Recombinant Synechocystis sp. Chaperone protein DnaJ 2 (dnaJ2)

Basic Research Questions

• What is the functional significance of DnaJ proteins in Synechocystis sp. PCC6803?

DnaJ proteins in Synechocystis sp. PCC6803 are essential molecular chaperones that play critical roles in protein folding, prevention of aggregation, and stress response. The main DnaJ protein encoded by sll0897 has been shown to be essential for cell survival . These proteins function as co-chaperones for DnaK (Hsp70) proteins, forming a highly conserved chaperone system that facilitates proper protein folding and maintains cellular proteostasis. While the N‐terminally located J‐ and G/F‐domains are sufficient for fundamental in vivo functions, the presence of the remaining domains becomes vital under stress conditions such as elevated temperatures .

Methodological approach for functional studies:

• Generate conditional knockout strains through targeted gene deletion

• Assess cell viability under various growth conditions

• Monitor protein aggregation profiles using biochemical fractionation

• Conduct complementation studies with specific domains to determine minimal functional requirements

• Use growth assays at normal versus elevated temperatures to assess stress-specific functions

• How are DnaJ proteins classified in Synechocystis, and what methods can identify their structural domains?

In Synechocystis sp. PCC6803, seven DnaJ co-chaperones have been identified, categorized into three classes:

• Class A: Contains the J-domain, G/F-domain, and cysteine-rich region (1 protein - Sll0897)

• Class B: Contains the J-domain and G/F-domain but lacks the cysteine-rich region (2 proteins - Slr0093 and Sll1933)

• Class C: Contains only the J-domain (4 proteins - Sll0909, Sll1011, Sll1384, and Sll1666)

Methodological approach for domain identification:

• Perform sequence analysis using domain prediction tools

• Generate truncated protein constructs for functional testing

• Use site-directed mutagenesis to disrupt specific domains

• Apply bioinformatic approaches such as artificial neural networks to classify J-domains based on sequence features

• Conduct structural studies (X-ray crystallography, NMR) to confirm domain boundaries and interactions

• What experimental approaches can determine the essentiality of specific DnaJ proteins in Synechocystis?

The essentiality of DnaJ proteins can be determined through systematic genetic manipulation approaches:

Methodological approach:

• Generate targeted deletion constructs with antibiotic resistance markers

• Transform Synechocystis cells and select transformants on appropriate antibiotics

• Test genomic segregation through multiple rounds of selection with increasing antibiotic concentrations

• Verify segregation status using PCR amplification of the targeted genomic region

• Introduce complementation constructs at neutral genomic sites to confirm gene essentiality

Using this approach, researchers demonstrated that the complete deletion of sll0897 was not possible even after prolonged selection, indicating its essential nature. In contrast, when a complementing copy was introduced elsewhere in the genome, complete deletion became possible, confirming its essential status .

• How can researchers express and purify recombinant Synechocystis DnaJ proteins for in vitro studies?

Methodological approach:

• Clone the DnaJ gene into an appropriate expression vector with affinity tags (His-tag, MBP-tag)

• Express in heterologous systems:

  • E. coli-based expression for rapid production

  • Insect cell expression (Sf9) for proteins requiring eukaryotic processing, as demonstrated for His-dj2

• For class A DnaJs with CaaX motifs, enhance farnesylation by adding mevalonolactone to the culture medium

• Purify using sequential chromatography:

  • Nickel chelate affinity column for His-tagged proteins

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as a final polishing step

• What methods can be used to study DnaJ-DnaK interactions in Synechocystis?

Methodological approach:

• Co-immunoprecipitation assays to confirm physical interactions

• In vitro ATPase assays to measure stimulation of DnaK ATPase activity by DnaJ

• Surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics

• Yeast two-hybrid or bacterial two-hybrid systems for interaction mapping

• Site-directed mutagenesis of key residues in the J-domain to disrupt interaction

• Computational modeling and co-evolution analysis to identify interaction interfaces

Research has shown that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells .

Advanced Research Questions

• How can the specific domains of DnaJ proteins be functionally characterized in Synechocystis?

Methodological approach:

• Generate a series of truncated constructs expressing partial proteins:

  • J-domain only

  • J-domain + G/F-rich region

  • Full-length protein without cysteine-rich region

  • Full-length protein with mutated functional residues

• Express these constructs under native or inducible promoters

• Test functional complementation by attempting to delete the endogenous gene

• Assess growth phenotypes under normal and stress conditions

• Evaluate chaperone activity using functional assays

This approach revealed that the J- and G/F-domains of Sll0897 are sufficient for fundamental functions under normal growth conditions, but the cysteine-rich and C-terminal domains become essential under stress conditions like elevated temperatures .

• What techniques can assess the role of DnaJ proteins in protein folding and aggregation prevention?

Methodological approach:

• In vitro protein refolding assays:

  • Use denatured firefly luciferase as a model substrate

  • Monitor refolding by measuring recovery of enzymatic activity

  • Compare refolding efficiency with different DnaJ variants

• Aggregation prevention assays:

  • Heat-denature model proteins in the presence/absence of DnaJ

  • Measure aggregation by light scattering or centrifugation

• In vivo approaches:

  • Express aggregation-prone proteins (e.g., mutant huntingtin) with fluorescent tags

  • Quantify aggregate formation with/without DnaJ overexpression

  • Use microscopy and biochemical fractionation to monitor aggregation

Research in mammalian systems has demonstrated that manipulation of DNAJ chaperones can suppress aggregation and ameliorate polyglutamine toxicity in cellular and animal models of Huntington's disease .

• How can researchers investigate the specific role of DnaJ2 in circadian rhythm regulation in Synechocystis?

Methodological approach:

• Generate targeted mutations in kaiA, kaiB, and kaiC genes based on known free-running period (FRP) mutations in S. elongatus

• Create reporter strains with luciferase fused to clock-controlled promoters

• Monitor circadian rhythms using automated bioluminescence recording systems

• Assess the impact of DnaJ2 overexpression or depletion on circadian period and amplitude

• Perform co-immunoprecipitation experiments to identify interactions with clock proteins

• Use chromatin immunoprecipitation (ChIP) to determine if clock proteins regulate DnaJ2 expression

Researchers have successfully translated FRP mutations from S. elongatus to Synechocystis, generating mutant strains with altered circadian periods ranging from ~23h to ~28h .

• What approaches can be used to study the substrate specificity of different DnaJ proteins in Synechocystis?

Methodological approach:

• Immunoprecipitation coupled with mass spectrometry to identify binding partners

• Comparative substrate binding assays using purified proteins

• Peptide array screening to identify binding motifs

• In vitro competition assays between different DnaJ proteins

• Differential proteomics in strains lacking specific DnaJ proteins

• Cross-linking studies to capture transient interactions

Understanding substrate specificity is crucial as different DnaJ proteins may have distinct roles in protein quality control. For example, the cysteine-rich region of bacterial DnaJ is important for binding to chemically denatured luciferase .

• How can genomic and bioinformatic approaches be used to study the evolution of DnaJ proteins across species?

Methodological approach:

• Sequence collection and multi-sequence alignment of J-domains from diverse organisms

• Phylogenetic analysis using maximum likelihood or Bayesian methods

• Application of Artificial Neural Networks (ANNs) to classify J-domains based on sequence features

• Identification of discriminatory sequence positions through machine learning interpretability

• Comparative analysis of domain architectures across evolutionary lineages

• Co-evolution analysis to identify coordinated changes with DnaK partners

This approach has revealed that J-domains carry sufficient discriminatory information to predict with high reliability the phylogeny, localization, and domain composition of the corresponding full-length protein .

Table 1: Comparison of DnaJ Protein Classes in Synechocystis sp. PCC6803

• What methods can determine if the J-domain alone is sufficient for functional interaction with DnaK in Synechocystis?

Methodological approach:

• Express isolated J-domains from different DnaJ proteins

• Perform in vitro ATPase stimulation assays with cognate DnaK proteins

• Use isothermal titration calorimetry to measure binding affinities

• Create chimeric proteins with J-domains swapped between different DnaJs

• Test functional complementation in vivo using truncated constructs

• Apply structural biology techniques to determine binding interfaces

Research in E. coli has shown that the N-terminal 108 amino acids of DnaJ, containing the J-domain and G/F-rich region, are sufficient to partially support DnaK function in vivo , suggesting similar approaches could be productive in Synechocystis.

• How can researchers investigate the role of post-translational modifications in DnaJ function?

Methodological approach:

• Mass spectrometry-based proteomic analysis to identify modifications

• Site-directed mutagenesis of potential modification sites

• Expression systems that enhance specific modifications:

  • Addition of mevalonolactone to enhance farnesylation of DnaJs with CaaX motifs

  • Use of eukaryotic expression systems for phosphorylation or other modifications

• Comparative functional analysis of modified versus unmodified proteins

• Subcellular localization studies to determine if modifications affect targeting

• Inhibitor studies targeting specific modification pathways

Class A DnaJ proteins like Ydj1p and dj2 contain CaaX prenylation motifs at their C-termini and undergo farnesyl modification post-translationally , which may affect their localization and function.