Abstract
Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an abb'xa' structural organization in which the a and a' domains have CGHC active sites, the b and b' domains are involved with substrate binding, and x is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether this unfolding-refolding cycle is a substrate-induced process or an intrinsic physical property of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal denaturation. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54 °C unfolding transition temperature (Tm) and regained 85% of its native structure after nearly complete thermal denaturation. Oxidized PDI had a lower Tm of 48-50 °C and regained 70% of its native conformation after 75% denaturation. Both reduced PDI and oxidized PDI were functional after refolding from these denatured states. Additional studies documented increased stability of a PDI construct lacking the a' domain and decreased thermal stability of a construct lacking the a domain. Furthermore, oxidation of the a domain limited the ability of PDI to refold. The stability and conformational resilience of PDI are thus linked to both redox-dependent and domain-specific effects. These findings document previously unrecognized properties of PDI and provide insight into the physical foundation of its biological function.