title: Aggregation as the basis for complex behaviour of cutinase in different denaturants.
abstract: We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.
authors.affiliation: Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg DK- 9000, Denmark. dao@bio.aau.dk
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Otzen
authors.forename: Lise
authors.initials: L
authors.lastname: Giehm
authors.forename: Ricardo P
authors.initials: RP
authors.lastname: Baptista
authors.forename: Søren R
authors.initials: SR
authors.lastname: Kristensen
authors.forename: Eduardo P
authors.initials: EP
authors.lastname: Melo
authors.forename: Shona
authors.initials: S
authors.lastname: Pedersen
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D019791:Guanidine