Recognition of protein substrates by protein-disulfide isomerase - A sequence of the b ' domain responds to substrate binding

Refolding of partially folded mitochondrial malate de hydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold m olar excess of PDI over denatured protein (0.1 mu M) accelerates the recove ry of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 mu M . The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation c onstant of 0.2 mu M. It is shown that the fluorescence probe is covalently attached to a SH resi due located in the b' domain. Based on the fluorescence measurements of nat ive and derivatized PDI, it is suggested that recognition of the unfolded s ubstrate involves conformational changes propagated to several domains of P DI.