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Conformational Resilience of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) is a 57 kDa protein with both oxidoreductase and chaperone activities that is mainly localized to the endoplasmic reticulum. It is a U‐shaped protein with an abb'xa’ structural organization where the a and a′ domains have conserved CGHC active sites, the b and b′ domains are involved with substrate binding, and x is an unstructured linker. PDI exhibits substantial structural 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 the unfolding‐refolding cycle of PDI is a substrate‐induced process or an intrinsic physical propensity of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal stress. 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 84% of its native structure after 70% thermal denaturation. Oxidized PDI had a lower Tm of 48°C and only regained 44% of its native conformation after 70% denaturation. Additional studies documented increased thermal stability of a PDI deletion construct lacking the a′ domain and decreased thermal stability of a construct lacking the a domain. Both constructs were able to regain a portion of their native conformation after exposure to thermal stress and, like full‐length PDI, were more stable in the reduced form than the oxidized form. A bb'x PDI construct lacking both a and a′ domains was highly stable, with a 60°C Tm in the reduced state and a 56°C Tm in the oxidized state. Both reduced and oxidized forms of the bb'x construct could return to the native state after heating to 90°C. These findings provide new insight into the physical foundation of the biological function of PDI.