title: Cleaved serpin refolds into the relaxed state via a stressed conformer.
abstract: Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Gakuencho 1-2, Nakaku, Sakai 599-8570, Japan. onda@b.s.osakafu-u.ac.jp
authors.forename: Maki
authors.initials: M
authors.lastname: Onda
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Nakatani
authors.forename: Sayaka
authors.initials: S
authors.lastname: Takehara
authors.forename: Mika
authors.initials: M
authors.lastname: Nishiyama
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Masaaki
authors.initials: M
authors.lastname: Hirose
mesh_terms: D000818:Animals; D000838:Anions; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002645:Chickens; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism; D004529:Egg White; D007700:Kinetics; D008956:Models, Chemical; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D015843:Serpins / Q000737:chemistry*
chemical_list: D000838:Anions; D015843:Serpins