Recombinant Synechococcus sp. Chaperone protein dnaK2 (dnaK2), partial

What is DnaK2 and what is its role in cyanobacteria?

DnaK2 is a 70-kDa heat shock response protein belonging to the Hsp70 family with ATPase activity. It plays essential roles in basic cellular processes including folding of newly synthesized proteins, preventing protein aggregation, protein transport and translocation, and regulative control of proteins in cyanobacteria . In Synechococcus sp. PCC7942, DnaK2 is one of three DnaK homologues (along with DnaK1 and DnaK3), and gene disruption experiments have demonstrated that DnaK2 is essential for normal growth, unlike DnaK1 . DnaK2 also exhibits a typical heat shock response, with its synthesis increasing upon temperature upshift .

How does DnaK2 differ functionally from other DnaK homologues in cyanobacteria?

The three DnaK homologues in Synechococcus sp. PCC7942 (DnaK1, DnaK2, and DnaK3) show distinct functional characteristics:

• Essentiality: DnaK2 and DnaK3 are essential for normal growth, whereas DnaK1 is not essential .

• Heat shock response: DnaK2 exhibits a typical heat shock response with increased synthesis upon temperature upshift, similar to GroEL. In contrast, DnaK1 and DnaK3 do not respond to heat shock; the DnaK1 protein level actually decreases under heat stress .

• Functional complementation: When expressed in an E. coli dnaK756 mutant, only DnaK2 can suppress the growth deficiency at nonpermissive temperature, while DnaK1 and DnaK3 cannot. Conversely, overproduction of DnaK1 or DnaK3 results in growth inhibition at the permissive temperature .

• Morphological effects: Overproduction of DnaK1 or DnaK2 in E. coli results in defects in cell septation and formation of cell filaments, while overproduction of DnaK3 leads to swollen and twisted cell morphology .

What is the relationship between DnaK2 and Rubisco assembly?

DnaK2 plays a critical role in the assembly of Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase), particularly in heterologous expression systems. Synechocystis sp. PCC 6803 Rubisco shows limited solubility and a lack of assembly in the Escherichia coli expression system. Research has demonstrated that upon introduction of Synechocystis DnaK2 to the E. coli system, RbcL (the large subunit of Rubisco) is produced in soluble form .

DnaK2 is responsible for RbcL peptide recognition, and this recognition is enhanced when specific DnaJ proteins (such as Sll1384) are present . The cooperation between DnaK and DnaJ has been proven necessary for efficient biosynthesis of functional cyanobacterial Rubisco both through in vitro binding constant measurements and in vivo analysis of Synechocystis PCC 6803 knockout mutants .

What are the optimal expression systems for producing recombinant DnaK2?

For recombinant DnaK2 production, E. coli is commonly used as an expression host, though careful consideration must be given to the construct design. When expressing cyanobacterial DnaK2 in E. coli, researchers should consider the following methodological approaches:

• Expression vector selection: Inducible expression systems such as those using the trc promoter have been successfully used for DnaK expression . For example, a fragment containing lacIq, trc promoter, and dnaK3 has been isolated and recloned into integration vectors for controlled expression .

• Host strain considerations: When expressing DnaK2 in E. coli, it's important to note that overproduction can result in defects in cell septation and formation of cell filaments, potentially affecting yield . Therefore, controlled expression (e.g., through tight promoter regulation) is recommended.

• Codon optimization: Since there are codon usage differences between cyanobacteria and E. coli, codon optimization of the dnaK2 sequence may improve expression levels, particularly if high yields are required.

• Growth conditions: For optimal expression, E. coli cultures should be maintained at appropriate temperatures (typically 30-37°C depending on the construct) and induced at mid-log phase.

What molecular techniques are necessary for creating recombinant DnaK2 constructs for different experimental applications?

Creating functional recombinant DnaK2 constructs requires careful molecular cloning strategies:

• Gene amplification and cloning: The dnaK2 gene can be amplified from Synechococcus sp. genomic DNA using PCR with specific primers. For partial constructs, design primers that amplify the specific domain of interest.

• Vector selection: For protein expression, vectors with appropriate promoters (like trc or T7) and affinity tags (His, GST, etc.) should be selected based on the experimental goals.

• Fusion protein design: For structural or functional studies, consider fusion proteins with fluorescent tags. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) have been characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002 .

• Genome integration: For expression in cyanobacteria, genome integration tools have been developed, including several putative neutral sites for genome integration in Synechococcus sp. PCC 7002. The minimum homology arm length for genome integration has been determined to be approximately 250 bp .

• Construction method: For genome integration, prepare linear integration cassettes with appropriate homology arms. As an example, a construct with the structure "NS2_5′-Prbc-FP-Trbc-KmR-NS2_3′" has been successfully used, where NS2_5′ and NS2_3′ are homology arms for neutral site 2, Prbc is the promoter, FP is the gene of interest, Trbc is the terminator, and KmR is the kanamycin resistance cassette .

How can researchers determine the binding specificity of DnaK2 to client proteins?

Several methodological approaches can be used to determine the binding specificity of DnaK2 to client proteins:

• In vitro binding assays: Measure binding constants between purified DnaK2 and potential client proteins (such as RbcL) using techniques like surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or fluorescence anisotropy. These methods provide quantitative data on binding affinities and kinetics.

• Co-immunoprecipitation (Co-IP): This technique can identify protein-protein interactions in cell lysates. Antibodies against DnaK2 can be used to pull down the protein along with its binding partners, which can then be identified using Western blotting or mass spectrometry.

• Yeast two-hybrid screening: This approach can identify novel interaction partners of DnaK2 in a high-throughput manner. Though not native conditions, it provides valuable insights into potential binding partners.

• Crosslinking coupled with mass spectrometry: This approach can capture transient interactions between DnaK2 and its clients, providing structural information on the binding interface.

• Functional complementation assays: Express DnaK2 in systems lacking endogenous DnaK (like E. coli dnaK756 mutant) to assess its ability to recognize and process specific client proteins .

What experimental approaches are most effective for analyzing DnaK2's role in Rubisco assembly?

To analyze DnaK2's role in Rubisco assembly, researchers can employ several complementary approaches:

• Heterologous co-expression systems: Co-express cyanobacterial RbcL with DnaK2 (with or without specific DnaJ proteins) in E. coli and analyze Rubisco solubility, folding, and assembly. This approach has demonstrated that introduction of Synechocystis DnaK2 to the E. coli system enables RbcL to be produced in soluble form, and this effect is enhanced by the addition of specific DnaJ proteins (e.g., Sll1384) .

• In vitro reconstitution: Purify the components (DnaK2, DnaJ, GrpE, and RbcL) and attempt to reconstitute the assembly process in vitro under controlled conditions (ATP, temperature, etc.).

• Gene knockout/complementation in cyanobacteria: Generate conditional DnaK2 mutants in cyanobacteria and assess the impact on Rubisco assembly and activity. This approach has confirmed that DnaK2 is indispensable for RbcL biosynthesis .

• Structural analysis: Use techniques like cryo-EM or X-ray crystallography to determine the structure of DnaK2 alone or in complex with substrates, providing insights into the molecular mechanism of substrate recognition.

• Site-directed mutagenesis: Identify key residues in DnaK2 (e.g., the EEV motif found in E. coli DnaK) that might be involved in RbcL recognition and mutate them to verify their importance .

How can DnaK2 be engineered for improved Rubisco assembly in heterologous hosts?

Engineering DnaK2 for improved Rubisco assembly involves several advanced approaches:

• Domain swapping: Create chimeric proteins by swapping domains between DnaK2 and other DnaK homologues to identify which regions are critical for Rubisco recognition and folding.

• Directed evolution: Implement directed evolution strategies to select for DnaK2 variants with enhanced ability to fold Rubisco in heterologous hosts. This could involve creating libraries of randomly mutagenized DnaK2 and selecting for improved Rubisco assembly or activity.

• Co-evolution analysis: Analyze the co-evolution patterns between DnaK2 and RbcL across different cyanobacterial species to identify potentially important interaction interfaces that could be optimized.

• Rational design based on structural data: Once structural information on the DnaK2-RbcL interaction is available, rationally design DnaK2 variants with improved binding or processing capabilities.

• Optimization of chaperone systems: Develop optimized expression systems that co-express not only DnaK2 but also appropriate co-chaperones (DnaJ and GrpE) and downstream chaperonins (GroEL/ES) in the correct stoichiometry to maximize Rubisco assembly efficiency.

What are the implications of the essential nature of DnaK2 for genome engineering in cyanobacteria?

The essential nature of DnaK2 in cyanobacteria has significant implications for genome engineering approaches:

• Conditional mutation strategies: Since direct knockout of DnaK2 is lethal , researchers must use conditional mutation strategies (e.g., inducible promoters, temperature-sensitive alleles) to study its function.

• Careful design of genome integration constructs: When designing genome modifications, researchers should ensure that integration constructs do not disrupt DnaK2 expression. Neutral sites for genome integration have been identified in Synechococcus sp. PCC 7002, including regions between SYNPCC7002_A0932 and SYNPCC7002_A0933 (neutral site 1—NS1), between SYNPCC7002_A1202 and SYNPCC7002_A1203 (neutral site 2—NS2), and between SYNPCC7002_A1778 and SYNPCC7002_A1779 (neutral site 3—NS3) .

• Homology arm considerations: For targeted genome modifications, homology arms of at least 250 bp should be used, with longer arms improving transformation efficiency .

• Complementation approaches: When studying DnaK2 function, express a wild-type copy from a neutral site before attempting to modify the endogenous gene.

• Species-specific considerations: Researchers should note that while DnaK2 is essential in Synechococcus sp. PCC7942, its essentiality might vary in other cyanobacterial species. Therefore, preliminary studies should confirm its status in the specific strain being engineered.

What are common challenges in expressing functional recombinant DnaK2, and how can they be addressed?

Researchers often encounter several challenges when expressing recombinant DnaK2:

• Low solubility: DnaK2 may form inclusion bodies when overexpressed.

  • Solution: Optimize expression conditions (lower temperature, reduced inducer concentration), use solubility-enhancing fusion tags (MBP, SUMO), or employ refolding protocols if inclusion bodies form.

• Reduced ATPase activity: Recombinant DnaK2 may show lower ATPase activity than the native protein.

  • Solution: Ensure proper folding by co-expressing with appropriate co-chaperones or optimizing purification protocols to maintain native conformation.

• Difficulty in purification: Contaminating proteins or degradation products may co-purify with DnaK2.

  • Solution: Design constructs with cleavable affinity tags and implement multi-step purification protocols (ion exchange, size exclusion chromatography after initial affinity purification).

• Toxicity in expression hosts: Overexpression of DnaK2 in E. coli can result in defects in cell septation and formation of cell filaments .

  • Solution: Use tightly regulated promoters, adjust induction conditions, or consider alternative expression hosts.

• Improper folding: Without its natural co-chaperones, DnaK2 may not fold correctly.

  • Solution: Co-express with cognate DnaJ and GrpE proteins from the same organism.

What experimental controls are crucial when studying DnaK2's interaction with Rubisco and other client proteins?

When studying DnaK2's interactions with client proteins, several crucial controls should be included:

• ATPase activity controls:

  • Test DnaK2 ATPase activity with and without potential client proteins

  • Include non-client proteins as negative controls

  • Test ATPase-deficient DnaK2 mutants (e.g., mutations in the ATP binding site)

• Binding specificity controls:

  • Include closely related but non-interacting proteins

  • Use DnaK1 or DnaK3 as alternative chaperones to demonstrate specificity

  • Perform competition assays with known substrates

• Co-chaperone dependency controls:

  • Test DnaK2 function with and without appropriate DnaJ proteins

  • Include non-cognate DnaJ proteins to demonstrate specificity

  • Test the effect of nucleotide exchange factors (GrpE)

• Functional outcome controls:

  • When studying Rubisco assembly, measure not only protein solubility but also enzymatic activity

  • Compare native Rubisco assembly with in vitro or heterologous assembly

• Domain function controls:

  • Create and test truncated versions of DnaK2 to identify essential domains

  • Test substrate binding domain and nucleotide binding domain separately

  • Create point mutations in key residues to validate functional importance

How do the functional properties of DnaK1, DnaK2, and DnaK3 from Synechococcus sp. compare?

The three DnaK homologues in Synechococcus sp. PCC7942 display distinct functional characteristics that can be summarized in the following comparative table:

This comparative analysis demonstrates the functional divergence of these homologues despite their structural similarities, suggesting they have evolved distinct cellular roles .

What structural features distinguish DnaK2 from other molecular chaperones in cyanobacteria?

DnaK2 possesses several structural features that distinguish it from other molecular chaperones in cyanobacteria:

• Domain organization: Like other Hsp70 family members, DnaK2 has a conserved domain structure with an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD) connected by a flexible linker. This structure is distinct from other chaperone families like GroEL/ES (Hsp60) or small heat shock proteins.

• Substrate recognition elements: DnaK2 contains specific substrate recognition elements that enable it to recognize and bind RbcL. The E. coli DnaK protein contains a glutamic acid-glutamic acid-valine (EEV) motif that may be involved in substrate recognition, and similar motifs may be present in cyanobacterial DnaK2 .

• Interaction interfaces: DnaK2 has specific interfaces for interaction with co-chaperones (DnaJ and GrpE) that modulate its activity. These interfaces differ from those in other chaperone systems.

• ATP binding and hydrolysis site: The ATPase domain of DnaK2 contains conserved residues that are critical for its function and distinguish it from non-Hsp70 chaperones.

• Allosteric regulation mechanisms: DnaK2 undergoes ATP-dependent conformational changes that regulate substrate binding and release, a mechanism distinct from other chaperone families.

Understanding these structural features is crucial for engineering DnaK2 for specific applications or developing strategies to modulate its activity in vivo.