Kinetic and thermodynamic analysis of the conformational folding process of SS‐reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability

Arai, Kenichiro; Kumakura, Fumio; Iwaoka, Michio

doi:10.1016/j.fob.2012.04.001

Cited by 16 publications

(21 citation statements)

References 61 publications

(116 reference statements)

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“…These des intermediates, which are the metastable key folding intermediates of HEL, were unambiguously characterized by the thermolysin digestion (see Figures S2–S4 and Tables S2–S4). Since no DHS ox was left in the solution and the intermolecular SS exchange reaction among oxidative folding intermediates must proceed much slower than the corresponding intramolecular process [11], the des intermediates of HEL should not be generated from 2S but from 3S via intramolecular SS rearrangement ( i.e. , 3S→des).…”

Section: Resultsmentioning

confidence: 99%

“…To estimate the exact populations of the des intermediates and N generated in the folding solutions, reduction pulse experiments [11,13] were performed. In the experiments, an excess amount of DTT red was added before quenching the folding reaction by AEMTS so as to rapidly reduce the intermediates without rigid folded structure ( i.e ., 1S–4S) back to R (and/or 1S).…”

Section: Resultsmentioning

confidence: 99%

“…To identify the effective precursors of N in the oxidative folding pathways of HEL, oxidation pulse experiments [11] were performed. After incubation of the reaction mixture of R and three equivalents of DHS ox for 300 min, the generated SS intermediates were forced to be oxidized with 1.5 equivalents of DHS ox .…”

Section: Resultsmentioning

confidence: 99%

“…, oxidation) proceeds much faster than SS rearrangement. This allows accumulation of 3S prior to the rearrangement to the des intermediates [9,11]. …”

Section: Discussionmentioning

confidence: 99%

“…DHS ox enables rapid, irreversible, and stoichiometric SS formation in various polythiol peptides in an aqueous medium [10]. For example, when this analytical method was applied to the oxidative folding of bovine pancreatic RNase A having four SS linkages in the native state, the SS formation and SS rearrangement phases were clearly separated from each other, and the oxidative folding pathways could be easily characterized [11,12]. Encouraged by the success, we have applied herein the DHS ox strategy to HEL to characterize the precursors of the des intermediates as well as to identify the major folding pathways at various temperatures.…”

Section: Introductionmentioning

confidence: 99%

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Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

Arai

Shibagaki

Shinozaki

et al. 2013

IJMS

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It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64–80] and des[6–127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5–45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1–4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1–4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76–94] was populated in the largest amount, but the oxidation to N was slower than that of des[64–80] and des[6–127]. At 35 °C, on the other hand, des[64–80] and des[6–127] were no longer stable, and only des[76–94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…, oxidation) proceeds much faster than SS rearrangement. This allows accumulation of 3S prior to the rearrangement to the des intermediates [9,11]. …”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

Arai

Shibagaki

Shinozaki

et al. 2013

IJMS

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Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

2019

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Bovine β‐lactoglobulin (BLG) is a major whey protein with unique structural characteristics: it possesses a free Cys thiol (SH) and two disulfide (SS) bonds and consists of a β‐barrel core surrounded by one long and several short α helices. Although SS‐intact conformational folding has been studied in depth, the oxidative folding pathways and accompanying SS formation/rearrangement are poorly understood. In this study, we used trans‐3,4‐dihydroxyselenolane oxide, a water‐soluble selenoxide reagent which undergoes rapid and quantitative SS formation, to determine the oxidative folding pathways of BLG variant A (BLGA) at pH 8.0 and 25 °C. This was done by characterizing two key one‐SS intermediates, a particular folding intermediate having a Cys66–Cys160 SS bond (I‐1) and a particular folding intermediate having a Cys106–Cys119 SS bond (I‐2), which have a native Cys66–Cys160 and Cys106–Cys119 SS bond, respectively. In the major folding pathway, the reduced protein (R) with abundant α helices was oxidized to I‐1, which was then transformed to I‐2 through SS rearrangement. The native protein (N) was formed by oxidation of I‐2. The redundant Cys121 thiol facilitates SS rearrangement. N is also generated from an ensemble of folding intermediates having two SS bonds (2SS) intermediates with scrambled SS bonds through SS rearrangement, but this minor pathway is deteriorative due to aggregation or overoxidation of 2SS. During oxidative folding of BLGA, α→β conformational transition occurred as previously observed in SS‐intact folding. These findings are informative not only for elucidating oxidative folding pathways of other members of the β‐lactoglobulin family, but also for understanding the roles of a redundant Cys thiol in the oxidative folding process of a protein with odd Cys residues.

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An Amphiphilic Selenide Catalyst Behaves Like a Hybrid Mimic of Protein Disulfide Isomerase and Glutathione Peroxidase 7

Arai

Moriai

Ogawa

et al. 2014

Chemistry An Asian Journal

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Protein disulfide isomerase (PDI) and glutathione peroxidase 7 (GPx7) cooperatively promote the oxidative folding of disulfide (SS)-containing proteins in endoplasmic reticulum by recognizing the nascent proteins to convert them into the native folds by means of SS formation and SS isomerization and by catalyzing reoxidation of reduced PDI with H2O2, respectively. In this study, new amphiphilic selenides with a long-chain alkyl group were designed as hybrid mimics of PDI and GPx7 and were applied to the refolding of reduced hen egg-white lysozyme (HEL-R). Competitive SS formation at pH 4 using HEL-R and glutathione (GSH) in the presence of the selenide catalyst and H2O2 showed that the amphiphilic selenides can preferentially catalyze SS formation of HEL-R, probably on account of hydrophobic interactions between the protein and the catalyst. In contrast, simple water-soluble selenides did not exhibit such behavior. In addition, when the pH of the solution was adjusted to 8.5 after the SS formation, surviving GSH promoted the SS isomerization of misfolded HEL to recover the native SS linkages. Thus, the amphiphilic selenides designed here could mimic the function of the PDI-GPx7 system. The combination of a water-soluble selenide and a long-chain alkyl group would be a useful motif in designing medicines for both protein misfolding diseases and antioxidant therapy.

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Kinetic and thermodynamic analysis of the conformational folding process of SS‐reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability

Cited by 16 publications

References 61 publications

Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

An Amphiphilic Selenide Catalyst Behaves Like a Hybrid Mimic of Protein Disulfide Isomerase and Glutathione Peroxidase 7

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