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Daniel Otzen

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

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Unfolding and partial refolding of a cellulase from the SDS-denatured state : From β-sheet to α-helix and back. / Rasmussen, Helena Ø.; Enghild, Jan J; Otzen, Daniel E et al.

In: B B A - General Subjects, Vol. 1864, No. 1, 129434, 01.2020, p. 129434 1-12.

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Rasmussen HØ, Enghild JJ, Otzen DE, Pedersen JS. Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back. B B A - General Subjects. 2020 Jan;1864(1):129434 1-12. 129434. Epub 2019 Sep 13. doi: 10.1016/j.bbagen.2019.129434

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@article{bc07763ce84a4c1abeab472ac6af4be4,
title = "Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back",
abstract = "Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, and about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.",
keywords = "Cellulase, Charge neutralisation, Refolding, SAXS, Surfactant, Unfolding",
author = "Rasmussen, {Helena {\O}.} and Enghild, {Jan J} and Otzen, {Daniel E} and Pedersen, {Jan Skov}",
note = "Copyright {\textcopyright} 2019. Published by Elsevier B.V.",
year = "2020",
month = jan,
doi = "10.1016/j.bbagen.2019.129434",
language = "English",
volume = "1864",
pages = "129434 1--12",
journal = "Biochimica et Biophysica Acta (BBA) - General Subjects",
issn = "0304-4165",
publisher = "Elsevier BV",
number = "1",

}

RIS

TY - JOUR

T1 - Unfolding and partial refolding of a cellulase from the SDS-denatured state

T2 - From β-sheet to α-helix and back

AU - Rasmussen, Helena Ø.

AU - Enghild, Jan J

AU - Otzen, Daniel E

AU - Pedersen, Jan Skov

N1 - Copyright © 2019. Published by Elsevier B.V.

PY - 2020/1

Y1 - 2020/1

N2 - Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, and about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.

AB - Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, and about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.

KW - Cellulase

KW - Charge neutralisation

KW - Refolding

KW - SAXS

KW - Surfactant

KW - Unfolding

U2 - 10.1016/j.bbagen.2019.129434

DO - 10.1016/j.bbagen.2019.129434

M3 - Journal article

C2 - 31525408

VL - 1864

SP - 1294341

EP - 1294312

JO - Biochimica et Biophysica Acta (BBA) - General Subjects

JF - Biochimica et Biophysica Acta (BBA) - General Subjects

SN - 0304-4165

IS - 1

M1 - 129434

ER -