Merck

S-Nitrosation destabilizes glutathione transferase P1-1.

Biochemistry (2013-11-26)
David Balchin, Stoyan H Stoychev, Heini W Dirr
ABSTRACT

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

MATERIALS
Product Number
Brand
Product Description

Supelco
L-Cysteine, certified reference material, TraceCERT®
Sigma-Aldrich
L-Cysteine, produced by Wacker Chemie AG, Burghausen, Germany, ≥98.0%
Sigma-Aldrich
L-Cysteine, BioUltra, ≥98.5% (RT)
Sigma-Aldrich
L-Cysteine, from non-animal source, BioReagent, suitable for cell culture, ≥98%
SAFC
L-Cysteine
Sigma-Aldrich
Glutathione S-Transferase from equine liver, lyophilized powder, ≥25 units/mg protein
Sigma-Aldrich
L-Cysteine, 97%
Sigma-Aldrich
L-Cysteine, ≥97%, FG