis During Syntrophy:

  • CrcB-mediated ion transport likely contributes to maintaining proper electrochemical gradients during syntrophic electron transfer

  • The protein may help regulate intracellular pH when P. carbinolicus participates in interspecies hydrogen transfer with methanogens such as Methanospirillum hungatei

• Metabolic Integration With Alcohol Oxidation:

  • During ethanol metabolism, P. carbinolicus employs NAD+-dependent alcohol dehydrogenase activity and multiple acetaldehyde-oxidizing pathways

  • CrcB may interact with these pathways by:

    • Controlling ion fluxes that influence redox balance

    • Potentially responding to metabolic intermediates or end products

    • Contributing to membrane integrity under varying growth conditions

• Genomic Context Analysis:

  • The genomic neighborhood of crcB (Pcar_0981) may provide clues to its metabolic integration

  • Potential functional relationships with comproportionating hydrogenases (gene loci Pcar_1602-Pcar_1605 and Pcar_1633-Pcar_1936) should be investigated

  • Comparative expression analysis between syntrophic and non-syntrophic growth conditions could reveal regulatory patterns

While direct experimental evidence linking CrcB to syntrophic metabolism is limited, the protein's presumed ion transport function suggests it may play a role in maintaining cellular homeostasis during the energetically challenging process of syntrophic growth, where energy conservation is critical for survival.

How might post-translational modifications affect CrcB homolog function in P. carbinolicus?

Post-translational modifications (PTMs) likely play critical roles in regulating CrcB homolog function in P. carbinolicus, with several potential mechanisms:

• Phosphorylation:

  • Potential phosphorylation sites include serine, threonine, and tyrosine residues in cytoplasmic loops

  • Phosphorylation could modulate:

    • Channel gating kinetics

    • Protein-protein interaction affinity

    • Subcellular localization

  • Response to environmental signals may involve phosphorylation cascades similar to those identified in REC protein family members

• S-Palmitoylation and Lipid Modifications:

  • Cysteine residues in membrane-proximal regions could undergo S-palmitoylation

  • Such modifications would:

    • Alter membrane microdomain targeting

    • Affect protein stability in the membrane

    • Potentially regulate oligomerization

• Redox-Based Modifications:

  • Under anaerobic conditions characteristic of P. carbinolicus growth, redox-sensitive modifications may occur

  • Oxidation/reduction of thiol groups could serve as a sensing mechanism for metabolic status

  • This would align with the organism's role in syntrophic metabolism

Experimental approaches to investigate these modifications should include mass spectrometry-based PTM mapping, site-directed mutagenesis of potential modification sites, and functional assays comparing wild-type and modification-deficient protein variants. Understanding these PTMs could provide insights into how CrcB activity is integrated with the organism's broader metabolic networks and response to environmental changes.

What are the major technical challenges in expressing and purifying functional P. carbinolicus CrcB for structural studies?

Researchers face several significant technical challenges when expressing and purifying P. carbinolicus CrcB for structural studies:

• Expression Yield Optimization:

  • Challenge: Membrane proteins typically express at lower levels than soluble proteins

  • Solution approaches:

    • Screen multiple expression systems (bacterial, yeast, insect, mammalian)

    • Optimize codon usage for expression host

    • Test fusion partners (MBP, SUMO, GFP) to improve folding

    • Employ controlled expression systems with tunable promoters

• Membrane Extraction and Stability:

  • Challenge: Maintaining protein stability during extraction from membranes

  • Solution approaches:

    • Systematic detergent screening (ranging from harsh SDS to mild DDM or LMNG)

    • Implement detergent exchange strategies during purification

    • Consider styrene-maleic acid lipid particles (SMALPs) for native membrane extraction

    • Incorporate stabilizing lipids throughout purification process

• Functional Validation:

  • Challenge: Ensuring the purified protein maintains native activity

  • Solution approaches:

    • Develop robust activity assays specific to ion transport function

    • Implement liposome reconstitution to assess channel activity

    • Monitor structural integrity through biophysical techniques (CD, thermostability)

    • Compare properties with better-characterized CrcB homologs

• Crystallization Barriers:

  • Challenge: Obtaining diffraction-quality crystals

  • Solution approaches:

    • Screen truncation constructs to remove disordered regions

    • Utilize lipidic cubic phase crystallization methods

    • Consider antibody fragment co-crystallization to provide crystal contacts

    • Explore cryo-EM as an alternative to crystallography

The recombinant protein's storage buffer composition (Tris-based buffer with 50% glycerol) provides a starting point for stability optimization, but systematic screening will be required to identify conditions that maintain both structural integrity and functional activity throughout the purification process .

How can researchers investigate the potential role of CrcB in P. carbinolicus stress response and environmental adaptation?

To investigate CrcB's role in stress response and environmental adaptation, researchers should implement a multi-faceted experimental strategy:

• Gene Expression Analysis Under Stress Conditions:

  • Method: Quantitative RT-PCR and RNA-seq analysis of crcB expression under various stressors:

    • Fluoride exposure (0.1-100 mM NaF)

    • pH stress (pH 5.0-9.0)

    • Oxidative stress (H₂O₂, paraquat)

    • Syntrophic vs. non-syntrophic growth conditions

  • Expected outcomes: Identification of specific stressors that modulate crcB expression, suggesting functional relevance

• Genetic Manipulation Approaches:

  • Method: Development of crcB knockout and overexpression strains in P. carbinolicus

    • CRISPR-Cas9 or traditional homologous recombination for gene deletion

    • Inducible expression systems for controlled overexpression

  • Phenotypic analysis of mutants under various growth conditions

  • Expected outcomes: Direct evidence of CrcB's role in specific stress responses

• Interspecies Comparative Genomics:

  • Method: Analysis of crcB conservation, genomic context, and evolutionary patterns across related species

    • Comparison with P. acetylenicus and other syntrophic bacteria

    • Correlation with ecological niches and stress resistance profiles

  • Expected outcomes: Evolutionary insights into CrcB's role in environmental adaptation

• Metabolomic and Physiological Assessments:

  • Method: Comparative metabolomic analysis of wild-type and crcB mutant strains

    • GC-MS or LC-MS metabolite profiling

    • Membrane potential measurements

    • Ion flux quantification

  • Expected outcomes: Identification of metabolic pathways affected by CrcB function

This approach would bridge the gap between the known molecular characteristics of CrcB (123 amino acids, transmembrane structure) and its potential ecological roles in P. carbinolicus adaptation to challenging environments, particularly in the context of syntrophic relationships with methanogens .

What computational approaches can predict functional interactions between CrcB homolog and other proteins in P. carbinolicus metabolic networks?

Advanced computational approaches can provide valuable insights into CrcB's functional interactions within P. carbinolicus metabolic networks:

These computational approaches should be integrated with experimental validation, particularly focusing on predicted interactions with the ethanol oxidation pathway and syntrophic metabolism components identified in P. carbinolicus . Such integration would provide testable hypotheses regarding CrcB's role in this metabolically specialized bacterium.

How does the CrcB homolog compare with other fluoride channels in terms of selectivity and transport mechanisms?

Comparative analysis of P. carbinolicus CrcB homolog with other fluoride channels reveals distinct features in selectivity and transport mechanisms:

The selectivity mechanism in CrcB proteins typically involves:

• A narrow pore diameter (~2.5-3.0 Å) that excludes larger anions

• Strategically positioned polar residues that coordinate F⁻

• Electropositive microenvironment from partial positive charges of helix dipoles

While the exact transport mechanism of P. carbinolicus CrcB remains to be experimentally determined, sequence analysis suggests it likely functions similarly to other bacterial CrcB proteins as a passive fluoride channel rather than an active transporter. The conserved amino acid sequence pattern in transmembrane regions (rich in glycine and alanine) provides the structural framework for the ion permeation pathway .

What insights can comparative genomics provide about the evolutionary history of CrcB in Pelobacter and related genera?

Comparative genomics analysis of CrcB across Pelobacter and related genera reveals important evolutionary patterns:

• Phylogenetic Distribution and Conservation:

  • CrcB homologs are widespread across Deltaproteobacteria, including Pelobacter, Geobacter, and Desulfovibrio

  • Core sequence motifs are highly conserved, particularly in transmembrane regions

  • Sequence identity between P. carbinolicus CrcB and other Deltaproteobacteria ranges from 40-85%

  • The highest conservation occurs with closely related syntrophic organisms

• Genomic Context Evolution:

  • In P. carbinolicus, crcB (Pcar_0981) exists in a genomic neighborhood that differs from non-syntrophic relatives

  • Syntrophic species show evidence of genomic rearrangements around crcB, suggesting adaptive evolution

  • Gene neighborhood analysis reveals potential co-evolution with specific metabolic genes

• Selective Pressure Analysis:

  • Transmembrane regions show stronger sequence conservation (purifying selection)

  • Loop regions display higher variability, suggesting different selective pressures

  • dN/dS ratios indicate primarily purifying selection, with potential episodic diversifying selection similar to patterns observed in REC domain proteins

• Horizontal Gene Transfer Assessment:

  • Codon usage analysis suggests potential horizontal gene transfer events in some lineages

  • GC content comparison between crcB and genomic averages reveals integration history

  • No clear evidence of recent horizontal acquisition in P. carbinolicus

These evolutionary patterns suggest that CrcB represents an ancient protein family that has been maintained throughout bacterial evolution with functional constraints on the transmembrane regions responsible for ion selectivity and permeation. The protein's conservation across diverse bacterial lineages, including specialists like P. carbinolicus, underscores its fundamental importance in cellular physiology, likely extending beyond simple fluoride resistance to roles in broader ion homeostasis during specialized metabolic processes .

How does the research on P. carbinolicus CrcB inform our understanding of bacterial adaptation to extreme environments?

Research on P. carbinolicus CrcB provides valuable insights into bacterial adaptation to extreme environments through several key mechanisms:

• Anaerobic Niche Specialization:

  • P. carbinolicus inhabits strictly anaerobic environments where syntrophic relationships with methanogens are essential for survival

  • CrcB likely contributes to membrane integrity and ion homeostasis under these specialized conditions

  • The protein may facilitate adaptation to the low energy yield characteristic of syntrophic metabolism

  • This represents a distinct adaptive strategy compared to aerobic extremophiles

• Stress Response Integration:

  • CrcB's presumed function in fluoride export represents an ancient detoxification mechanism

  • This function may be repurposed in P. carbinolicus for broader ion homeostasis during:

    • Fluctuating salt concentrations

    • pH changes common in anaerobic sediments

    • Metabolite accumulation during syntrophic growth

• Metabolic Adaptation Mechanisms:

  • P. carbinolicus demonstrates specialized metabolic pathways for acetaldehyde degradation and ethanol oxidation

  • CrcB may play supporting roles in these pathways by:

    • Maintaining optimal intracellular conditions for enzyme function

    • Contributing to energy conservation through proper membrane potential

    • Potentially interacting with specialized enzyme systems like tungsten-dependent enzymes

• Evolutionary Implications:

  • The conservation of CrcB across diverse bacterial lineages suggests fundamental importance

  • P. carbinolicus represents a case study in how ancient protein families can be integrated into highly specialized metabolic systems

  • This exemplifies the concept of "tinkering" in evolution, where existing components are repurposed for new functions

The study of P. carbinolicus CrcB extends our understanding beyond traditional extremophiles (thermophiles, halophiles, acidophiles) to include organisms adapted to energy-limited anaerobic niches, highlighting how membrane transport systems contribute to bacterial survival in diverse challenging environments .

What potential biotechnological applications could emerge from research on P. carbinolicus CrcB homolog?

Research on P. carbinolicus CrcB homolog could enable several innovative biotechnological applications:

• Bioremediation Enhancement:

  • Development of engineered bacteria with modified CrcB for improved halogen contamination remediation

  • Creation of biosensors using CrcB-based detection systems for environmental fluoride monitoring

  • Enhancement of syntrophic consortia for degradation of recalcitrant pollutants by optimizing ion transport

• Synthetic Biology Tools:

  • Design of genetic circuits using CrcB as a selective marker for fluoride-based selection systems

  • Development of membrane protein expression platforms optimized for challenging membrane proteins

  • Creation of customized ion channels with altered selectivity based on CrcB structure

• Anaerobic Bioprocessing:

  • Enhancement of syntrophic microbial communities for biofuel production

  • Improvement of methane generation in anaerobic digesters through engineered interspecies interactions

  • Development of novel fermentation processes for specialized biochemicals

• Structural Biology Advances:

  • Use of CrcB as a model system for studying membrane protein folding and stability

  • Platform for testing novel membrane protein crystallization methods

  • Template for computational design of synthetic ion channels

Implementation would involve genetic engineering of P. carbinolicus or heterologous expression of modified CrcB variants in industrial strains, with experimental validation focusing on enhanced ion transport properties and integration with metabolic engineering approaches for optimized performance in specific applications.

How might research on P. carbinolicus CrcB contribute to understanding microbial community dynamics in anaerobic environments?

Research on P. carbinolicus CrcB offers significant potential for understanding complex microbial community dynamics in anaerobic environments:

• Syntrophic Relationship Mechanics:

  • CrcB may influence the stability and efficiency of syntrophic partnerships between P. carbinolicus and methanogens like Methanospirillum hungatei

  • Investigation of ion transport during interspecies electron transfer could reveal:

    • Critical homeostatic mechanisms that enable syntrophy

    • Molecular adaptations that facilitate efficient energy conservation

    • Potential signaling mechanisms between syntrophic partners

• Ecological Niche Differentiation:

  • Comparative analysis of CrcB variants across related species may explain:

    • Distinct substrate preferences (e.g., P. carbinolicus vs. P. acetylenicus)

    • Adaptation to specific microenvironments within sediments

    • Competitive advantages in diverse anaerobic communities

• Community Resilience Factors:

  • Understanding CrcB's role in stress response could help explain:

    • Community stability during environmental fluctuations

    • Recovery mechanisms after perturbation

    • Succession patterns in anaerobic ecosystems

• Methodological Approaches:

  • Functional genomics: Correlate crcB expression with community composition

  • Meta-transcriptomics: Track crcB expression across community members

  • Stable isotope probing: Link CrcB function to specific metabolic processes

  • Fluorescence in situ hybridization: Visualize spatial relationships between species

This research direction would connect molecular mechanisms to ecosystem-level processes, potentially revealing how fundamental cellular functions like ion transport contribute to the emergent properties of complex microbial communities in environments ranging from freshwater sediments to anaerobic digesters and the human gut microbiome.

What emerging technologies might advance our understanding of membrane proteins like P. carbinolicus CrcB in the next decade?

Several emerging technologies promise to revolutionize our understanding of membrane proteins like P. carbinolicus CrcB in the coming decade:

• Structural Biology Breakthroughs:

  • Cryo-electron microscopy advancements:

    • Improved detectors and processing algorithms pushing resolution below 2Å

    • Microcrystal electron diffraction (MicroED) for small membrane protein crystals

    • Time-resolved cryo-EM capturing conformational changes during transport

  • Integrative structural biology approaches:

    • Combining NMR, X-ray crystallography, and computational methods

    • In-cell structural determination techniques

    • Advanced EPR methods for membrane protein dynamics

• Advanced Computational Methods:

  • AI-driven structure prediction:

    • Further refinement of AlphaFold and RoseTTAFold for membrane proteins

    • Specialized neural networks for transmembrane domain interactions

    • Accurate prediction of protein-lipid interfaces

  • Molecular dynamics simulations:

    • Quantum mechanics/molecular mechanics (QM/MM) for ion coordination

    • Enhanced sampling techniques for rare transport events

    • Whole-cell simulations incorporating membrane protein function

• Single-Molecule Technologies:

  • Advanced fluorescence techniques:

    • Super-resolution microscopy below 10nm resolution

    • Single-molecule FRET for conformational dynamics

    • Correlative light and electron microscopy (CLEM) for contextual information

  • Electrical recording advances:

    • High-throughput patch-clamp arrays

    • Solid-state nanopores for single-channel recording

    • Graphene-based sensing platforms for ion transport

• Synthetic Biology and Engineering:

  • Cell-free membrane protein expression systems:

    • Customized lipid environments for optimal folding

    • Direct incorporation into nanodiscs or liposomes

    • High-throughput screening platforms

  • Genetically encoded sensors:

    • Ion-specific fluorescent reporters based on channel architecture

    • Optogenetic control of membrane protein function

    • In vivo monitoring of transport activity

• Multi-omics Integration:

  • Systems biology approaches connecting:

    • Proteomics of membrane microdomains

    • Lipidomics of protein-associated lipids

    • Metabolomics of transported substrates

    • Transcriptomics of regulatory networks

These technological advances will likely resolve long-standing questions about membrane protein function and regulation, potentially revealing unexpected roles for proteins like CrcB beyond their currently understood functions in ion transport .