ERp46 substitutes for the a domain of PDI and increases conformational flexibility in a shuttle cysteine, Cys99, of Ero1-α. ¹³C NMR spectra of the constitutively active Ero1-α(C104A/C131A) labeled with ¹³C selectively at the carbonyl carbons of cysteine residues (Ero1*). (A–E) Spectra were measured in the absence (A) and presence of equimolar amounts of PDI (B), PDI(a′) (C), ERp46 (D), or PDI(a′) and ERp46 (E). Spectrum of the unlabeled protein has already been subtracted. PDI(a′) or ERp46 had no apparent effect on the mobilization of Cys99. The asterisk indicates the peak originating from Cys99. A.U., arbitrary unit.