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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 1; p. 129434
• Main Authors: Rasmussen, Helena Ø., Enghild, Jan J., Otzen, Daniel E., Pedersen, Jan Skov
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.01.2020
• Subjects: beta-lactoglobulin; calorimetry; Cellulase; Charge neutralisation; circular dichroism spectroscopy; detergents; endo-1,4-beta-glucanase; enzyme activity; gel chromatography; Humicola insolens; industry; micelles; neutralization; nonionic surfactants; Refolding; SAXS; small-angle X-ray scattering; Surfactant; titration; ultraviolet radiation; Unfolding; Unfolding; Charge neutralisation; Surfactant; Cellulase; Refolding; SAXS
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.129434

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, 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. •Cellulase Cel7b is stable against SDS at high and neutral pH but unfolds at low pH.•SDS converts the secondary structure of Cel7b from β-sheet to mainly α-helical.•Part of the enzyme population is refolded by adding the nonionic surfactant C12E8.•Part of Cel7b population is refolded by addition of the nonionic surfactant C12E8.•Cel7b partially regains its original cellulase activity upon refolding with C12E8.