Oxidative Folding of Proteins: The "Smoking Gun" of Glutathione

Abstract

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found "not guilty". Now, new surprising evidence repeats its request to be the "smoking gun" which reopens the criminal trial revealing the crucial involvement of this tripeptide.

Keywords: cysteine reactivity; glutathione; glutathionylation; nitrosylation; oxidative folding; ribosomal exit tunnel; transient complex.

Figure 1

Hyper-reactivity of structural cysteines in five different proteins. Hyper-reactivity of Cys75, Cys94, Cys95, Cys1, Cys148 and Cys197 toward GSSG found in albumin, lysozyme, ribonuclease A, chymotrypsinogen, and trypsinogen, respectively. Pseudo first-order kinetic constants were normalized to that of an unperturbed protein cysteine.

Figure 2

Dependence of the second-order kinetic constants (αk_RS-) on pK_a for the reaction of several thiols with different pK_a with different disulfides at pH 7.4 (modified from Ref. [9]). The red arrow marks the maximum value of the bell-shaped graph. The pK_a of the unperturbed protein cysteine is labelled with the green arrow. The maximum implement of reaction rate due to a lowered pK_a was found to be 3 times.

Figure 3

Reactivity of protein cysteines toward natural disulfides. The enhanced reactivity represents the second-order kinetic constants normalized to that of GSH. All proteins did not show any evident hyper-reactivity except the small enhanced reactivity found in Lysozyme toward cystine (65 times) which is small compared to the one of Cys94 toward GSSG (about 3000 times).

Figure 4

Visualization of ribosomal 50S subunit at the interface of endoplasmic reticulum with a nascent polypeptide chain. Modified cysteines of the bovine γB-crystallin found in the ribosomal exit tunnel during its nascent phase, as demonstrated in Ref. [15]. On the right, an “imaginary joke structure” of the glutathionylated protein, which represents the “smoking gun” for glutathione in the early scenario of the oxidative folding (the β-barrel structure represents the revolver grip, while the coiled coil is the revolver barrel).

Figure 5

Schematization of modified cysteines on a polypeptide. The upper side represents the nitrosylation due to GSNO. The lower side represents the glutathionylation due to GSSG.