Abstract
In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.
Highlights
To understand how protein folding occurs in living cells we need to be able to describe protein folding pathways in the presence of cellular folding factors, and to characterize interactions between these factors and their unfolded and partly-folded protein substrates in thermodynamic, kinetic and structural terms
In this paper we have selected unfolded, partly-folded and folded species corresponding to stages on a well-defined oxidative protein folding pathway and characterized in physico-chemical terms their interaction with protein disulfide-isomerase (PDI), the major catalyst of oxidative protein folding in the endoplasmic reticulum of eukaryotic cells
The observed mass of the refolded wild-type corresponds to the theoretical mass of fully-oxidised wild-type basic pancreatic trypsin inhibitor (BPTI) containing 3 disulfide bonds (6643 Da), while that of the refolded double mutant corresponds to the mass expected for the species containing 2 disulfide bonds (6613 Da)
Summary
To understand how protein folding occurs in living cells we need to be able to describe protein folding pathways in the presence of cellular folding factors, and to characterize interactions between these factors and their unfolded and partly-folded protein substrates in thermodynamic, kinetic and structural terms. The process occurs in several cellular compartments [1], but has been most extensively studied in relation to proteins secreted by eukaryotic cells, whose oxidative folding occurs primarily in the endoplasmic reticulum [2]. Generation of native disulfides involves both disulfide formation and disulfide isomerization steps; rate-determining steps frequently involve the concomitant formation of ‘correct’ buried disulfide bonds and of the native conformation [3,10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
