title: Conformational plasticity of DM43, a metalloproteinase inhibitor from Didelphis marsupialis: chemical and pressure-induced equilibrium (un)folding studies.
abstract: We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ 21045-900, Brazil. henk@ioc.fiocruz.br
authors.forename: Alex
authors.initials: A
authors.lastname: Chapeaurouge
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Martins
authors.forename: Oliver
authors.initials: O
authors.lastname: Holub
authors.forename: Surza L G
authors.initials: SL
authors.lastname: Rocha
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
authors.forename: Ana G C
authors.initials: AG
authors.lastname: Neves-Ferreira
authors.forename: Sérgio T
authors.initials: ST
authors.lastname: Ferreira
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales