Effect of protein disulfide isomerase on the rate‐determining steps of the folding of bovine pancreatic ribonuclease A

Shin, Hang-Cheol; Song, Myeong-Cheol; Scheraga, Harold A.

doi:10.1016/s0014-5793(02)02825-9

Cited by 14 publications

(11 citation statements)

References 28 publications

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“…PDI catalyzes oxidative folding of many substrates including proteins with well-defined oxidative folding pathways such as ribonuclease [14], [15], lysozyme [16] and basic pancreatic trypsin inhibitor (BPTI) [17]–[19]. In each case, PDI catalyzes formation and isomerization of disulfides, both in early unstructured intermediates, and in later rate-determining steps in which relatively structured intermediates undergo disulfide isomerization to form stable conformation and stable native disulfides.…”

Section: Introductionmentioning

confidence: 99%

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

et al. 2014

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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.

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Section: Introductionmentioning

confidence: 99%

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

et al. 2014

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“…Therefore, the current experimental design may allow us to probe the interactions of PDI/PDIp with unfolded RNase containing the original native disulfide bonds. The results of our present study offer mechanistic insights into the earlier observations that PDI can efficiently catalyze the conversion of kinetically-trapped substrate intermediates containing native disulfide bonds into on-pathway intermediates during the oxidative refolding of RNase and BPTI [7, 13–14, 27, 29]. …”

Section: Introductionmentioning

confidence: 54%

“…Notably, stable disulfide-linked PDI-substrate complexes have also been observed during the refolding of some of these substrate proteins [8, 21, 23–25] and these complexes were suggested to be intermediates during PDI-catalyzed protein folding [26]. Besides non-native disulfide bonds, there are also evidence suggesting that native disulfide bonds, either in native proteins [13, 15–16] or in folding intermediates [7, 14, 27–28], if accessible to PDI, are also subject to its attack. In these cases, although PDI was hypothesized to also form disulfide-linked PDI-substrate complexes [15], there has been no direct evidence presented thus far for the existence of such complexes [7, 14–16, 27–28].…”

Section: Introductionmentioning

confidence: 99%

“…Besides non-native disulfide bonds, there are also evidence suggesting that native disulfide bonds, either in native proteins [13, 15–16] or in folding intermediates [7, 14, 27–28], if accessible to PDI, are also subject to its attack. In these cases, although PDI was hypothesized to also form disulfide-linked PDI-substrate complexes [15], there has been no direct evidence presented thus far for the existence of such complexes [7, 14–16, 27–28]. Therefore, whether PDI can really form disulfide-linked complexes with native disulfide bond-containing substrate proteins remains an unanswered question.…”

Section: Introductionmentioning

confidence: 99%

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Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes

Zhu

2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.

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“…The refolding process to obtain the native disulfide bonds is known to be very difficult. Protein disulfide isomerase (PDI) catalyzes disulfidecoupled folding of proteins and its mechanism is well-studied (Shin and Scheraga 2000;Shin et al 2002). In this study, PDI was used as a fusion partner to produce an active EKL and it was anticipated that PDI helps EKL acquire native disulfide linkages during oxidation period.…”

Section: Expression Of Pdi-eklmentioning

confidence: 99%

Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli

et al. 2011

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Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.

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Effect of protein disulfide isomerase on the rate‐determining steps of the folding of bovine pancreatic ribonuclease A

Cited by 14 publications

References 28 publications

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes

Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli

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