DnaK Lid Covering Substrate E.Coli
Description
Introduction to DnaK Lid Covering Substrate in Escherichia coli
The DnaK Lid Covering Substrate is a critical structural and functional component of the bacterial Hsp70 chaperone DnaK, which plays a central role in protein folding, refolding, and proteostasis. This α-helical subdomain (residues 508–638) forms a "lid" that dynamically regulates the accessibility of the substrate-binding cleft in the C-terminal substrate-binding domain (SBD) of DnaK . Its conformational state is tightly coupled to nucleotide binding (ATP/ADP) in the N-terminal nucleotide-binding domain (NBD), enabling allosteric control of substrate binding kinetics .
Allosteric Regulation
The lid’s conformation is modulated by ATP/ADP binding in the NBD:
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ATP-bound state: Lid opens, releasing substrate and enabling rapid exchange .
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ADP-bound state: Lid closes, trapping substrates in the SBD cleft .
Key Allosteric Coupling
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Lid-Interdomain Linker Interaction: Disruption of the latch between the lid and β-sandwich subdomain destabilizes substrate binding .
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Conformational Heterogeneity: Single-molecule FRET studies reveal dynamic lid fluctuations in ADP-bound states but homogeneity in ATP-bound states .
Kinetic Parameters
| Parameter | Value | Condition | Source |
|---|---|---|---|
| K<sub>d</sub> (ADP) | 0.93–11 μM | Closed lid state | |
| Substrate dissociation rate | 440–2500-fold increase with ATP | Allosteric regulation |
Biological Significance
The lid enables DnaK to perform distinct roles:
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Protein Folding: Prevents aggregation of nascent polypeptides by stabilizing folding intermediates .
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Refolding: Slows refolding kinetics by retaining substrates in the SBD, allowing proper folding .
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Proteostasis: Collaborates with cochaperones (e.g., DnaJ, GrpE) to regulate substrate release and ATP hydrolysis .
Comparative Analysis of Lidless Mutants
Product Specs
MNEDEIQKMV RDAEANAEAD RKFEELVQTR NQGDHLLHST RKQVEEAGDK LPADDKTAIESALTALETAL KGEDKAAIEA KMQELAQVSQ KLMEIAQQQH AQQQTAGADASANNAKDDDVVDAEFEEVKDKK.
Product Science Overview
The DnaK protein, also known as Heat Shock Protein 70 (HSP70), is a molecular chaperone found in Escherichia coli (E. coli). It plays a crucial role in the folding and assembly of newly synthesized polypeptide chains and in preventing the aggregation of stress-denatured proteins. The DnaK protein is essential for the survival of bacteria under stress conditions, such as high temperatures.
The DnaK protein consists of several domains, including the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The SBD is responsible for binding to unfolded or partially folded polypeptides, while the NBD binds and hydrolyzes ATP, providing the energy required for the chaperone activity.
The DnaK Lid Covering Substrate domain, specifically, refers to a segment of the DnaK protein that acts as a lid covering the substrate-binding cleft. This lid is composed of residues 508-638 and is α-helical in structure . The lid plays a critical role in regulating the binding and release of substrate proteins.
Recombinant DnaK Lid Covering Substrate domain is produced in E. coli as a single, non-glycosylated polypeptide chain containing 132 amino acids and having a molecular mass of 14.6 kDa . The recombinant protein is purified to apparent homogeneity using conventional column chromatography techniques . The protein is typically formulated in a buffer containing 25mM Tris-HCl (pH 7.5), 100mM NaCl, 5mM DTT, and 10% glycerol .
The recombinant DnaK Lid Covering Substrate domain is used in various research applications, including:
- Protein Folding Studies: Understanding the mechanisms of protein folding and the role of molecular chaperones.
- Stress Response Research: Investigating how bacteria respond to stress conditions and the role of heat shock proteins in stress tolerance.
- Drug Development: Screening for potential inhibitors of DnaK activity, which could serve as antibacterial agents.
The recombinant DnaK Lid Covering Substrate domain should be stored at 4°C if used within 2-4 weeks. For longer storage periods, it should be frozen at -20°C, and it is recommended to add a carrier protein (0.1% HSA or BSA) to prevent degradation . Multiple freeze-thaw cycles should be avoided to maintain protein integrity.
