DnaK SBD
Description
Mechanism of Substrate Binding
DnaK SBD binds hydrophobic regions of unfolded proteins through a sequence of conformational changes:
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Peptide Recognition:
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α-Lid Dynamics:
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Competitive Inhibition:
Allosteric Regulation
DnaK SBD activity is tightly coupled to the nucleotide-binding domain (NBD) via an allosteric network:
| Parameter | NBD State (ATP/ADP) | SBD Conformation | Substrate Affinity |
|---|---|---|---|
| ATP-bound | Open NBD | α-Lid open | Low |
| ADP-bound | Closed NBD | α-Lid closed | High |
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ATP Hydrolysis: NBD ATPase activity triggers SBD conformational shifts, enabling substrate release .
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Linker Role: The flexible linker (residues 502–508) transmits allosteric signals between NBD and SBD, enabling coordinated domain movement .
Functional Collaboration with Other Chaperones
DnaK SBD interacts with auxiliary chaperones to coordinate substrate handling:
In E. coli, DnaK binds ~700 nascent polypeptides, transferring those requiring specialized folding to GroEL . Single-molecule studies show DnaK SBD binds collapsed intermediates with high affinity, preventing aggregation .
Research Findings and Data
Recent structural and biochemical studies highlight DnaK SBD’s versatility:
Table 1: Binding Affinities of Synthetic Peptides to DnaK SBD
| Peptide | Sequence | Binding Affinity (Kd) | Orientation | Source |
|---|---|---|---|---|
| NRLLLTG | NRLLLTG | ~50 nM | N→C | |
| l-PYR (short) | LLLFLLY | 5.5 nM | N→C | |
| proPhoA Site A | Signal sequence | 5 nM | C→N |
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Peptide Competition: l-PYR outcompetes natural substrates by extending substrate interactions into the SBD channel .
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Backbone Flexibility: DnaK SBD accommodates both N→C and C→N peptide orientations, allowing diverse substrate recognition .
Evolutionary Conservation
DnaK SBD shares structural homology with eukaryotic Hsp70s but exhibits distinct sequence preferences:
| Organism | Key Difference | Functional Impact |
|---|---|---|
| E. coli | Broad hydrophobic binding motif | Generalist chaperone activity |
| M. tuberculosis | Enhanced SBD flexibility | Adaptation to host stress environments |
Product Specs
MDVKDVLLLD VTPLSLGIET MGGVMTTLIA KNTTIPTKHS QVFSTAEDNQ SAVTIHVLQG ERKRAADNKS LGQFNLDGIN PAPRGMPQIE VTFDIDADGI LHVSAKDKNS GKEQKITIKA SSGLNEDEIQ KMVRDAEANA EADRKFEELV QTRNQGDHLL HST.
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 protein folding, assembly, and preventing the aggregation of stress-denatured proteins. The DnaK Substrate Binding Domain (SBD) is a specific region of the DnaK protein that is essential for its chaperone activity.
The DnaK protein consists of three main domains:
- Nucleotide Binding Domain (NBD): Responsible for ATP binding and hydrolysis.
- Substrate Binding Domain (SBD): Binds to unfolded or partially folded polypeptides.
- C-terminal Domain: Involved in the regulation of the chaperone activity.
The Substrate Binding Domain (SBD) of DnaK, specifically residues 508-638, is α-helical and acts as a lid covering the substrate binding cleft . This domain is critical for recognizing and binding to unfolded or misfolded proteins, thereby preventing their aggregation and assisting in their proper folding.
Recombinant DnaK Substrate Binding Domain is produced in E. coli as a single, non-glycosylated polypeptide chain. The recombinant protein typically contains 163 amino acids and has a molecular mass of approximately 17.7 kDa . The production process involves cloning the gene encoding the DnaK SBD into an expression vector, transforming E. coli cells with this vector, and inducing protein expression. The recombinant protein is then purified using conventional column chromatography techniques .
The recombinant DnaK Substrate Binding Domain has several applications in biochemical and biophysical research:
- Protein Folding Studies: Used to study the mechanisms of protein folding and the role of molecular chaperones.
- Protein-Protein Interactions: Investigating interactions between DnaK and its substrate proteins.
- Drug Discovery: Screening for potential inhibitors or modulators of DnaK activity, which could be useful in developing treatments for diseases related to protein misfolding and aggregation.
The recombinant DnaK SBD is typically formulated in a buffer containing Tris-HCl, β-mercaptoethanol (B-ME), and dithiothreitol (DTT) to maintain its stability . It should be stored at 4°C for short-term use (2-4 weeks) and at -20°C for long-term storage. To prevent degradation, it is recommended to avoid multiple freeze-thaw cycles and to add a carrier protein such as human serum albumin (HSA) or bovine serum albumin (BSA) for long-term storage .
