Introduction to Bacterial Stress Response and Protein Folding

Bacteria respond to environmental stress—such as heat shock, oxidative stress, or nutrient deprivation—by synthesizing a group of specialized proteins known as stress proteins or heat shock proteins (HSPs). These proteins are essential for maintaining cellular homeostasis, preventing protein aggregation, and ensuring proper protein folding. Among the most crucial stress proteins in bacteria are DnaK (Hsp70), DnaJ (Hsp40), GroEL (Hsp60), GroES (Hsp10), and GrpE (nucleotide exchange factor). Together, they form a sophisticated chaperone network that assists in the folding of newly synthesized or misfolded proteins147.

Overview of Key Stress Proteins

• DnaK (Hsp70): An ATP-binding protein that binds to unfolded or misfolded polypeptides, preventing aggregation and promoting proper folding.

• DnaJ (Hsp40): A co-chaperone that targets substrate proteins to DnaK and stimulates its ATPase activity.

• GroEL/GroES (Hsp60/Hsp10): A chaperonin system that encapsulates partially folded proteins in a protected environment, facilitating their folding.

• GrpE: A nucleotide exchange factor that accelerates the replacement of ADP with ATP on DnaK, enabling substrate release and folding.

The ATP-Driven Cycle of DnaK and DnaJ in Protein Folding

The DnaK/DnaJ/GrpE system operates through a tightly regulated ATP-driven cycle to assist in protein folding:

1. Substrate Recognition and Binding:

   • DnaJ identifies and binds to exposed hydrophobic regions of unfolded or misfolded proteins.

   • DnaJ delivers the substrate to DnaK and stimulates the hydrolysis of ATP bound to DnaK234.

   • Upon ATP hydrolysis, DnaK undergoes a conformational change, trapping the substrate in a stable complex.

2. Substrate Release and Folding:

   • GrpE facilitates the exchange of ADP for ATP on DnaK, causing DnaK to release the substrate.

   • Released substrates can either fold spontaneously or be transferred to downstream chaperones like GroEL/GroES for further folding assistance467.

3. Repeated Cycles:

   • The cycle of binding, ATP hydrolysis, and substrate release is repeated, preventing aggregation and increasing the yield of properly folded proteins.

Evaluating the Statements on Protein Folding

Let’s analyze each statement in the context of the DnaK/DnaJ/GrpE chaperone system:

1. The affinity of DnaK to the polypeptide upon hydrolysis of the ATP to ADP.

• Correct. When DnaK hydrolyzes ATP to ADP, its affinity for the polypeptide increases, resulting in a stable DnaK-substrate complex34. This is a critical step in the chaperone cycle.

2. DnaJ is an exchange factor that replaces ADP to ATP in DnaK.

• Incorrect. DnaJ is a co-chaperone that targets substrates to DnaK and stimulates ATP hydrolysis. The nucleotide exchange factor that replaces ADP with ATP is GrpE234.

3. ATP hydrolysis is required for the phosphorylation of DnaJ.

• Incorrect. ATP hydrolysis is required for DnaK’s chaperone activity, but DnaJ is not phosphorylated in this process. Phosphorylation of DnaJ is not part of the bacterial stress response.

4. ATP hydrolysis is required for the phosphorylation of GrpE.

• Incorrect. GrpE is a nucleotide exchange factor and is not phosphorylated. ATP hydrolysis is essential for DnaK’s function, but not for GrpE phosphorylation.

Detailed Mechanism of DnaK/DnaJ/GrpE Action

Substrate Targeting and ATP Hydrolysis

• DnaJ recognizes hydrophobic patches on unfolded or misfolded proteins and delivers them to DnaK.

• DnaJ stimulates the hydrolysis of ATP bound to DnaK, converting DnaK to the ADP-bound state234.

• DnaK in the ADP-bound state has high affinity for the substrate, trapping it in a stable complex.

Substrate Release and Folding

• GrpE binds to DnaK and accelerates the exchange of ADP for ATP.

• ATP binding causes DnaK to release the substrate, allowing it to fold or be transferred to other chaperones46.

• Repeated cycles of binding and release prevent aggregation and increase the yield of properly folded proteins.

Synergy with Other Chaperones

• GroEL/GroES acts downstream of DnaK/DnaJ/GrpE, providing a protected environment for proteins that require additional folding assistance67.

• Small heat shock proteins (e.g., IbpA, IbpB) and proteases (e.g., ClpB) may also collaborate with the DnaK system to resolve aggregates and degrade irreversibly misfolded proteins1.

Biological Significance of the Chaperone System

The DnaK/DnaJ/GrpE system is essential for bacterial survival under stress because it:

• Prevents protein aggregation: By binding to unfolded or misfolded proteins, chaperones prevent the formation of toxic aggregates.

• Promotes proper folding: The ATP-driven cycle ensures that substrates are released in a state conducive to folding.

• Facilitates protein quality control: Misfolded proteins that cannot be refolded are targeted for degradation.

Key Concepts and Keywords

• Bacterial stress response: Cellular reaction to environmental stress, involving the synthesis of stress proteins.

• Heat shock proteins (HSPs): Proteins like DnaK, DnaJ, GroEL, GroES, and GrpE that assist in protein folding.

• DnaK (Hsp70): ATP-binding chaperone that binds and stabilizes unfolded proteins.

• DnaJ (Hsp40): Co-chaperone that targets substrates to DnaK and stimulates ATP hydrolysis.

• GrpE: Nucleotide exchange factor that accelerates ADP/ATP exchange on DnaK.

• GroEL/GroES: Chaperonin system that facilitates protein folding in a protected environment.

• ATP hydrolysis: Energy-dependent process that drives the chaperone cycle.

• Protein folding: The process by which polypeptides attain their functional three-dimensional structure.

• Protein aggregation: The clumping of misfolded proteins, which can be toxic to the cell.

• Nucleotide exchange: The replacement of ADP with ATP on DnaK, mediated by GrpE.

• Substrate release: The step in the chaperone cycle where the substrate is released for folding or transfer.

• Chaperone network: The coordinated action of multiple chaperones to ensure proper protein folding and quality control.

Frequently Asked Questions

Q: What is the role of DnaK in bacterial protein folding?
A: DnaK binds to unfolded or misfolded proteins, prevents aggregation, and promotes proper folding through an ATP-driven cycle.

Q: How does DnaJ contribute to the chaperone system?
A: DnaJ targets substrates to DnaK and stimulates the hydrolysis of ATP, increasing DnaK’s affinity for the substrate.

Q: What is the function of GrpE?
A: GrpE is a nucleotide exchange factor that accelerates the replacement of ADP with ATP on DnaK, enabling substrate release.

Q: Do DnaJ or GrpE require phosphorylation for their function?
A: No, neither DnaJ nor GrpE is phosphorylated as part of the bacterial stress response.

Q: How does the chaperone system prevent protein aggregation?
A: By repeatedly binding and releasing unfolded or misfolded proteins, the chaperone system prevents their aggregation and increases the yield of properly folded proteins.

Conclusion

The bacterial stress response relies on a sophisticated chaperone network centered around DnaK, DnaJ, and GrpE. DnaK binds to unfolded or misfolded proteins, and upon ATP hydrolysis (stimulated by DnaJ), its affinity for the substrate increases, trapping it in a stable complex. GrpE then facilitates the exchange of ADP for ATP, causing DnaK to release the substrate for folding or transfer to other chaperones. Among the provided statements, only the increase in DnaK’s affinity for the polypeptide upon ATP hydrolysis to ADP accurately describes a step in the protein folding process.

Correct Answer:
(1) The affinity of DnaK to the polypeptide upon hydrolysis of the ATP to ADP.