Recombinant Synechocystis sp. Chaperone protein ClpB 1 (clpB1), partial

What is the molecular structure of ClpB1 in Synechocystis sp. PCC 6803?

ClpB1 in Synechocystis sp. PCC 6803 is a heat shock protein that functions as a molecular chaperone with a molecular weight of approximately 95 kDa . The functional form of ClpB is a hexamer that requires ATP for stability and activity . The protein contains distinct structural domains that contribute to its function:

• N-terminal domain: Critical for substrate interaction and recognition

• Middle domain: Contains nucleotide-binding sites essential for ATP hydrolysis

• C-terminal domain: Important for oligomerization and self-association

Unlike many proteins, ClpB1 in Synechocystis has an internal translation initiation site that can produce alternative forms of the protein with molecular weights of 85.4 kDa in addition to the full-length 95 kDa form . This structural complexity may contribute to its functional versatility in responding to thermal stress.

How does ClpB1 differ from other chaperones in the Clp/Hsp100 family?

ClpB1 belongs to the Clp/Hsp100 family of proteins that act to remodel or disassemble protein complexes and aggregates using ATP energy . Unlike other members of this family, ClpB1 specializes in disaggregation rather than proteolysis. The key differences include:

Unlike the Arabidopsis ClpB homologs, which have diversified into specialized roles in different cellular compartments, Synechocystis ClpB1 appears to have retained a more generalized cytoplasmic function primarily related to heat shock response . Additionally, while some Clp proteins work as part of proteolytic complexes, ClpB1 functions specifically in protein remodeling without degradation activity.

What are the optimal expression systems for producing functional recombinant ClpB1?

The expression of recombinant ClpB1 requires careful consideration of several factors to ensure the production of functional protein. Based on published methodologies, the following approach is recommended:

Expression vector selection: pET-based expression systems have been successfully used for ClpB homologs, with pET-20b being particularly effective when the gene is inserted between NdeI and XhoI restriction sites . For Synechocystis ClpB1, it's important to use a template that lacks the internal translation initiation site to prevent production of truncated proteins, unless specific variants are desired for study .

Expression conditions:

• Host strain: E. coli BL21(DE3) or similar strains with reduced protease activity

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: Lower induction temperature (16-20°C) for 16-20 hours can improve solubility

• Media supplements: Addition of 5% glycerol and 1% glucose can reduce inclusion body formation

It's worth noting that the large size and complex oligomeric structure of ClpB1 can present challenges for heterologous expression. Using native promoters like rbcL or psbA2 has proven effective for overexpression directly in Synechocystis, as demonstrated in studies where ClpB1 was overproduced by 16-fold under the control of the rbcL promoter .

What purification strategy yields the highest activity of recombinant ClpB1?

A multi-step purification approach is recommended to obtain highly active recombinant ClpB1:

• Initial clarification:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 2 mM β-mercaptoethanol, and 10% glycerol

  • Include protease inhibitors such as PMSF (1 mM) and Complete EDTA-free protease inhibitor cocktail

  • Clarify by centrifugation at 30,000 × g for 30 minutes at 4°C

• Chromatography sequence:

  • Initial capture: Anion exchange chromatography (Q-Sepharose)

  • Intermediate purification: ATP-affinity chromatography exploits the natural ATP-binding property of ClpB1

  • Polishing step: Size exclusion chromatography (Superdex 200) to isolate properly oligomerized ClpB1

• Activity preservation:

  • Include 5-10% glycerol in all buffers to stabilize oligomeric structure

  • Maintain ATP (0.1-0.5 mM) in final storage buffer to preserve hexameric state

  • Flash-freeze aliquots in liquid nitrogen and store at -80°C

The purification strategy should account for the ATP-dependent oligomerization of ClpB1. The C-terminal domain plays a crucial role in self-association, as C-terminally truncated variants show decreased affinity for ATP and reduced ATPase and chaperone activity . Therefore, monitoring oligomeric state throughout purification is essential for obtaining functionally active protein.

How should researchers design assays to evaluate the chaperone activity of ClpB1?

Evaluating the chaperone activity of ClpB1 requires assays that measure its ability to disaggregate proteins and work cooperatively with other chaperones. Four complementary approaches are recommended:

• Protein concentration-dependent self-association assay:

  • Method: Analytical ultracentrifugation or size exclusion chromatography

  • Analysis: Measure the equilibrium between monomeric and oligomeric forms at varying ClpB1 concentrations

  • Significance: Self-association is essential for chaperone function and is mediated by the C-terminal domain

• ATP-induced self-association assay:

  • Method: Light scattering or native PAGE

  • Analysis: Monitor changes in oligomeric state upon ATP addition

  • Significance: ATP binding drives functional hexamer formation

• Casein-stimulated ATPase activity assay:

  • Method: Colorimetric or NADH-coupled ATPase assay with casein as a model substrate

  • Analysis: Measure phosphate release in the presence and absence of substrate

  • Significance: Substrate-stimulated ATPase activity correlates with chaperone function

• Protein reactivation assay:

  • Method: Thermal inactivation and refolding of firefly luciferase or other model substrates

  • Analysis: Measure recovery of enzymatic activity in the presence of ClpB1 alone and with the DnaK chaperone system

  • Significance: This assay directly measures the functional outcome of chaperone activity

When using these assays, it's important to note that N-terminal mutations in ClpB can produce proteins that appear normal in vitro but show impaired function in vivo . This suggests that current biochemical assays may not capture all essential aspects of ClpB1 function, and complementary in vivo approaches should be considered for comprehensive characterization.

What experimental approaches can distinguish between the activities of different ClpB domains?

Understanding domain-specific functions in ClpB1 requires systematic analysis using truncation and mutation strategies. Based on current research, the following approaches are effective:

• Truncation analysis:

  • Create systematic truncations: Full-length ClpB (1-857), N-terminal truncation (149-857), C-terminal truncation (1-769), and double truncation (149-769)

  • Compare activities using standardized assays to isolate domain contributions

  • Results interpretation: C-terminal truncation primarily affects oligomerization and ATP affinity, while N-terminal truncation affects substrate interaction

• Point mutation analysis:

  • Target conserved residues in each domain

  • Use site-directed mutagenesis to create variants

  • Assess functional impact through in vitro and in vivo assays

• Domain swap experiments:

  • Exchange domains between ClpB homologs (e.g., between ClpB1 and ClpB2)

  • Create chimeric proteins to identify domain-specific functions

  • Analyze which functions are preserved or altered

A comprehensive analysis revealed that both terminal regions of ClpB are essential for chaperone activity, but their functions differ: the N-terminal region is involved in substrate interaction, while the C-terminal region supports protein self-association . These findings demonstrate the previously unknown function of the PDZ-like SSD domain in ClpB and highlight the importance of analyzing both regions when characterizing ClpB1 function.

How does ClpB1 overexpression affect thermotolerance in cyanobacteria?

Constitutive overexpression of ClpB1 in Synechocystis sp. PCC 6803 has significant protective effects against heat stress. Detailed research findings show:

• Quantitative improvement in thermotolerance:

  • 20-fold increase in cell survival when cultures were heated rapidly (1°C/s) to 50°C

  • Delayed cell death by an average of 3 minutes during incubation at high temperatures (>46°C)

  • Further enhancement of survival when ClpB1 is co-overexpressed with DnaK2

• Cellular mechanisms:

  • ClpB1 overexpression maintains protein aggregate size during heat treatment, whereas control strains show apparent protein aggregation

  • ClpB1 is dispersed throughout the cytoplasm but concentrates near thylakoid membranes

  • Despite significant protection, ClpB1 overproduction does not alter cellular morphology, chlorophyll content, or photosystem ratio

The research demonstrates that constitutive ClpB1 overproduction allows an earlier response to heat shock and specifically protects against rapid temperature increases . This suggests that ClpB1 acts as a first-response protein during thermal stress, with particular importance in natural environments where temperature fluctuations can be sudden.

What is the relationship between ClpB1 and other heat shock proteins in stress response pathways?

ClpB1 functions within a coordinated network of heat shock proteins to provide comprehensive cellular protection. The interactions and synergies include:

• Cooperative action with the DnaK system:

  • ClpB1 works synergistically with DnaK2 in resolubilizing damaged proteins

  • Co-overexpression of ClpB1 and DnaK2 provides enhanced thermotolerance compared to overexpression of either protein alone

  • This cooperation suggests a "bi-chaperone network" where ClpB disaggregates large protein complexes, and DnaK assists in refolding

• Hierarchical activation:

  • clpB is among the first genes induced after heat shock

  • The rapid induction of ClpB1 positions it as an early responder in the heat shock response

  • Timing of expression suggests ClpB1 prepares the cellular environment for the action of other heat shock proteins

• Functional specialization:

  • ClpB1 and ClpB2 in Synechocystis show different induction patterns under heat stress

  • While ClpB1 is strongly induced during heat shock, ClpB2 is not induced under these conditions (at least in Synechococcus sp. PCC 7942)

  • This specialization suggests that ClpB1 has evolved specifically to address thermal stress, while ClpB2 may serve other functions

This interrelated chaperone network highlights the complexity of cellular stress responses and suggests that optimal protection requires balanced expression of multiple heat shock proteins working in coordination.

What imaging techniques best reveal the subcellular localization and dynamics of ClpB1?

Advanced imaging techniques provide crucial insights into the subcellular distribution and functional dynamics of ClpB1:

• Immunoelectron microscopy:

  • Technique: Fixed cells embedded in resin, sectioned and labeled with anti-ClpB1 antibodies followed by gold-conjugated secondary antibodies

  • Results: Revealed that ClpB1 is dispersed throughout the cytoplasm but concentrated in specific areas and more prevalent near thylakoid membranes

  • Advantages: Provides nanometer-scale resolution of protein localization in relation to cellular structures

• Fluorescence microscopy with tagged variants:

  • Technique: Expression of ClpB1-GFP fusion proteins for live-cell imaging

  • Application: Enables tracking of protein redistribution during heat shock

  • Considerations: Verify that GFP tags do not interfere with hexamer formation or chaperone function

• Super-resolution microscopy:

  • Techniques: PALM, STORM, or STED microscopy

  • Benefits: Overcomes diffraction limit to resolve protein clusters and interactions at nanoscale

  • Application: Can reveal dynamic reorganization of ClpB1 in response to stress conditions

• FRET-based interaction studies:

  • Approach: Use fluorescence resonance energy transfer to study ClpB1 interactions with substrate proteins or other chaperones

  • Advantage: Provides real-time information about protein-protein interactions in living cells

  • Implementation: Requires careful selection of fluorophore pairs and controls

When implementing these techniques, researchers should consider that ClpB1 localization may change dramatically during heat shock, and experimental designs should include appropriate temperature controls and time-course analyses to capture the dynamic nature of ClpB1 redistribution during stress response.

How can researchers effectively analyze the ATP-dependent disaggregation mechanism of ClpB1?

Unraveling the ATP-dependent disaggregation mechanism of ClpB1 requires sophisticated biochemical and biophysical approaches:

• Single-molecule analysis:

  • Technique: Optical tweezers or magnetic tweezers coupled with fluorescence detection

  • Application: Measure force generation during protein disaggregation

  • Advantage: Provides direct mechanical insights into the protein remodeling process

• Cryo-electron microscopy:

  • Approach: Capture ClpB1 hexamers in different nucleotide-bound states

  • Resolution: Near-atomic resolution structures can reveal conformational changes during the ATP hydrolysis cycle

  • Impact: Helps identify substrate threading mechanisms and channel dimensions

• FRET-based conformational studies:

  • Strategy: Introduce donor-acceptor fluorophore pairs at strategic positions to monitor domain movements

  • Analysis: Track distance changes during ATP binding, hydrolysis, and substrate processing

  • Benefit: Provides real-time information about conformational dynamics

• Site-directed spin labeling and EPR spectroscopy:

  • Method: Introduce spin labels at specific residues and measure distances by electron paramagnetic resonance

  • Application: Map conformational changes in the ClpB1 hexamer during the ATPase cycle

  • Advantage: Works in solution without crystallization requirements

• Hydrogen-deuterium exchange mass spectrometry:

  • Technique: Monitor the accessibility of protein regions to solvent exchange during different functional states

  • Utility: Identifies regions involved in substrate binding and conformational changes

  • Benefit: Can be performed with relatively small amounts of protein

These advanced approaches can reveal the mechanical details of how ClpB1 converts ATP hydrolysis into the physical work of protein disaggregation, providing deeper insights into its molecular mechanism than conventional biochemical assays alone.

What strategies can overcome common difficulties in expressing functional ClpB1?

Expressing functional recombinant ClpB1 presents several challenges that can be addressed with targeted strategies:

• Addressing protein solubility issues:

  • Challenge: ClpB1 tends to form inclusion bodies when overexpressed

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.3 mM), and include solubility enhancers like sorbitol (0.5 M) and betaine (1 mM) in the culture medium

  • Alternative approach: Consider using solubility-enhancing fusion tags like MBP or SUMO, with appropriate protease cleavage sites

• Managing internal translation sites:

  • Challenge: The internal translation initiation site in ClpB1 produces multiple protein products

  • Solution: Use a mutated template that eliminates the internal start site , or design primers that specifically include or exclude this site depending on experimental needs

  • Verification method: Western blot analysis using anti-ClpB1 antibodies that can detect both the 95 kDa and 85.4 kDa forms

• Preserving oligomeric structure:

  • Challenge: The functional hexameric structure may dissociate during purification

  • Solution: Include ATP (0.2-0.5 mM) in purification buffers to stabilize the hexamer

  • Monitoring method: Use dynamic light scattering or native PAGE to assess oligomeric state throughout purification

• Optimizing for homologous expression:

  • Challenge: Heterologous expression may not fully recapitulate native protein function

  • Solution: Consider expression in Synechocystis using strong endogenous promoters like rbcL or psbA2

  • Advantage: Ensures proper folding and post-translational modifications in the native cellular environment

These strategies can be adapted based on specific experimental goals and available resources, with the understanding that optimizing ClpB1 expression often requires iterative refinement of conditions.

How can researchers differentiate between the functional contributions of ClpB1 and other chaperones in vivo?

Distinguishing the specific contributions of ClpB1 from other chaperones requires carefully designed genetic and biochemical approaches:

• Genetic manipulation strategies:

  • Knockout approach: Generate clean deletion mutants of clpB1 (slr1641) in Synechocystis

  • Complementation testing: Reintroduce wild-type or mutant variants of ClpB1 to assess functional recovery

  • Double knockout analysis: Create combinations of chaperone deletions (e.g., clpB1 with dnaK2) to assess synthetic phenotypes and functional overlap

  • Challenge: Complete deletion of some chaperones like ClpB2 may not be obtainable due to essential functions

• Phenotypic analysis methods:

  • Temperature shift experiments: Compare survival rates at various heating rates (1°C/s vs. gradual heating)

  • Protein aggregation assessment: Use differential centrifugation to isolate and quantify aggregated proteins

  • Specific substrate monitoring: Track the refolding of known ClpB1 substrates using activity assays or solubility analysis

• Temporal dissection of chaperone contributions:

  • Inducible expression systems: Use tetracycline-inducible or similar promoters to control timing of chaperone expression

  • Time-course analysis: Monitor cellular responses at different timepoints after heat shock

  • Translation inhibition experiments: Use protein synthesis inhibitors to differentiate between existing and newly synthesized chaperone contributions

• Biochemical isolation of specific activities:

  • Immunodepletion: Selectively remove specific chaperones from cell lysates to assess remaining disaggregation activity

  • Reconstitution experiments: Add purified chaperones to depleted lysates to restore specific functions

  • Native complex isolation: Use tandem affinity purification to isolate intact chaperone complexes and identify components

These approaches collectively provide a framework for dissecting the specific roles of ClpB1 while accounting for the functional redundancy and cooperation inherent in chaperone networks.