Recombinant Propionibacterium freudenreichii subsp. shermanii Chaperone protein ClpB (clpB), partial

What is the ClpB chaperone protein in Propionibacterium freudenreichii?

ClpB is a member of the AAA+ (ATPases Associated with various cellular Activities) superfamily of proteins found in P. freudenreichii. It functions as a molecular chaperone that helps protect cells from protein inactivation and aggregation, particularly during extreme stress conditions. The protein forms a hexameric ring structure and contains two nucleotide-binding sites per monomer, enabling ATP-dependent protein disaggregation and remodeling activities . In P. freudenreichii, ClpB collaborates with the DnaK chaperone system to dissolve protein aggregates and restore protein functionality following stress exposure.

Similar to homologs like E. coli ClpB and yeast Hsp104, P. freudenreichii ClpB is essential for cellular survival during extreme conditions, particularly heat stress. The protein's ability to disaggregate and reactivate previously aggregated proteins makes it critical for maintaining cellular homeostasis in this beneficial bacterium, which is increasingly studied for its probiotic potential .

How does the ClpB protein interact with the DnaK chaperone system?

The ClpB protein functions synergistically with the DnaK chaperone system in a collaborative mechanism to remodel proteins and dissolve aggregates. This interaction represents a sophisticated protein quality control system that enhances cellular resilience to stress. Experimental evidence demonstrates that while ClpB alone can perform protein remodeling under specific conditions (when its ATPase activity is asymmetrically slowed), the addition of the DnaK system significantly amplifies its remodeling capacity .

For functional collaboration between ClpB and the DnaK system, several requirements must be met:

• ATP must be available as an energy source

• Both nucleotide-binding sites of ClpB must be capable of hydrolyzing ATP

• The structural integrity of both proteins must be maintained for proper interaction

Studies utilizing protein remodeling assays with varying nucleotide conditions have confirmed that nonphysiological conditions (such as mixtures of ATP and ATPγS) can elicit remodeling activity by ClpB alone, but the maximum disaggregation efficiency is achieved only through proper collaboration with the DnaK system .

What molecular techniques are used to study ClpB function in P. freudenreichii?

Studying ClpB function in P. freudenreichii requires a multifaceted methodological approach spanning molecular biology, biochemistry, and advanced proteomics. Key techniques include:

Genetic manipulation techniques:

• Site-directed mutagenesis to create specific variants (e.g., nucleotide-binding domain mutations)

• Homologous recombination using suicide plasmids for gene knockout (similar to approaches used for slpB mutations in P. freudenreichii)

• Complete genome DNA sequencing to confirm genetic modifications and assess potential genomic rearrangements

Protein analysis methods:

• Recombinant protein expression and purification

• In vitro ATPase activity assays to measure nucleotide hydrolysis rates

• Protein remodeling assays using model substrates

• Surface extractable protein analysis using guanidine extraction followed by nanoLC-MS/MS

Functional characterization:

• Growth monitoring in various stress conditions (measuring OD650nm and CFU counts)

• Protein aggregation and disaggregation assays

• Co-immunoprecipitation to study protein-protein interactions

These methodologies provide complementary data that collectively illuminate the complex functions of ClpB in maintaining protein homeostasis in P. freudenreichii.

How can recombinant P. freudenreichii ClpB be expressed and purified for functional studies?

Expression and purification of recombinant P. freudenreichii ClpB requires careful optimization due to the protein's large size and tendency to form oligomeric structures. The following methodological approach is recommended:

Expression system selection:

• E. coli BL21(DE3) typically provides high-yield expression for P. freudenreichii proteins

• Expression vectors containing T7 promoters (pET series) with appropriate affinity tags (His6 or Strep-tag)

• Growth at lower temperatures (16-18°C) after induction to enhance proper folding

Purification protocol:

• Cell lysis using sonication or French press in buffer containing appropriate protease inhibitors

• Initial purification using affinity chromatography (Ni-NTA for His-tagged constructs)

• Size exclusion chromatography to separate hexameric forms from aggregates

• Ion exchange chromatography for final polishing

Functional validation:

• ATPase activity assay using colorimetric detection of released phosphate

• Oligomerization state verification using analytical ultracentrifugation or dynamic light scattering

• Protein disaggregation assays using model substrates like heat-denatured luciferase

Critical considerations:

• Maintain ATP in buffers during purification to stabilize hexameric structure

• Include reducing agents to prevent oxidation of cysteine residues

• Verify protein activity immediately after purification, as storage may affect functionality

This protocol yields functionally active recombinant ClpB protein suitable for in vitro biochemical and structural studies.

What approaches can be used to investigate ClpB mutations in P. freudenreichii?

Investigating ClpB mutations in P. freudenreichii requires a comprehensive strategy combining genetic, biochemical, and phenotypic analyses. Based on approaches used for similar proteins like SlpB in P. freudenreichii, the following methodological framework is recommended:

Generation of ClpB mutants:

• Site-specific mutagenesis targeting conserved motifs in nucleotide-binding domains

• Gene disruption using homologous recombination with suicide plasmids carrying antibiotic resistance markers

• Verification of mutations through whole genome sequencing to confirm site-specific integration without affecting adjacent genes

Phenotypic characterization:

• Growth curve analysis under normal and stress conditions (heat, acid, oxidative stress)

• Cell surface physicochemical property measurements (ζ-potential, surface hydrophobicity)

• Stress tolerance assays comparing wild-type and mutant strains

Molecular analysis:

• Whole-cell proteomics using high-definition mass spectrometry to identify differentially expressed proteins

• RNA sequencing to detect changes in global gene expression patterns

• In silico analysis to determine if the clpB gene is part of an operon and potential polar effects of mutation

This integrated approach provides insights into the pleiotropic effects of ClpB mutations, similar to the comprehensive analysis performed for SlpB mutations in P. freudenreichii .

How does ATP hydrolysis affect ClpB function in protein disaggregation?

ATP hydrolysis is central to ClpB function, providing the energy required for protein disaggregation through a precisely coordinated mechanism. Research demonstrates that both nucleotide-binding domains (NBDs) of ClpB must be capable of ATP hydrolysis for optimal collaboration with the DnaK system in protein remodeling .

Key aspects of ATP hydrolysis in ClpB function:

• Asymmetric ATP hydrolysis: Studies show that when ClpB's ATPase activity is asymmetrically slowed (either by providing a mixture of ATP and ATPγS or by inactivating one NBD through mutation), the protein can perform limited remodeling independently of the DnaK system .

• Synergistic collaboration: Maximum disaggregation efficiency requires both:

  • ATP hydrolysis at both NBDs of ClpB

  • Functional interaction with the DnaK chaperone system

• Conformational changes: ATP binding and hydrolysis drive significant structural rearrangements within the hexameric ClpB complex, creating the mechanical force needed to extract polypeptides from aggregates.

Experimental evidence supporting ATP requirements:

• Protein remodeling assays show that non-hydrolyzable ATP analogs inhibit disaggregation activity

• Mutations in Walker A or Walker B motifs of either NBD significantly impair chaperone function

• Kinetic analysis reveals that optimal disaggregation requires coordinated ATP hydrolysis cycles

These findings highlight the critical role of ATP hydrolysis in powering the molecular machinery of ClpB, enabling its essential function in protein quality control within P. freudenreichii.

How do proteomic approaches reveal the impact of ClpB dysfunction in P. freudenreichii?

Proteomic analysis provides powerful insights into the pleiotropic effects of chaperone protein dysfunction in P. freudenreichii. Based on studies of similar proteins like SlpB, whole-cell quantitative proteomics using high-definition mass spectrometry can reveal global changes following ClpB mutation .

Recommended proteomic workflow:

• Sample preparation:

  • Harvest cells in stationary phase (approximately 76h, 2×10^9 CFU/mL)

  • Extract proteins using optimized lysis buffers containing protease inhibitors

  • Perform tryptic digestion and peptide purification

• Mass spectrometry analysis:

  • Utilize nanoLC-MS/MS with high-definition mass spectrometry (HDMSE)

  • Employ data-independent acquisition for comprehensive proteome coverage

  • Implement label-free quantification for comparative analysis

• Data analysis:

  • Identify proteins using established databases (UniProt, NCBI)

  • Apply stringent statistical analysis to determine significantly altered proteins

  • Utilize pathway enrichment analysis to identify affected cellular processes

Expected outcomes based on similar studies:
A comprehensive analysis would likely detect approximately 1,200-1,300 quantifiable proteins, representing ~50-55% of the theoretical proteome based on genome sequence data . In the case of SlpB mutation in P. freudenreichii, 97 proteins showed significant differential expression between wild-type and mutant strains, affecting various cellular processes including signaling, metabolism, and DNA repair/replication .

Protein functional categorization:
Based on patterns observed in similar studies, affected proteins after ClpB mutation might include:

This proteomic profiling would illuminate the central role of ClpB in cellular homeostasis and stress response networks in P. freudenreichii .

What are the structural dynamics of ClpB during protein disaggregation cycles?

The structural dynamics of ClpB during protein disaggregation cycles represent a complex choreography of conformational changes driven by ATP binding and hydrolysis. Advanced biophysical techniques reveal the molecular mechanism of this AAA+ protein as it engages and remodels protein aggregates.

Key structural transitions during the disaggregation cycle:

• Initial substrate recognition:

  • In collaboration with the DnaK system, ClpB recognizes and binds exposed hydrophobic regions in aggregated proteins

  • The N-terminal domain exhibits flexibility, allowing initial substrate engagement

• ATP-driven conformational changes:

  • ATP binding induces oligomerization into the active hexameric ring structure

  • Sequential ATP hydrolysis at the two nucleotide-binding domains (NBDs) generates conformational changes that create a power stroke

  • These power strokes apply mechanical force to extract polypeptides from aggregates

• Substrate translocation:

  • The central channel of the hexameric ring provides the pathway for substrate translocation

  • Conserved tyrosine residues in the central channel grip the substrate

  • Sequential ATP hydrolysis drives a peristaltic movement that pulls the substrate through the channel

Methodologies for studying ClpB structural dynamics:

Research indicates that the functional collaboration between ClpB and the DnaK system requires both nucleotide-binding sites of ClpB to hydrolyze ATP, suggesting a precisely coordinated structural mechanism . These structural transitions represent the molecular basis for ClpB's essential role in protein quality control within P. freudenreichii.

How does ClpB deficiency impact cellular stress response pathways in P. freudenreichii?

ClpB deficiency profoundly alters cellular stress response pathways in P. freudenreichii, triggering compensatory mechanisms and revealing the interconnected nature of protein quality control networks. Drawing parallels from studies on similar proteins and CLPB deficiency in other systems, we can predict several significant cellular impacts .

Global transcriptomic and proteomic changes:
ClpB deficiency would likely trigger comprehensive reprogramming of gene expression and protein levels, particularly affecting:

• Upregulation of alternative chaperone systems:

  • Increased expression of small heat shock proteins

  • Enhanced production of other AAA+ proteins to compensate for ClpB loss

  • Elevated levels of DnaK system components

• Metabolic adaptations:

  • Shift toward energy conservation pathways

  • Altered carbon metabolism

  • Reduced growth rate and biomass production

• Stress response modulation:

  • Heightened sensitivity to thermal stress

  • Compromised acid stress resistance

  • Reduced tolerance to oxidative damage

Molecular consequences of ClpB deficiency:
Studies of CLPB variants in human systems provide insights into potential molecular mechanisms of dysfunction . Variants affecting mRNA splicing, like the c.1257+5G>A variant identified in human CLPB, highlight the importance of proper protein expression . Similar splicing defects in P. freudenreichii ClpB would likely result in truncated or absent protein, abolishing disaggregation activity.

Practical implications for P. freudenreichii as a probiotic:
The compromised stress response in ClpB-deficient strains would likely impact:

What methodologies can detect functional interactions between ClpB and other molecular chaperones?

Detecting and characterizing functional interactions between ClpB and other molecular chaperones, particularly the DnaK system, requires sophisticated methodological approaches spanning biochemical, biophysical, and cellular techniques. These methods collectively reveal the synergistic collaboration essential for protein disaggregation.

In vitro interaction analysis techniques:

• Co-immunoprecipitation (Co-IP):

  • Precipitate ClpB using specific antibodies and detect co-precipitating chaperones

  • Alternatively, precipitate DnaK system components and identify bound ClpB

  • Western blotting confirms specific interactions between chaperone systems

• Surface plasmon resonance (SPR):

  • Immobilize ClpB on sensor chips and measure binding kinetics of DnaK, DnaJ, and GrpE

  • Determine association and dissociation rates under various nucleotide conditions

  • Quantify how mutations affect binding affinity between chaperone systems

• Förster resonance energy transfer (FRET):

  • Label ClpB and DnaK system components with compatible fluorophores

  • Monitor real-time interactions through changes in fluorescence

  • Track dynamic association during protein disaggregation reactions

Functional collaboration assays:

• Protein disaggregation assays:

  • Measure reactivation of heat-aggregated model substrates (e.g., luciferase, GFP)

  • Compare activity of individual chaperones versus combined systems

  • Test effects of varying ATP:ATPγS ratios on disaggregation efficiency

• ATP hydrolysis coupling:

  • Measure ATPase activity using malachite green or NADH-coupled assays

  • Determine how substrate and co-chaperone presence affects hydrolysis rates

  • Compare wild-type ClpB with nucleotide-binding site mutants

Cellular visualization methods:

• Bimolecular fluorescence complementation:

  • Express ClpB and DnaK tagged with complementary fragments of a fluorescent protein

  • Interaction brings fragments together, generating fluorescence

  • Visualize interactions in living P. freudenreichii cells

• Proximity ligation assay:

  • Use antibodies against ClpB and DnaK system components

  • Secondary antibodies linked to oligonucleotides enable signal amplification

  • Fluorescent signals indicate proteins in close proximity (<40nm)

Research using these methodologies has demonstrated that ClpB and the DnaK system act synergistically, with both nucleotide-binding sites of ClpB requiring ATP hydrolysis capability for functional collaboration . These approaches provide a comprehensive toolkit for investigating the molecular mechanisms underlying chaperone cooperation in protein quality control systems.

What are the future research directions for P. freudenreichii ClpB studies?

The investigation of Propionibacterium freudenreichii ClpB protein presents numerous promising research avenues that could significantly advance our understanding of protein quality control systems and probiotic mechanisms. Based on current knowledge gaps and emerging technologies, several key research directions warrant focused attention.

Structural biology approaches:
Advanced structural studies using cryo-electron microscopy and integrative structural biology techniques could reveal the precise molecular architecture of P. freudenreichii ClpB and its conformational changes during the disaggregation cycle. These studies would illuminate species-specific features that might differentiate P. freudenreichii ClpB from better-characterized homologs in E. coli and yeast.

Substrate specificity investigations:
Determining which P. freudenreichii proteins depend on ClpB for disaggregation would provide insights into the specific cellular processes most vulnerable to aggregation in this organism. Proteomic identification of ClpB-dependent substrates under various stress conditions could reveal previously unknown connections between protein quality control and probiotic functionality.

Genetic regulation studies:
Understanding the transcriptional and post-transcriptional regulation of ClpB expression in P. freudenreichii would elucidate how this beneficial bacterium modulates its protein quality control systems in response to environmental changes. This knowledge could inform strategies for enhancing stress tolerance in probiotic formulations.

Translational applications:
Exploring the relationship between ClpB function and probiotic properties could lead to rational strain development for enhanced therapeutic applications. Similar to findings with SlpB protein, where mutation revealed pleiotropic effects on probiotic properties , engineered ClpB variants might offer improved stress resistance or novel functional characteristics.

System-level integration:
Investigating how ClpB cooperates with other chaperone systems beyond the DnaK system would provide a more comprehensive understanding of the protein quality control network in P. freudenreichii. This systems biology approach could reveal redundancies and specializations within the chaperone network that contribute to this organism's remarkable stress tolerance.

These research directions collectively address fundamental questions about ClpB function while offering practical insights for probiotic applications. The multidisciplinary nature of these approaches highlights the need for collaborative research combining expertise in structural biology, proteomics, microbiology, and synthetic biology.

How can knowledge of ClpB function contribute to improved P. freudenreichii-based probiotics?

Understanding ClpB function in Propionibacterium freudenreichii offers strategic opportunities to enhance the development and efficacy of P. freudenreichii-based probiotics. The central role of ClpB in stress tolerance and protein homeostasis directly impacts probiotic viability and functionality in therapeutic applications.

Process optimization:
Understanding how ClpB contributes to survival during manufacturing processes can inform optimization strategies. Specific stress preconditioning regimens that upregulate ClpB expression prior to lyophilization or encapsulation could enhance survival rates and extend shelf-life of probiotic formulations.

Genetic improvement strategies:
Targeted enhancement of ClpB expression or activity through precision genetic engineering could produce P. freudenreichii strains with superior stress tolerance. This approach might include:

• Promoter engineering to enhance ClpB expression under specific conditions

• Codon optimization to improve translation efficiency

• Point mutations based on structure-function knowledge to enhance disaggregation activity

Synbiotic formulations:
Knowledge of how ClpB function affects metabolic capabilities could guide the development of synbiotic combinations that provide substrates supporting optimal ClpB activity, thereby enhancing probiotic survival and functionality in the gastrointestinal environment.

Clinical applications:
Understanding the relationship between ClpB function and specific probiotic properties could enable the development of specialized P. freudenreichii strains for targeted health applications. For example, strains with optimized ClpB activity might offer enhanced immunomodulatory properties similar to those observed with P. freudenreichii CIRM-BIA 129, which has shown promising effects in inflammatory bowel disease models .