Thermostability of Refolded Ovalbumin andS-Ovalbumin

Abstract

Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (DeltaT(m)=8 degrees C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small. To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and S-ovalbumin were correctly refolded into the original non-denatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization.

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

“…The turbidity measurements showed that these peptides did not affect the aggregation kinetics of OVA at 80°C. 32 IAIMSA 37 of hB induced the formation of long straight fibrils of OVA without significantly changing the aggregation kinetics, an effect on OVA fibril formation similar to that caused by reduction of the disulfide bond between Cys 73 and Cys 120 . Therefore, the effect of 32 IAIMSA 37 on OVA aggregation could be explained by the interaction of 32 IAIMSA 37 and hB to promote a conformational change.…”

“…32 IAIMSA 37 of hB induced the formation of long straight fibrils of OVA without significantly changing the aggregation kinetics, an effect on OVA fibril formation similar to that caused by reduction of the disulfide bond between Cys 73 and Cys 120 . Therefore, the effect of 32 IAIMSA 37 on OVA aggregation could be explained by the interaction of 32 IAIMSA 37 and hB to promote a conformational change. A previous study on the loop insertion of serpin showed that serpin polymerization may be blocked by peptide fragments of the reactive center loop (54).…”

“…For example, the transformation of an ␣-helical segment of a monomeric prion to a ␤-strand conformation is the key event in the formation of aggregates (60), and an ␣-to-␤ transition is also a key step in the amyloid ␤-protein fibrillogenesis (61). The change in fibril morphology of OVA may be explained by the fluctuating conformation of the N-terminal helical region in SH-OVA (40) 32 IAIMSA 37 and hB of OVA to promote the ␣-to-␤ transition in this region.…”

“…10, long straight fibrils were formed when OVA was co-incubated with 32 IAIMSA 37 (1:15 molar ratio), and they were distinct from semiflexible fibrils formed in the absence of this peptide fragment. In addition, 32 IAIMSA 37 of hB had a slight effect on the aggregation kinetics of OVA (Fig. 2a, open square plot).…”

“…The partial loop-inserted model has previously been proposed to explain the formation of a thermostabilized form of OVA (S-OVA) from native conformation under elevated pH conditions (15,32,33). Although this model was subsequently excluded by analyzing the crystal structure of S-OVA (34), the P1-P1Ј-cleaved and thermostabilized OVA mutant has been shown by crystallography to assume the fully loop-inserted conformation (35,36).…”