Abstract
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.
Highlights
The oxidative folding of proteins containing disulfide bridges is a critical process in the pathway leading to native conformations [1]
This paper discovers that the reduced ribonuclease (rRNase) in a molten globule like conformation, representing the early proof of its native conformation, shows one particular cysteine hyper-reactive toward GSSG, which is estimated to be thousand times more reactive than an unperturbed protein cysteine
This study discloses for the first time the existence, in the rRNase, of a curious and extraordinary hyper-reactivity of one cysteine toward GSSG and a parallel relevant hyper-reactivity of five other cysteines (3500 and 45 times more reactive when compared to an unperturbed protein cysteine, respectively) (Table 1, Figures 2 and 5)
Summary
The oxidative folding of proteins containing disulfide bridges is a critical process in the pathway leading to native conformations [1]. Disulfide bonds have co-evolved with protein sequences to minimize protein misfolding and propensity to form potentially toxic aggregates [5]; and understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent these diseases [6]. In this context, the discovery of new kinetic properties of structural cysteines in the nascent structure of a protein may be of paramount interest and it represents the main novelty of the present study
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