Recombinant Hydrogenobaculum sp. Protein CrcB homolog (crcB)

How do expression systems for recombinant Hydrogenobaculum sp. CrcB affect protein yield and activity?

Expression of Hydrogenobaculum sp. CrcB is typically performed in E. coli expression systems due to their efficiency and cost-effectiveness . When expressing this thermophilic membrane protein, several methodological considerations impact yield and activity:

For optimal activity retention, expression temperature should be maintained between 18-25°C after induction, as higher temperatures may cause protein misfolding, particularly important for this thermophilic protein . Using specific detergents (DDM or LMNG at 1-2%) during purification is critical for maintaining proper folding and activity of this membrane protein.

What purification strategies are most effective for Hydrogenobaculum sp. CrcB homolog?

Purification of recombinant Hydrogenobaculum sp. CrcB requires specific approaches due to its membrane protein nature:

• Initial extraction: Membrane fractionation followed by solubilization using mild detergents (DDM 1%, LMNG 1%, or C12E8 0.5%)

• Affinity chromatography: Most preparations utilize His-tagged versions for initial purification via Ni-NTA or TALON resin

• Secondary purification: Size exclusion chromatography using Superdex 200 columns in buffers containing 0.05-0.1% detergent, 150 mM NaCl, 20 mM Tris pH 8.0

• Storage optimization: The protein should be stored in Tris-based buffer with 50% glycerol at -20°C or -80°C to maintain stability

For maximum purity (>95%), a combination of affinity chromatography followed by size exclusion is recommended, with yields typically reaching 2-5 mg of purified protein per liter of culture.

How can fluoride transport activity of recombinant CrcB be accurately measured in experimental systems?

Measuring fluoride transport activity of recombinant CrcB requires specialized methodologies:

• Liposome-based fluorescence assays: Reconstitute purified CrcB into liposomes loaded with a fluoride-sensitive fluorophore (PBFI or SNAFL derivatives). Fluoride transport is measured as changes in fluorescence upon addition of external fluoride .

• Electrode-based measurements: Using fluoride-selective electrodes to measure fluoride transport across proteoliposomes reconstituted with purified CrcB.

• Cell-based assays: E. coli or yeast strains deficient in endogenous fluoride exporters can be complemented with CrcB to assess functional activity by measuring growth in fluoride-containing media.

The most reliable methodology involves a combination of approaches, with the liposome-based assay providing quantitative kinetic parameters (Km ~0.2-0.5 mM for fluoride, Vmax ~20-50 nmol/min/mg protein) , while cell-based assays confirm physiological relevance.

What are the critical considerations when designing site-directed mutagenesis experiments for Hydrogenobaculum sp. CrcB?

When designing site-directed mutagenesis experiments for Hydrogenobaculum sp. CrcB:

• Target residue selection:

  • Focus on conserved residues in fluoride-binding site (typically polar residues)

  • Target transmembrane helices at positions 25-45 and 90-110 in the amino acid sequence

  • Consider residues unique to thermophilic adaptation (compared to mesophilic homologs)

• Mutagenesis strategy:

  • Use PCR-based methods (QuikChange or Q5 site-directed mutagenesis)

  • Design primers with a minimum of 15-20 nucleotides flanking each side of the mutation

  • Consider codon optimization for E. coli expression

• Validation methodology:

  • Sequencing verification of the entire coding region

  • Expression level comparison (Western blot)

  • Localization assessment (membrane fraction verification)

  • Activity assays (fluoride transport)

• Control mutations:

  • Include known inactive mutants (e.g., Asn to Ala in conserved sites)

  • Include conservative substitutions as controls

This systematic approach ensures reliable structure-function relationships can be established for this thermophilic membrane transporter.

How should researchers approach crystallization trials for structural studies of Hydrogenobaculum sp. CrcB?

Crystallization of membrane proteins like CrcB presents significant challenges. A methodical approach includes:

• Pre-crystallization optimization:

  • Assess protein homogeneity by SEC-MALS and negative-stain EM

  • Screen detergents using thermal stability assays (TSA/CPM)

  • Test protein stability in various buffers (pH 6.0-8.5) and salt concentrations (100-500 mM)

• Crystallization strategy:

  • Vapor diffusion (hanging or sitting drop) with 1:1 protein:reservoir ratio

  • Lipidic cubic phase (LCP) methodology using monoolein or other lipid matrices

  • Initial screening at both 4°C and 20°C

• Recommended screening conditions:

  • Detergent: DDM (0.03-0.05%), LMNG (0.01%), or replacement with amphipols

  • Protein concentration: 5-15 mg/ml

  • Additives: Fluoride analogs (0.5-5 mM) or transport inhibitors

  • Specialized screens: MemGold, MemSys, MemStart

• Optimization approaches:

  • Microseeding from initial crystals

  • Dehydration protocols

  • Lipid:protein ratio optimization in LCP

Successful crystallization typically requires screening hundreds of conditions and optimizing promising leads through multiple rounds of refinement.

How does the thermostability of Hydrogenobaculum sp. CrcB compare to mesophilic homologs, and what structural features contribute to thermoadaptation?

The Hydrogenobaculum sp. CrcB homolog exhibits significant thermostability compared to mesophilic homologs, reflecting adaptations to the thermophilic lifestyle of its source organism :

Structural features contributing to thermostability include:

• Amino acid composition: Higher proportion of charged residues (Arg, Glu) forming salt bridges in thermophilic CrcB

• Secondary structure elements: More compact transmembrane helices with enhanced hydrophobic packing

• Loop regions: Shorter connecting loops between transmembrane domains (reduced by 2-4 amino acids compared to mesophilic homologs)

• Core interactions: Increased number of ionic interactions stabilizing the protein fold

These adaptations allow Hydrogenobaculum sp. CrcB to maintain structural integrity and function at the elevated temperatures of its native geothermal habitat, without compromising the conformational flexibility needed for transport activity .

What approaches are recommended for studying protein-lipid interactions of Hydrogenobaculum sp. CrcB?

Studying protein-lipid interactions of CrcB requires specialized techniques:

• Molecular dynamics (MD) simulations:

  • Coarse-grained simulations to identify preferential lipid binding sites

  • All-atom simulations with specific lipid compositions

  • Parameters should reflect thermophilic conditions (50-80°C)

• Experimental approaches:

  • Native mass spectrometry to identify co-purifying lipids

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map lipid-protein interfaces

  • Fluorescence-based assays using labeled lipids to determine binding affinities

• Functional assessment:

  • Reconstitution in defined lipid compositions to correlate lipid environment with transport activity

  • Systematic evaluation of lipid headgroups and acyl chain lengths

  • Temperature-dependent activity in different lipid environments

• Data analysis framework:

  • Plot lipid-dependence curves for activity vs. lipid composition

  • Calculate apparent binding constants for specific lipids

  • Develop thermodynamic models of lipid-protein interactions

This multi-faceted approach provides a comprehensive understanding of how membrane environment affects CrcB structure and function, particularly important for this thermophilic protein that likely operates in specialized lipid environments in vivo.

How can researchers integrate computational and experimental approaches to elucidate the fluoride transport mechanism of CrcB?

An integrated computational-experimental approach for elucidating CrcB's transport mechanism includes:

• Computational methods:

  • Homology modeling based on known CrcB structures

  • Molecular dynamics simulations of ion permeation

  • Free energy calculations for fluoride binding and transport

  • Markov state modeling of transport cycles

• Key experimental validations:

  • Site-directed mutagenesis of predicted key residues

  • Ion selectivity measurements (F⁻ vs. Cl⁻ vs. other ions)

  • pH-dependence profiles of transport activity

  • Voltage-dependence using electrophysiology

• Integration framework:

  • Use experimental constraints to refine computational models

  • Predict new mutations based on computational insights

  • Iteratively improve models through experimental validation

Example workflow table:

This iterative approach has successfully elucidated transport mechanisms in similar channel proteins and can be applied to understand CrcB's unique fluoride transport properties.

What NIH Guidelines requirements apply to research with recombinant Hydrogenobaculum sp. CrcB?

Research involving recombinant Hydrogenobaculum sp. CrcB homolog must adhere to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. The specific requirements depend on the experimental details:

• Risk assessment classification:

  • CrcB expression typically falls under Section III-D or III-E of the NIH Guidelines

  • Hydrogenobaculum sp. is considered a Risk Group 1 organism (not associated with disease in healthy adults)

• Institutional approval requirements:

  • Institutional Biosafety Committee (IBC) registration and approval required prior to initiation

  • Experiments must be registered with the IBC through appropriate institutional channels

• Containment requirements:

  • Standard work with CrcB typically requires Biosafety Level 1 (BSL-1) containment

  • Large-scale production (>10 liters) requires additional review per Section III-D-6

• Documentation requirements:

  • Detailed experimental protocols

  • Risk assessment documentation

  • Training records for laboratory personnel

Researchers should consult their institution's IBC for specific guidance, as requirements may vary slightly between institutions while still maintaining compliance with NIH Guidelines .

What are the most effective detergents and conditions for solubilizing recombinant Hydrogenobaculum sp. CrcB while maintaining function?

Effective solubilization of functional CrcB requires careful selection of detergents and conditions:

Critical buffer components for function preservation:

• pH range: Optimal stability at pH 7.5-8.0

• Salt concentration: 150-300 mM NaCl provides optimal stability

• Stabilizing additives:

  • Glycerol (10-20%) for short-term storage

  • 50% glycerol for long-term storage at -20°C/-80°C

  • 5-10 mM fluoride (substrate) can improve stability

Solubilization protocol optimization:

• Temperature: Perform extraction at 4°C despite thermostability

• Time: 1-2 hours for efficient extraction

• Agitation: Gentle rotation rather than vortexing

Functional assays should be performed immediately after purification to confirm that the protein retains transport activity following the solubilization process.

How can researchers effectively troubleshoot expression and purification issues with recombinant Hydrogenobaculum sp. CrcB?

When encountering challenges with recombinant CrcB expression and purification, systematic troubleshooting approaches include:

• Low expression yield troubleshooting:

  Issue	Possible Causes	Solutions
  Poor growth	Toxicity of overexpressed membrane protein	Use C41/C43 E. coli strains; reduce IPTG concentration to 0.1-0.2 mM
  Inclusion body formation	Rapid expression overwhelming membrane insertion	Lower induction temperature (16-18°C); use slower promoters (trc vs. T7)
  Degradation	Protease activity	Add protease inhibitors; optimize extraction speed

• Purification challenges:

  Issue	Diagnostic Signs	Recommended Solutions
  Poor solubilization	Low protein in supernatant after detergent treatment	Try alternative detergent mixtures; increase extraction time to 3-4 hours
  Low binding to affinity resin	Target protein in flow-through	Verify tag accessibility; adjust imidazole in binding buffer (10-20 mM)
  Impurities/aggregation	Multiple bands or high MW smear on SDS-PAGE	Add secondary purification step (ion exchange or SEC)

• Functional activity issues:

  Problem	Verification Test	Remediation Strategy
  Loss of activity	Fluoride transport assay	Adjust detergent type/concentration; verify protein folding by circular dichroism
  Poor reconstitution	Proteoliposome incorporation efficiency	Optimize lipid:protein ratio; try different reconstitution detergents
  Unstable storage	Activity loss over time	Add glycerol and reducing agent; aliquot and avoid freeze-thaw cycles

This systematic approach helps identify the specific issues in the expression and purification pipeline, allowing targeted interventions to obtain functional recombinant Hydrogenobaculum sp. CrcB.

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) be optimized for studying conformational dynamics of Hydrogenobaculum sp. CrcB?

HDX-MS optimization for CrcB membrane protein analysis requires specialized approaches:

• Sample preparation considerations:

  • Detergent selection: Use MS-compatible detergents (DDM or LMNG at minimum concentrations)

  • Deuterium labeling conditions: Temperature-controlled labeling (4°C) with time points from 10 seconds to 4 hours

  • Quenching optimization: pH 2.5, 0°C conditions with reducing agents

• Digestion optimization:

  • Protease selection: Use pepsin immobilized on beads for optimal digestion

  • Online digestion: Flow rate of 50-100 μL/min through protease column

  • Optimize organic solvent percentage (typically 10-15% acetonitrile) to improve coverage

• MS analysis parameters:

  • Back-exchange correction: Use fully deuterated controls

  • Peptide identification coverage: Target >85% sequence coverage

  • Data acquisition: Use high-resolution MS (>60,000 resolution)

• Data interpretation for membrane proteins:

  • Correct for detergent effects on exchange rates

  • Compare exchange patterns in different functional states

  • Map data onto structural models of CrcB

For CrcB specifically, focus HDX-MS analysis on regions predicted to undergo conformational changes during transport cycle, particularly the cytoplasmic and periplasmic loop regions connecting transmembrane helices.

What are the recommended approaches for studying oligomerization states of Hydrogenobaculum sp. CrcB under various conditions?

Comprehensive analysis of CrcB oligomerization states requires multiple complementary techniques:

• In-solution techniques:

  • Analytical ultracentrifugation (AUC): Sedimentation velocity and equilibrium experiments

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Native mass spectrometry with careful detergent optimization

• Visualization methods:

  • Negative-stain electron microscopy

  • Atomic force microscopy of membrane-reconstituted protein

  • Blue native PAGE for initial oligomerization screening

• Chemical crosslinking approaches:

  • Homobifunctional crosslinkers (DSS, BS3) at varying concentrations

  • MS/MS analysis of crosslinked peptides to identify interaction interfaces

  • In vivo crosslinking to capture physiologically relevant states

Experimental conditions to test should include:

• Temperature variations (25°C, 37°C, 50°C, 65°C)

• Lipid environment effects (reconstitution in different lipid compositions)

• Ligand/substrate (fluoride) concentration effects

• pH effects (5.5-8.0 range)

These approaches collectively provide robust determination of the oligomeric state of CrcB and how it may change under different physiological conditions, which is critical for understanding its transport mechanism.

How can advanced bioinformatics approaches assist in analyzing the evolutionary relationships of CrcB homologs across thermophilic and mesophilic organisms?

Advanced bioinformatics approaches for evolutionary analysis of CrcB homologs include:

• Sequence-based analyses:

  • Multiple sequence alignment using MAFFT or T-Coffee with transmembrane-specific parameters

  • Phylogenetic tree construction using maximum likelihood (RAxML) or Bayesian methods (MrBayes)

  • Calculation of selection pressures (dN/dS ratios) across different lineages

  • Conservation analysis using ConSurf or similar tools

• Structure-informed evolutionary analyses:

  • Homology modeling of multiple CrcB homologs

  • Structural alignment to identify conserved structural elements

  • Analysis of co-evolving residue networks using methods like GREMLIN or EVcouplings

  • Integration of structural constraints in phylogenetic analyses

• Thermoadaptation-specific analyses:

  • Amino acid composition bias analysis between thermophilic and mesophilic homologs

  • Identification of signature positions correlated with thermostability

  • Calculation of stability energy differences between homologs

  • Machine learning approaches to identify thermostability determinants

• Data visualization and interpretation:

  • Mapping evolutionary data onto structural models

  • Creating heat maps of conservation across different functional domains

  • Network analysis of co-evolving residue clusters

  • Statistical validation of identified patterns