Recombinant Chaperone protein ClpB 2 (clpB2), partial

What is Chaperone protein ClpB 2 and what is its primary function in cellular systems?

ClpB2 is a molecular chaperone belonging to the AAA+ (ATPases associated with various cellular activities) family of proteins. It functions as a disaggregase that collaborates with the DnaK/Hsp70 chaperone system to rescue proteins from aggregated states. ClpB's primary function is resolving protein aggregates formed during stress conditions, particularly heat shock, by using ATP hydrolysis to extract and unfold proteins from these aggregates .

Unlike standard chaperones that prevent aggregation, ClpB specializes in recovering already aggregated proteins, making it crucial for cellular stress tolerance and thermotolerance. In bacterial systems like E. coli, ClpB works synergistically with the DnaK-DnaJ-GrpE chaperone team to disaggregate and reactivate insoluble aggregated proteins that form during stress conditions .

How is the ClpB2 protein structured, and how does this structure facilitate its function?

ClpB has a modular structure consisting of multiple domains that contribute to its function:

• N-terminal domain: Involved in substrate binding and interaction with aggregated proteins

• Two ATP-binding domains (NBD1 and NBD2): Arranged in separate rings in the hexameric structure

• Middle domain (M-domain): Unique to Hsp100 disaggregases, critical for interaction with DnaK/Hsp70

• C-terminal domain: Important for oligomerization and proper assembly

The full-length ClpB forms hexameric ring structures in an ATP-dependent manner. These hexamers are the functional units that thread aggregated proteins through their central channel during the disaggregation process .

Table 1: Functional Domains of ClpB and Their Roles

What are the optimal conditions for expressing soluble recombinant ClpB2 in E. coli?

Expressing soluble recombinant ClpB2 requires optimization of several parameters. The two-step procedure has been shown to be more effective than one-step approaches for many recombinant proteins including ClpB .

Optimal Two-Step Procedure:

• Initial expression phase: Induce with appropriate concentration of IPTG

• Folding period: Add chloramphenicol to inhibit protein synthesis while allowing folding for 2 hours at 20°C

• Co-expression with chaperones: Particularly effective combinations include KJE (DnaK, DnaJ, GrpE), ClpB, and ELS (GroEL, GroES)

Temperature control is critical, with lower temperatures (20-30°C) generally favoring proper folding. The choice of E. coli strain can also significantly impact soluble expression, with BL21(DE3) and derivatives being common choices .

How can co-expression of chaperones enhance the solubility of recombinant ClpB2?

Co-expression of complementary chaperone systems has been demonstrated to dramatically improve the solubility and proper folding of recombinant proteins. For ClpB2 specifically, the following approaches have shown success:

• Co-expression with the DnaK-DnaJ-GrpE chaperone team significantly enhances solubility. Even a modest excess (two to three times the wild-type level) can prevent aggregation .

• Co-expression with GroEL-GroES can also be effective, though it typically requires higher expression levels (5-10 times the wild-type level) .

• Combined co-expression of both chaperone teams (DnaK-DnaJ-GrpE and GroEL-GroES) often provides synergistic effects, particularly for preventing aggregation .

The optimal strategy involves controlled, regulated expression of these chaperone systems. Using separate promoters that can be independently induced allows fine-tuning of chaperone expression levels relative to the target protein .

What are the recommended storage conditions to maintain the activity of purified recombinant ClpB2?

For optimal preservation of ClpB2 activity, the following storage conditions are recommended:

• Storage temperature: -20°C/-80°C

• Buffer composition: Addition of 5-50% glycerol as a cryoprotectant (50% glycerol is commonly used)

• Aliquoting: Divide into small aliquots to avoid repeated freeze-thaw cycles

• Short-term storage: Working aliquots can be maintained at 4°C for up to one week

• Long-term stability: Lyophilized form typically maintains activity for 12 months at -20°C/-80°C, while liquid form is stable for approximately 6 months

Repeated freezing and thawing should be avoided as it can lead to protein denaturation and activity loss. For maximum stability, the protein should be stored in appropriate buffer conditions with stabilizing agents .

What assays can be used to measure the chaperone and ATPase activity of recombinant ClpB2?

Several established assays can be used to assess different aspects of ClpB2 function:

ATPase Activity Assays:

• Malachite green assay: Quantifies inorganic phosphate released during ATP hydrolysis

• Coupled enzyme assays: Using pyruvate kinase and lactate dehydrogenase to measure ATP consumption through NADH oxidation

• Casein-stimulated ATPase assay: Measures the activation of ClpB ATPase activity by casein

Disaggregation Activity Assays:

• GFP reactivation assay: Heat-inactivated GFP is used as a substrate, with recovery of fluorescence indicating successful disaggregation

• Luciferase reactivation assay: Heat-denatured firefly luciferase is used as a substrate, with recovery of luminescence activity indicating successful disaggregation and refolding

• MDH (malate dehydrogenase) disaggregation assay: Measures the recovery of enzymatic activity of aggregated MDH

• RepA activation assay: Measures the conversion of inactive RepA dimers to active DNA-binding monomers

Oligomerization Assays:

• Size-exclusion chromatography: Assesses ATP-dependent hexamer formation

• Analytical ultracentrifugation: Determines oligomeric state and association constants

• Dynamic light scattering: Measures changes in particle size upon oligomerization

How can I determine if truncated variants of ClpB2 retain functional activity?

To assess the functionality of truncated ClpB2 variants:

• Compare ATPase activity: Measure basal and substrate-stimulated ATPase activity of the truncated variant versus full-length protein. C-terminal truncation typically decreases ATPase activity, while N-terminal truncation may not affect basal ATPase but reduces casein-stimulated activity .

• Evaluate oligomerization: Use size-exclusion chromatography or analytical ultracentrifugation to determine if the truncated variant can form proper oligomeric structures. C-terminal truncation significantly impairs self-association and hexamer formation .

• Assess chaperone activity: Test the ability of the truncated variant to reactivate model substrates like heat-denatured luciferase or GFP. Both N- and C-terminal truncations typically impair chaperone activity but for different mechanistic reasons .

• Examine cooperation with the DnaK system: Test whether the truncated variant can still function synergistically with the DnaK-DnaJ-GrpE system in disaggregation assays .

Research has shown that both terminal regions of ClpB are essential for chaperone activity, but they contribute differently: the N-terminal region is primarily involved in interactions with protein substrates, while the C-terminal region supports protein self-association .

How does ClpB2 utilize ATP during the protein disaggregation process?

The mechanism of ATP utilization by ClpB during protein disaggregation is complex and adaptable:

• ATP binding induces conformational changes that promote oligomerization into hexameric rings.

• ATP hydrolysis drives the mechanical force needed to extract polypeptides from aggregates.

• Mode of ATP utilization varies depending on:

  • Substrate properties

  • Presence or absence of the DnaK system

Research using heterohexamers of wild-type and ATPase-deficient ClpB mutants has revealed two distinct mechanisms:

• Probabilistic mechanism: When working alone, ClpB can function with approximately three active and three inactive protomers, with optimal activity when there are ~6 active ATPase sites distributed throughout the hexamer .

• Sequential mechanism: When working with the DnaK system on aggregated substrates, ClpB appears to utilize ATP in a sequential or semi-sequential manner. In this case, incorporation of even a single inactive subunit significantly impairs disaggregation activity .

This adaptable ATP utilization mechanism may allow ClpB to address different substrate needs efficiently. The location of active ATP sites within the hexamer appears less important than the total number of active sites available .

What is the precise role of the N-terminal and C-terminal domains in ClpB2 function?

Research comparing full-length and truncated ClpB variants has provided clear insights into the distinct roles of the terminal domains:

N-terminal domain:

• Functions as a substrate-binding interface for aggregated proteins

• Critical for the activation of ClpB ATPase by substrates like casein

• Deletion of this domain does not inhibit self-association or basal ATPase activity

• N-terminally truncated variants show decreased ability to respond to substrate proteins

C-terminal domain:

• Essential for proper oligomerization and hexamer formation

• Supports ATP binding and subsequent ATPase activity

• Deletion severely impairs protein self-association

• C-terminally truncated variants show significantly decreased affinity for ATP and reduced ATPase activity

These findings indicate that while both domains are essential for full chaperone activity, they contribute through different mechanisms: the N-terminal domain primarily mediates substrate interactions, while the C-terminal domain enables the formation of functional oligomeric structures required for activity .

How does the cooperation between ClpB and the DnaK chaperone system function at the molecular level?

The cooperation between ClpB and the DnaK chaperone system is a sophisticated molecular mechanism that enhances protein disaggregation efficiency:

• Initial aggregate recognition: The DnaK system (DnaK, DnaJ, GrpE) binds to exposed hydrophobic regions on protein aggregates.

• Partial extraction: DnaK performs initial remodeling of the aggregate surface, loosening the structure and creating binding sites for ClpB.

• ClpB recruitment: DnaK directly interacts with the M-domain of ClpB, recruiting it to the aggregate surface.

• Coordinated disaggregation: ClpB and DnaK work synergistically:

  • ClpB uses ATP hydrolysis to extract polypeptides from the aggregate

  • DnaK assists in both extraction and subsequent refolding of the substrate

• Polypeptide handover: Extracted polypeptides are transferred between the chaperone systems during the process.

This cooperation is highly specific, as demonstrated by experiments showing that both chaperone teams (DnaK-DnaJ-GrpE and ClpB) are critically involved in protein refolding but in distinct ways. While ClpB can perform some remodeling activities independently, the presence of the DnaK system dramatically enhances its effectiveness in disaggregating stable protein aggregates .

Experimental evidence suggests these systems have synergistic roles in preventing protein aggregation, particularly important in ΔrpoH mutants that lack the heat shock response regulator .

How can recombinant ClpB2 be employed in biotechnology applications to enhance protein production?

Recombinant ClpB2, especially when used in coordination with other chaperone systems, offers several applications in biotechnology:

• Enhanced recombinant protein production: Co-expression of ClpB with the DnaK and GroEL chaperone systems can significantly increase yields of soluble, correctly folded recombinant proteins in bacterial expression systems .

• Protein disaggregation platform: ClpB-based systems can be developed for in vitro disaggregation and recovery of aggregated valuable proteins from inclusion bodies .

• Stress-resistant expression hosts: Engineering expression strains with regulated ClpB and complementary chaperone expression can create platforms for producing difficult-to-express proteins .

Research has shown that chaperone co-overproduction is successful in improving solubility for approximately 50% of tested recombinant proteins, with the combination of KJE, ClpB, and ELS (GroEL/ES) being the most effective .

Table 2: Effectiveness of Two-Step Procedure with Different Chaperone Combinations

Data compiled from research findings in .

What experimental considerations are important when designing studies involving ClpB2 and protein aggregation?

When designing experiments with ClpB2 for protein aggregation studies, several critical factors should be considered:

• ATP concentration and regeneration system:

  • Maintain sufficient ATP levels (typically 2-5 mM)

  • Include an ATP regeneration system (e.g., creatine phosphate/creatine kinase)

  • Consider testing different ATP:ADP ratios to understand nucleotide effects

• Chaperone ratios and concentrations:

  • Optimal ClpB:substrate ratios typically range from 1:2 to 1:10 (molar basis)

  • When using the DnaK system, maintain appropriate stoichiometry (typically DnaK:DnaJ:GrpE at 10:2:5)

  • Control expression levels carefully as excessive chaperone expression can sometimes reduce target protein yield

• Temperature conditions:

  • Perform disaggregation reactions at physiologically relevant temperatures (usually 30-37°C)

  • Consider temperature-cycling protocols for difficult aggregates

  • Use lower temperatures (20°C) during protein expression phases

• Buffer composition:

  • Include appropriate divalent cations (Mg²⁺ is essential for ATP hydrolysis)

  • Control pH carefully (typically pH 7.0-7.5)

  • Consider crowding agents to mimic cellular conditions

• Appropriate controls:

  • Include ATPase-deficient ClpB mutants as negative controls

  • Test individual chaperone components separately to establish baseline activities

  • Include non-aggregated protein controls to establish maximum recovery levels

Why might recombinant ClpB2 form inclusion bodies during expression, and how can this be prevented?

The formation of inclusion bodies during ClpB2 expression can result from several factors:

• High expression rates overwhelming the cell's folding capacity

• Improper folding due to insufficient chaperone availability

• Domain misfolding due to the complex multi-domain structure of ClpB

To prevent inclusion body formation:

Expression optimization strategies:

• Reduce expression temperature to 16-25°C

• Decrease inducer concentration to slow expression rate

• Use rich media supplements (e.g., glucose/glycerol mixes)

• Consider auto-induction media instead of IPTG induction

• Implement the two-step procedure with a dedicated folding period

Co-expression strategies:

• Co-express with DnaK-DnaJ-GrpE chaperones (moderate levels)

• Co-express with GroEL-GroES chaperones (higher levels may be needed)

• Consider including IbpA/IbpB heat shock proteins which can stabilize partially folded intermediates until disaggregating chaperones become available

Host strain selection:

• Consider ΔrpoH mutant strains (lacking the heat shock sigma factor) supplemented with controlled chaperone expression, which have shown promising results for difficult proteins

• Test protease-deficient strains if degradation is an issue

What approaches can be used to study the ATP-dependent oligomerization of ClpB2?

Studying the ATP-dependent oligomerization of ClpB2 requires specialized techniques:

• Size-exclusion chromatography (SEC):

  • Monitor hexamer formation in the presence of ATP or ATP analogs

  • Analyze concentration-dependent oligomerization

  • Compare oligomerization of wild-type versus mutant variants

• Analytical ultracentrifugation:

  • Determine sedimentation coefficients in the presence/absence of nucleotides

  • Calculate molecular weights of different oligomeric species

  • Measure association constants for hexamer formation

• Fluorescence-based approaches:

  • Tryptophan fluorescence can report on conformational changes (ClpB contains two Trp residues, W462 and W543)

  • Fluorescence anisotropy to measure binding of labeled nucleotides

  • FRET between labeled ClpB monomers to track oligomerization dynamics

• Subunit mixing experiments:

  • Mix wild-type and mutant ClpB variants to create heterohexamers

  • Analyze the composition using tags or specific detection methods

  • Correlate heterohexamer composition with activity

Research has shown that ATP binding induces significant conformational changes that promote hexamer formation. The C-terminal domain plays a critical role in this process, as C-terminally truncated variants show severely impaired self-association .

How can researchers effectively create and study heterohexameric ClpB complexes for mechanistic insights?

Creating and studying heterohexameric ClpB complexes has provided valuable insights into the mechanism of ClpB function. This approach involves:

• Design of complementary ClpB variants:

  • Walker A mutants (K→T substitution): Unable to bind ATP

  • Walker B mutants (E→Q substitution): Can bind but not hydrolyze ATP

  • Domain deletion variants: Missing N-terminal or C-terminal domains

• Mixing protocol for heterohexamer formation:

  • Combine purified variants at different molar ratios

  • Incubate to allow subunit exchange (may require 30-60 minutes)

  • The ClpB hexamer is dynamic with subunits reshuffling on a timescale of minutes

• Validation of heterohexamer formation:

  • Size-exclusion chromatography to confirm hexamer formation

  • Tag-based pull-down assays to verify subunit incorporation

  • Analytical ultracentrifugation to assess complex homogeneity

• Functional analysis approaches:

  • ATPase activity measurements to detect potential cooperativity effects

  • Protein remodeling assays using model substrates (GFP, luciferase)

  • Disaggregation assays with the DnaK system

Research using heterohexamers has revealed that:

• When working alone, ClpB functions optimally with approximately 3 active and 3 inactive protomers

• With the DnaK system, even a single inactive subunit can significantly impair activity

• The activity of wild-type subunits within heterohexamers can be stimulated up to 4-fold

These findings demonstrate the adaptable nature of ClpB and how its mechanism can shift between probabilistic and sequential modes depending on the substrate and co-chaperone context .

What recent advances have been made in understanding ClpB2's role in stress resistance and pathogenicity?

Recent research has expanded our understanding of ClpB's role beyond protein quality control to include stress resistance and virulence in various organisms:

Studies in Streptococcus agalactiae have demonstrated that ClpB is critical for bacterial survival under hostile conditions. Deletion of the clpB gene significantly alters the expression of multiple genes involved in stress response and metabolic pathways .

Key findings from recent studies include:

• ClpB plays a vital role in bacterial heat and acid stress resistance, which are important for survival during infection.

• ClpB contributes to virulence in pathogenic bacteria, with clpB deletion mutants showing attenuated pathogenicity in infection models.

• The protein affects the expression of various stress response genes and metabolic pathways, suggesting a regulatory role beyond its direct chaperone function.

These discoveries provide enhanced understanding of how ClpB homologues function in gram-positive bacteria and contribute to their survival strategies during infection. This research also suggests ClpB as a potential target for developing new antimicrobial strategies .

How does the functional cooperation between ClpB2 and other chaperone systems vary across different bacterial species?

The cooperation between ClpB and other chaperone systems shows both conservation and species-specific adaptations across bacterial lineages:

• Core functional conservation:

  • The fundamental cooperation between ClpB and the DnaK system is conserved across most bacteria

  • Both systems are typically heat-shock inducible and regulated by similar mechanisms

• Species-specific variations:

  • The exact stoichiometry and interaction dynamics vary between bacterial species

  • Some organisms show greater dependency on the ClpB-DnaK cooperation than others

  • The regulatory networks controlling chaperone expression differ substantially

• Cyanobacterial adaptations:

  • In cyanobacteria like Synechocystis PCC 6803, ClpB proteins show specialized functions

  • ClpB1 and ClpB2 (slr0156) in Synechocystis have distinct roles, with ClpB2 being essential and not involved in thermotolerance

• Pathogen-specific roles:

  • In Streptococcus agalactiae, ClpB contributes to both stress resistance and virulence

  • In Streptomyces avermitilis, ClpB2 may have adapted to specific environmental stresses

Understanding these species-specific adaptations is important for developing targeted approaches for protein expression in different bacterial hosts and potentially for designing antimicrobial strategies that target specific pathogen-essential chaperone systems .