A comparative study of the unfolding of the endoglucanase Ce145 from Humicola insolens in denaturant and surfactant

TY - JOUR

T1 - A comparative study of the unfolding of the endoglucanase Ce145 from Humicola insolens in denaturant and surfactant

AU - Otzen, Daniel E.

AU - Christiansen, Lars

AU - Schülein, Martin

PY - 1999/1/1

Y1 - 1999/1/1

N2 - Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Ce145 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Ce145 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Ce145 is unfolded by very low concentrations of C 12-LAS (1-4 mM). An analysis of 16 mutants of Ce145 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent- mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce' the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.

AB - Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Ce145 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Ce145 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Ce145 is unfolded by very low concentrations of C 12-LAS (1-4 mM). An analysis of 16 mutants of Ce145 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent- mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce' the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.

KW - Alkyl benzene sulfonate

KW - Denaturant

KW - Detergent

KW - Intermediate

KW - Protein engineering

KW - Protein folding

KW - Transition state

UR - http://www.scopus.com/inward/record.url?scp=0032871869&partnerID=8YFLogxK

U2 - 10.1110/ps.8.9.1878

DO - 10.1110/ps.8.9.1878

M3 - Journal article

C2 - 10493589

AN - SCOPUS:0032871869

SN - 0961-8368

VL - 8

SP - 1878

EP - 1887

JO - Protein Science

JF - Protein Science

IS - 9

ER -