A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant.
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 Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 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. Cel45 is unfolded by very low concentrations of C12-LAS (1-4 mM). An analysis of 16 mutants of Cel45 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.
Otzen
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DE
Enzyme Research, Novo Nordisk A/S, Bagsvaerd, Denmark.
Christiansen
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L
Schülein
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D000136:Acid-Base Equilibrium; D017738:Alkanesulfonic Acids / Q000737:chemistry*; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008956:Models, Chemical; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D013501:Surface-Active Agents / Q000737:chemistry*
D017738:Alkanesulfonic Acids; D013501:Surface-Active Agents; C030641:alkylbenzyl sulfonic acid; C097231:endoglucanase V; D002480:Cellulase; D019791:Guanidine