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The roles of conserved amino acids on substrate binding and conformational integrity of ClpB N-terminal domain

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Date
2006-07-18
ICR Author
Lee, Yin-Fai
Author
Tanaka, N
Tani, Y
Tada, T
Lee, Y-F
Kanaori, K
Kunugi, S
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Type
Journal Article
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Abstract
Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. ( 2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.
URI
https://repository.icr.ac.uk/handle/internal/2510
DOI
https://doi.org/10.1021/bi0606804
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  • Closed Research Teams
Research team
Cell Transformation
Language
eng
License start date
2006-07-18
Citation
BIOCHEMISTRY, 2006, 45 pp. 8556 - 8561
Publisher
AMER CHEMICAL SOC

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