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

Bocedi, Alessio; Cattani, Giada; Gambardella, Giorgia; Ticconi, Silvia; Cozzolino, Flora; Fusco, Ornella Di; Pucci, Piero; Ricci, Giorgio

doi:10.3390/ijms20215440

Cited by 6 publications

(26 citation statements)

References 33 publications

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“…By examining the reaction of the reduced molten globule-like structures of the four tested proteins with oxidized glutathione (GSSG), we found that a single specific cysteine in each protein displays thousands-times higher reactivity than an unperturbed protein cysteine (Table 1). This phenomenon has been observed in rBSA, rLyz, rRNase and rChTg, for Cys75, Cys94, Cys95 and Cys1, respectively [13][14][15][16]. This hyper-reactivity is specific for GSSG, because a normal reactivity has been found toward other natural disulfides like cystine and cystamine (see Table 1).…”

Section: Productive Transient Complexmentioning

confidence: 71%

“…This phenomenon is mainly due to a productive transient protein-GSSG complex, as proven by fluorescence and kinetic experiments which also defined the dissociation constants of this interaction (K D = 0.3 mM for rLyz, K D = 0.12 mM for rRNase and K D = 1.5 mM for rChTg) [13][14][15][16]. In other words, a specific region of the molten globule-like conformations of these proteins resembled an enzyme active site which was able to bind productively with GSSG in order to react with a selected cysteine.…”

Section: Productive Transient Complexmentioning

confidence: 78%

“…The circular dichroism (CD) spectra for albumin, lysozyme, ribonuclease and chymotrypsinogen, both in their native and in their reduced molten globule-like status, have been reported in previous studies [13][14][15][16]. Now, a more detailed analysis, performed using the BeStSel program [20], can give us further insights about the molten globule-like structures.…”

Section: Analyses and Ans Fluorescence Fulfill Further Insightsmentioning

confidence: 80%

“…As a third and more evident kinetic-enhancing factor, we discovered the specific interaction of GSSG with only one cysteine residue of each of these proteins. Here, the increased reactivity is evaluated in terms of thousand-times with a stringent specificity: no other small natural disulfides, like cystine and cystamine, show similar reactivities [13][14][15][16]. This hyper-reactivity toward GSSG could reveal a primordial event inside the oxidative folding of many disulfide containing proteins, given that GSSG is present at millimolar concentrations in the endoplasmic reticulum [23].…”

Section: Discussionmentioning

confidence: 99%

“…These finding could explain the absence of hyper-reactivity toward CDNB and NBD-Cl (Table 1). a The CD spectra reported in previous studies [13][14][15][16] were analyzed using the BeStSel program. Abbreviations: BSA, bovine serum albumin; ChTg, chymotrypsinogen; Lyz, lysozyme; RNase, ribonuclease.…”

Section: Analyses and Ans Fluorescence Fulfill Further Insightsmentioning

confidence: 99%

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New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures

Gambardella

Cattani

Bocedi

et al. 2020

IJMS

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Protein cysteines often play crucial functional and structural roles, so they are emerging targets to design covalent thiol ligands that are able to modulate enzyme or protein functions. Some of these residues, especially those involved in enzyme mechanisms—including nucleophilic and reductive catalysis and thiol-disulfide exchange—display unusual hyper-reactivity; such a property is expected to result from a low pKa and from a great accessibility to a given reagent. New findings and previous evidence clearly indicate that pKa perturbations can only produce two–four-times increased reactivity at physiological pH values, far from the hundred and even thousand-times kinetic enhancements observed for some protein cysteines. The data from the molten globule-like structures of ribonuclease, lysozyme, bovine serum albumin and chymotrypsinogen identified new speeding agents, i.e., hydrophobic/electrostatic interactions and productive complex formations involving the protein and thiol reagent, which were able to confer exceptional reactivity to structural cysteines which were only intended to form disulfides. This study, for the first time, evaluates quantitatively the different contributions of pKa and other factors to the overall reactivity. These findings may help to clarify the mechanisms that allow a rapid disulfide formation during the oxidative folding of many proteins.

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Section: Productive Transient Complexmentioning

confidence: 71%

Section: Productive Transient Complexmentioning

confidence: 78%

Section: Analyses and Ans Fluorescence Fulfill Further Insightsmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Analyses and Ans Fluorescence Fulfill Further Insightsmentioning

confidence: 99%

See 3 more Smart Citations

New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures

Gambardella

Cattani

Bocedi

et al. 2020

IJMS

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Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins

Bocedi

Gambardella

Cattani

et al. 2020

Sci Rep

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Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the “incipit” of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

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The unusual properties of lactoferrin during its nascent phase

Notari,

Gambardella,

Vincenzoni

et al. 2023

Sci Rep

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Lactoferrin, a multifunctional iron-binding protein containing 16 disulfides, is actively studied for its antibacterial and anti-carcinogenic properties. However, scarce information is nowadays available about its oxidative folding starting from the reduced and unfolded status. This study discovers unusual properties when this protein is examined in its reduced molten globule-like conformation. Using kinetic, CD and fluorescence analyses together with mass spectrometry, we found that a few cysteines display astonishing hyper-reactivity toward different thiol reagents. In details, four cysteines (i.e. 668, 64, 512 and 424) display thousands of times higher reactivity toward GSSG but normal against other natural disulfides. The formation of these four mixed-disulfides with glutathione probably represents the first step of its folding in vivo. A widespread low pKa decreases the reactivity of other 14 cysteines toward GSSG limiting their involvement in the early phase of the oxidative folding. The origin of this hyper-reactivity was due to transient lactoferrin-GSSG complex, as supported by fluorescence experiments. Lactoferrin represents another disulfide containing protein in addition to albumin, lysozyme, ribonuclease, chymotrypsinogen, and trypsinogen which shows cysteines with an extraordinary and specific hyper-reactivity toward GSSG confirming the discovery of a fascinating new feature of proteins in their nascent phase.

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

Cited by 6 publications

References 33 publications

New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures

New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins

The unusual properties of lactoferrin during its nascent phase

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