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Open AccessArticle

Ultra-Rapid Glutathionylation of Ribonuclease: Is This the Real Incipit of Its Oxidative Folding?

1
Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, 00133 Rome, Italy
2
CEINGE Biotecnologie Avanzate and Department of Chemical Science, University of Naples “Federico II”, 80126 Naples, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(21), 5440; https://doi.org/10.3390/ijms20215440
Received: 7 October 2019 / Revised: 28 October 2019 / Accepted: 29 October 2019 / Published: 31 October 2019
(This article belongs to the Collection Protein Folding)
Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40–50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5’-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease. View Full-Text
Keywords: ribonuclease; glutathionylation; oxidative folding; molten globule; cysteine reactivity ribonuclease; glutathionylation; oxidative folding; molten globule; cysteine reactivity
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Bocedi, A.; Cattani, G.; Gambardella, G.; Ticconi, S.; Cozzolino, F.; Di Fusco, O.; Pucci, P.; Ricci, G. Ultra-Rapid Glutathionylation of Ribonuclease: Is This the Real Incipit of Its Oxidative Folding? Int. J. Mol. Sci. 2019, 20, 5440.

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