DnaJ E.Coli

What is the structural organization of DnaJ in E. coli?

DnaJ in E. coli is a 376-amino acid protein composed of distinct structural domains. The N-terminal fragment (residues 2-108) contains the highly conserved J domain (residues 2-72), which is sufficient for many of the protein's activities. Using NMR spectroscopy of 13C/15N doubly labeled DnaJ-(2-108), researchers have identified four α-helices in polypeptide segments of residues 6-11, 18-31, 41-55, and 61-68 in the J domain .

The complete DnaJ protein contains several functional regions:

• J domain: Essential for interaction with the Hsp70 partner DnaK, leading to ATP hydrolysis

• G/F motif: Adjacent to the J domain, contributes to stimulation of ATP hydrolysis

• Zinc-finger domain (residues 144-200): Involved in substrate binding

• C-terminal domain (residues 201-376): Also participates in substrate recognition

This modular organization allows DnaJ to perform its diverse cellular functions in protein quality control .

How does DnaJ function as part of the cellular chaperone machinery?

DnaJ functions as an essential component of the DnaK/DnaJ/GrpE (Hsp70/Hsp40) chaperone system in E. coli. The process follows several steps:

• DnaJ recognizes and binds to unfolded or misfolded protein substrates through its zinc-finger and C-terminal domains

• DnaJ delivers these substrates to DnaK and stimulates DnaK's ATPase activity via its J domain

• This ATP hydrolysis leads to a conformational change in DnaK that locks the substrate into DnaK's binding cavity

• GrpE facilitates nucleotide exchange, allowing the cycle to continue

This coordinated action enables efficient protein folding, disaggregation, and remodeling of protein complexes. Deletion of the zinc-finger and C-terminal domains generates a fragment that contains only the J domain and G/F motif, which lacks substrate-binding activity and has significantly reduced ability to stimulate DnaK's ATPase activity .

What experimental approaches are most effective for purifying active DnaJ protein?

For researchers working with DnaJ, successful purification of active protein requires:

• Expression system selection: E. coli BL21(DE3) with pET-based vectors works efficiently for DnaJ overexpression

• Induction conditions: 0.5 mM IPTG at OD600 ≈ 0.6, with reduced temperature (30°C) post-induction to enhance solubility

• Lysis buffer optimization: 50 mM Tris-HCl pH 8.0, 100 mM KCl, 5 mM DTT, with protease inhibitors

• Purification strategy:

  • Ammonium sulfate precipitation (0-30% cut)

  • Anion exchange chromatography (Q-Sepharose)

  • ATP-agarose affinity chromatography

  • Size exclusion chromatography for final polishing

Activity assessment can be performed by measuring DnaJ's ability to stimulate DnaK's ATPase activity using colorimetric phosphate release assays. Care must be taken to prevent zinc loss from the zinc-finger domain during purification, which can affect substrate binding capability .

What is the peptide binding specificity of DnaJ, and how does it compare to DnaK?

DnaJ exhibits distinct peptide binding preferences that both overlap with and differ from DnaK's specificity:

• DnaJ-binding peptides are particularly rich in aromatic residues (Phe, Tyr, Trp) and show a preference for aliphatic residues (Leu, Ile, Val)

• Statistical analysis of 1633 peptides from 14 proteins revealed that 75% of DnaJ-binding peptides contain at least one aromatic residue, while only 37% of non-binding peptides contain aromatics

• DnaJ binding sites frequently overlap with DnaK binding sites (6 out of 7 DnaK-binding peptides had affinity for DnaJ, and 3 out of 4 DnaJ-binding peptides had affinity for DnaK)

• DnaJ and DnaK can compete for the same peptide substrates, as demonstrated with fluorescently labeled peptides derived from σ32

Interestingly, DnaJ binds to peptides with d- and l-amino acids with similar affinity and binding patterns, indicating that its interaction with peptides relies predominantly on side-chain contacts rather than specific backbone orientation .

How can researchers experimentally determine DnaJ binding specificity?

To determine DnaJ binding specificity, researchers can employ several complementary approaches:

• Cellulose-bound peptide scanning:

  • Generate 13-mer peptides overlapping by 10 residues

  • Incubate with purified DnaJ protein

  • Detect binding via immunodetection and fluoroimaging

  • This method has been validated for quantitative binding analysis

• Solution-based fluorescence spectroscopy:

  • Synthesize fluorescently labeled peptides (e.g., with IAANS)

  • Measure binding through changes in fluorescence intensity or anisotropy

  • Determine binding constants and kinetic parameters

• Competition assays:

  • Use a well-characterized fluorescent probe peptide

  • Measure displacement by unlabeled test peptides

  • Determine relative binding affinities

• Systematic variation of peptide sequences:

  • Test peptides with d- vs l-amino acids

  • Compare binding to authentic vs inverse sequences

  • Substitute individual residues to identify critical binding determinants

These approaches have revealed that DnaJ binding relies primarily on side-chain interactions rather than backbone conformation, as demonstrated by similar binding patterns to peptides with d- and l-amino acids .

What methodological approaches can be used to study DnaJ-substrate interactions in vivo?

For investigating DnaJ-substrate interactions within living E. coli cells, researchers can employ:

• In vivo crosslinking:

  • Photo-activatable crosslinkers incorporated into DnaJ

  • UV-activation followed by affinity purification

  • Mass spectrometry identification of crosslinked partners

• Co-immunoprecipitation with substrate-trapping DnaJ variants:

  • Mutations in the J domain that impair DnaK activation

  • Stabilization of DnaJ-substrate complexes

  • Identification of bound proteins by proteomics

• Proximity-based labeling:

  • Fusion of DnaJ to enzymes like BioID or APEX2

  • Biotinylation of proteins in proximity to DnaJ

  • Streptavidin pull-down and proteomics analysis

• Genetic approaches:

  • Synthetic lethality screening with ΔdnaKJ mutants

  • Multicopy suppressor analysis

  • Identification of genes that compensate for DnaJ absence

The in vivo interactome analysis of DnaK in E. coli revealed that at least 50% of central metabolism enzymes interact with the DnaK system, including several enzymes (LdhA, Lpd, PykF, TalB) that were later identified as multicopy suppressors of chaperone-deficient mutants .

How does DnaJ influence E. coli growth on different carbon sources?

DnaJ significantly impacts E. coli growth according to carbon source availability, demonstrating a profound connection between chaperone function and metabolic adaptation:

• Carbon source utilization patterns in ΔdnaKJ mutants fall into distinct classes:

  • Class I: Carbon sources that do not support growth (heat shock response is detrimental)

  • Class II: Carbon sources that support higher growth rates (heat shock response is beneficial)

  • Class III: Carbon sources that support growth of wild-type and rpoH(I54N) but not ΔdnaKJ

  • Class IV: Carbon sources on which ΔdnaKJ exhibits reduced growth rate

  • Class V: Carbon sources with no significant growth differences between strains

• DnaK impacts carbon source hierarchy:

  • Even carbon sources sharing the same entry point in central metabolism show different growth patterns

  • For example, D-galactose (class I) and D-glucose (class V) both enter at glucose-6-phosphate level

  • D-mannose (class I), D-sorbitol (class III), and NAG (class V) all enter at fructose-6-phosphate

• Specific effects independent of the heat shock response:

  • Class III and IV carbon sources reveal DnaK-specific roles independent of HSR regulation

  • These effects may involve regulation at the transport level or early metabolic steps

What experimental methods best reveal DnaJ's impact on central metabolism?

To investigate DnaJ's interaction with central metabolism, researchers can employ:

• Real-time carbon assimilation analysis:

  • NMR spectroscopy to track carbon source consumption

  • Monitoring of intracellular metabolite levels

  • Analysis of metabolic flux distribution

• Metabolic byproduct profiling:

  • Quantification of excreted metabolites like acetate and orotate

  • Calculation of molar yields relative to substrate consumption

  • Comparison between wild-type and ΔdnaKJ mutants

• Carbon source screening with defined entry points:

  • Growth assessment on carbon sources entering at specific metabolic nodes

  • Comparison between ΔdnaKJ and rpoH(I54N) mutants to differentiate HSR-dependent and independent effects

  • Complementation experiments with plasmid-encoded DnaKJ

• Metabolic suppressor analysis:

  • Identification of metabolic genes that suppress chaperone-deficient phenotypes

  • Examples include ackA, ldhA, lpd, pykF, talB, and csrC, which partially suppress growth defects

  • These approaches have revealed that DnaK significantly impacts carbon source utilization hierarchies, metabolic coproduct excretion, and flux distribution

How do DnaJ-deficient strains differ in their metabolic flux patterns?

DnaJ deficiency leads to significant alterations in metabolic flux patterns:

• Pyruvate node regulation:

  • ΔdnaKJ mutants accumulate less pyruvate compared to wild-type

  • This suggests more efficient pyruvate consumption in the absence of DnaK

  • Three identified multicopy suppressors (LdhA, Lpd, PykF) play roles in pyruvate production/utilization

• Coproduct excretion patterns:

  • Altered patterns of acetate and orotate excretion in ΔdnaKJ mutants

  • Different molar yields of byproducts relative to substrate consumption

  • These changes reflect global metabolic reprogramming in response to chaperone deficiency

• Metabolic enzyme expression changes:

  • Endogenous expression of at least ten central metabolism enzymes increases in ΔdnaKJ mutants

  • TalB and LdhA (identified suppressors) show significantly increased expression

  • AckA (another suppressor) exhibits lower endogenous levels in the absence of DnaKJ

These observations indicate that DnaJ functions as an important regulator of metabolic flux distribution, potentially through its chaperone activity affecting the folding, stability, or activity of key metabolic enzymes .

How does the J domain of DnaJ molecularly interact with DnaK to stimulate its ATPase activity?

The J domain of DnaJ stimulates DnaK's ATPase activity through a specific molecular mechanism:

• Structural basis of interaction:

  • The four α-helices in the J domain (residues 6-11, 18-31, 41-55, and 61-68) form a helix bundle

  • The highly conserved HPD motif (histidine-proline-aspartate) located in the loop between helices II and III is critical for DnaK activation

  • This motif interacts with the ATPase domain of DnaK, triggering conformational changes that accelerate ATP hydrolysis

• Functional coupling:

  • J domain-mediated stimulation of DnaK's ATPase activity is significantly enhanced when DnaJ binds a substrate

  • Deletion of substrate-binding domains (zinc-finger and C-terminal) reduces ATPase stimulation capacity

  • This suggests allosteric communication between the substrate-binding regions and the J domain

• G/F region contribution:

  • The glycine/phenylalanine-rich region adjacent to the J domain contributes to ATPase stimulation

  • This region may provide flexibility and proper positioning of the J domain relative to DnaK

  • The G/F region also appears to influence substrate handling properties

Understanding these interactions is critical for designing experiments to modulate chaperone activity or develop targeted therapeutics based on chaperone function .

What are the most effective experimental approaches for studying DnaJ structure?

Researchers investigating DnaJ structure can employ several complementary techniques:

• NMR spectroscopy:

  • Particularly effective for studying individual domains

  • Use of 13C/15N doubly labeled protein enables comprehensive resonance assignments

  • Has been successfully applied to determine the three-dimensional structure of the J domain

  • Example protocol: Expression in minimal media with 15NH4Cl and 13C-glucose as sole nitrogen and carbon sources, followed by sequential assignment experiments (CBCA(CO)NH, HNCACB, etc.)

• X-ray crystallography:

  • Challenging for full-length DnaJ due to flexible domains

  • More successful with individual domains or in complex with binding partners

  • Requires optimization of crystallization conditions (protein concentration, buffer, precipitants, additives)

• Cryo-electron microscopy:

  • Increasingly valuable for studying DnaJ in complex with DnaK

  • Can capture different conformational states during the chaperone cycle

  • Sample preparation often involves chemical crosslinking to stabilize interactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Provides information on structural dynamics and solvent accessibility

  • Useful for mapping interaction surfaces with substrates and partner proteins

  • Complements static structural methods by revealing conformational flexibility

The NMR structure determination of DnaJ-(2-108) has been particularly informative, revealing the arrangement of four α-helices in the J domain that form the functional unit interacting with DnaK .

How do mutations in different domains of DnaJ affect its function and substrate specificity?

Mutational analysis of DnaJ has revealed domain-specific effects on function:

• J domain mutations:

  • HPD motif mutations (H33Q, D35N) abolish DnaK ATPase stimulation without affecting substrate binding

  • Mutations in helix II and III disrupt the structure of the J domain and impair all DnaJ functions

  • These mutants can serve as dominant negatives in in vivo studies

• Zinc-finger domain alterations:

  • Mutations of zinc-coordinating residues reduce substrate binding affinity

  • Specifically affect binding to substrates rich in aromatic residues

  • Can differentiate between zinc-dependent and zinc-independent substrate interactions

• C-terminal domain mutations:

  • Alterations in the C-terminal domain affect substrate specificity rather than general binding capacity

  • Mutations can shift preference from one class of substrates to another

  • Important for understanding substrate triage within the cell

• G/F region variations:

  • Alterations in the glycine/phenylalanine-rich region affect the efficiency of DnaK activation

  • These mutations can fine-tune chaperone activity without abolishing function

  • Natural variations in this region among DnaJ homologs correlate with differences in substrate preference

These structure-function relationships provide valuable insights for designing DnaJ variants with altered specificities for biotechnological applications or as research tools to probe chaperone-dependent processes .

How does DnaJ contribute to the heat shock response in E. coli?

DnaJ plays multiple roles in the E. coli heat shock response:

• Regulation of heat shock transcription factor σ32:

  • DnaJ directly interacts with σ32 (the heat shock sigma factor)

  • Under non-stress conditions, DnaJ and DnaK target σ32 for degradation

  • During heat shock, this negative regulation is relieved, allowing σ32 to activate heat shock genes

  • Fluorescently labeled peptide studies with σ32-Q132-Q144-C-IAANS demonstrate direct binding

• Protection of cellular proteins:

  • DnaJ recognizes partially unfolded proteins that accumulate during heat stress

  • It prevents their aggregation through direct holding activity

  • DnaJ delivers these substrates to DnaK for refolding

• Stress-specific adaptation:

  • The absence of DnaJ affects growth in a stress-specific manner

  • ΔdnaKJ and rpoH(I54N) mutations show distinct growth patterns under different stressors

  • This indicates that DnaJ function extends beyond simply regulating the general heat shock response

• Metabolic remodeling during stress:

  • DnaJ influences central metabolism during stress adaptation

  • The expression of DnaJ itself is subject to catabolite repression via Crp

  • This links stress response to metabolic state and carbon source availability

What methodologies can researchers use to study DnaJ's role in protein aggregation prevention?

To investigate DnaJ's function in preventing protein aggregation, researchers can employ:

• In vitro aggregation assays:

  • Light scattering measurements to monitor aggregation kinetics

  • Model substrates like citrate synthase, rhodanese, or luciferase

  • Comparison of aggregation prevention by DnaJ alone vs. complete DnaK/DnaJ/GrpE system

  • Quantitative analysis of concentration-dependent effects

• Fluorescence-based techniques:

  • FRET pair-labeled substrates to monitor conformational changes

  • Fluorescence correlation spectroscopy to measure complex formation

  • Single-molecule approaches to observe chaperone-substrate interactions

• Cellular aggregation models:

  • Expression of aggregation-prone proteins (e.g., truncated luciferase) in ΔdnaJ strains

  • Microscopy-based visualization of inclusion body formation

  • Biochemical fractionation to quantify soluble vs. insoluble protein

  • Proteomics analysis of the aggregated proteome

• Genetic interaction mapping:

  • Synthetic lethality screening with ΔdnaJ and other chaperone mutations

  • Identification of genetic interactions reveals functional relationships

  • The synthetic lethality of ΔdnaJ and Δtig (trigger factor) at temperatures ≥30°C demonstrates overlapping functions in preventing aggregation of newly synthesized proteins

How can researchers experimentally differentiate between DnaJ-specific and general heat shock response effects?

Distinguishing DnaJ-specific effects from general heat shock response (HSR) effects requires careful experimental design:

• Comparative genetic approach:

  • Use of both ΔdnaKJ and rpoH(I54N) mutants

  • ΔdnaKJ lacks DnaJ but has constitutively active HSR

  • rpoH(I54N) has reduced HSR activation but intact DnaJ

  • Phenotypes shared by both mutants likely reflect general HSR effects

  • Phenotypes unique to ΔdnaKJ indicate DnaJ-specific functions

• Domain-specific DnaJ mutants:

  • J domain mutants that maintain substrate binding but lose DnaK activation

  • Substrate binding domain mutants that retain DnaK activation

  • These separate direct DnaJ holdase activity from DnaK-dependent functions

• Complementation analysis:

  • Introduction of plasmid-encoded DnaJ, DnaK, or both

  • Testing other Hsp40 family members for functional substitution

  • Identification of unique DnaJ functions not complemented by other chaperones

• Carbon source phenotyping:

  • Classification of carbon sources based on growth patterns of different mutants

  • Classes III and IV carbon sources (where DnaJ plays a specific role independently of HSR)

  • This approach has successfully identified metabolic pathways specifically dependent on DnaJ function

How do bacterial DnaJ homologs differ in structure and function across species?

DnaJ homologs across bacterial species show both conservation and diversity:

• Domain organization conservation:

  • The J domain is highly conserved across all bacterial DnaJ homologs

  • The HPD motif is nearly invariant and essential for DnaK interaction

  • The four-helix bundle structure of the J domain is maintained

  • Most bacterial DnaJ proteins contain all canonical domains: J domain, G/F region, zinc-finger domain, and C-terminal domain

• Functional specialization:

  • Some bacteria possess multiple DnaJ homologs with specialized functions

  • Class I homologs contain all canonical domains

  • Class II homologs lack the zinc-finger domain

  • Class III homologs only have the J domain with variable additional sequences

• Substrate specificity variations:

  • Different bacterial DnaJ homologs show variations in substrate binding preferences

  • These differences correlate with variations in the zinc-finger and C-terminal domains

  • The J domain's conservation ensures maintained interaction with respective Hsp70 partners

• Species-specific adaptations:

  • Thermophilic bacteria contain DnaJ variants with enhanced stability

  • Psychrophilic species have more flexible DnaJ proteins

  • Pathogenic bacteria may have DnaJ homologs involved in virulence or host interaction

Understanding these variations helps researchers choose appropriate model systems and interpret cross-species experimental data .

What experimental approaches are most effective for identifying and characterizing novel DnaJ-interacting proteins?

For identifying novel DnaJ-interacting proteins, researchers can employ:

• Affinity purification-mass spectrometry (AP-MS):

  • Expression of tagged DnaJ (His, FLAG, etc.)

  • Optimization of crosslinking conditions to capture transient interactions

  • Stringent controls including J domain mutants that abolish DnaK interaction

  • Quantitative proteomics to determine enrichment factors

• Yeast two-hybrid screening:

  • Using full-length DnaJ or specific domains as bait

  • Screening against E. coli genomic libraries

  • Validation of interactions using directed Y2H and alternative methods

• Peptide array screening:

  • Testing DnaJ binding to cellulose-bound peptide arrays

  • Arrays representing complete sequences of candidate interacting proteins

  • Quantitative analysis using fluorescence detection

  • This approach successfully identified DnaJ binding to 1633 peptides from 14 proteins

• Multicopy suppressor analysis:

  • Screening for genes that suppress chaperone deficiency when overexpressed

  • This approach identified metabolic genes (ackA, ldhA, lpd, pykF, talB, csrC) and others involved in transcription, protein synthesis, and oxidative stress that partially suppress growth defects in chaperone-deficient strains

  • Functional classification reveals cellular processes dependent on DnaJ function

How does cooperation between DnaJ and Trigger Factor affect protein folding and cellular function?

The cooperation between DnaJ and Trigger Factor (TF) in E. coli reveals sophisticated chaperone networking:

• Sequential action during translation:

  • TF is a ribosome-bound chaperone that facilitates folding of newly synthesized proteins

  • DnaJ/DnaK system acts downstream of TF for proteins requiring additional assistance

  • Approximately 30% of newly synthesized E. coli proteins require both TF and DnaK/DnaJ assistance

• Functional overlap and compensation:

  • Deletion of the tig gene (encoding TF) enables DnaK to interact with shorter nascent chains

  • This increases DnaK interactors by more than 35%, indicating significant substrate overlap

  • TF and DnaJ/DnaK sequentially interact with large multi-domain proteins to promote folding

• Synthetic lethality:

  • Simultaneous deletion of both tig and dnaK/dnaJ genes is synthetically lethal at temperatures ≥30°C

  • This results in strong accumulation of cytosolic protein aggregates containing over 1000 different proteins

  • The Δtig ΔdnaKJ mutant serves as a sensitive genetic tool for studying proteostasis networks

• Hierarchical chaperone network:

  • Data suggest a hierarchical organization of chaperone functions

  • TF acts co-translationally at the ribosome

  • DnaJ can recognize TF clients that require further assistance

  • This coordination ensures efficient folding of complex proteins

This functional interplay highlights the sophisticated chaperone network that maintains proteostasis in E. coli and provides a model for understanding chaperone cooperation in other organisms .