Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride
Ternström, Tomas LU ; Svendsen, A ; Akke, Mikael LU
and Adlercreutz, Patrick
LU
(2005)
In Biochimica et Biophysica Acta - Proteins and Proteomics
1748(1).
p.74-83
- Abstract
- We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfasuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (mu HiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by ACT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three... (More)
- We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfasuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (mu HiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by ACT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol(-1) and -2.6 kcal mol(-1) for GdnHCl and AOT, respectively. Hence, the AOT denatured state (D-AOT) is less destabilised than the GdnHCl denatured state (D-GdnHcl), relative to the native state in water. Far-UV CD spectroscopy revealed that DAOT retains some secondary structure, while D-GdnHCl is essentially unstructured. Similarly, fluorescence data suggest that DAOT is more compact than D-GdnHCl. Activity measurements reveal that both D-AOT and D-GdnHCl are practically inactive (catalytic activity < 1% of that of the native enzyme). The fluorescence spectrum of DAOT in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of DAOT in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data. (c) 2004 Elsevier B.V All rights reserved. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/151549
- author
- Ternström, Tomas
LU
; Svendsen, A
; Akke, Mikael
LU
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 2005
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochimica et Biophysica Acta - Proteins and Proteomics
- volume
- 1748
- issue
- 1
- pages
- 74 - 83
- publisher
- Elsevier
- external identifiers
-
- wos:000227735200010
- pmid:15752695
- scopus:14744295719
- ISSN
- 1570-9639
- DOI
- 10.1016/j.bbapap.2004.12.014
- language
- English
- LU publication?
- yes
- id
- 6a5b6aed-1ee6-433a-a3f0-fff14440034b (old id 151549)
- date added to LUP
- 2016-04-01 16:58:30
- date last changed
- 2022-01-28 23:28:29
@article{6a5b6aed-1ee6-433a-a3f0-fff14440034b,
abstract = {{We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfasuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (mu HiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by ACT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol(-1) and -2.6 kcal mol(-1) for GdnHCl and AOT, respectively. Hence, the AOT denatured state (D-AOT) is less destabilised than the GdnHCl denatured state (D-GdnHcl), relative to the native state in water. Far-UV CD spectroscopy revealed that DAOT retains some secondary structure, while D-GdnHCl is essentially unstructured. Similarly, fluorescence data suggest that DAOT is more compact than D-GdnHCl. Activity measurements reveal that both D-AOT and D-GdnHCl are practically inactive (catalytic activity < 1% of that of the native enzyme). The fluorescence spectrum of DAOT in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of DAOT in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data. (c) 2004 Elsevier B.V All rights reserved.}},
author = {{Ternström, Tomas and Svendsen, A and Akke, Mikael and Adlercreutz, Patrick}},
issn = {{1570-9639}},
language = {{eng}},
number = {{1}},
pages = {{74--83}},
publisher = {{Elsevier}},
series = {{Biochimica et Biophysica Acta - Proteins and Proteomics}},
title = {{Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride}},
url = {{http://dx.doi.org/10.1016/j.bbapap.2004.12.014}},
doi = {{10.1016/j.bbapap.2004.12.014}},
volume = {{1748}},
year = {{2005}},
}