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Accessibility explains preferred thiol-disulfide isomerization in a protein domain.

ABSTRACT: Disulfide bonds are key stabilizing and yet potentially labile cross-links in proteins. While spontaneous disulfide rearrangement through thiol-disulfide exchange is increasingly recognized to play an important physiological role, its molecular determinants are still largely unknown. Here, we used a novel hybrid Monte Carlo and Molecular Dynamics scheme to elucidate the molecular principles of thiol-disulfide exchange in proteins, for a mutated immunoglobulin domain as a model system. Unexpectedly, using simple proximity as the criterion for thiol-disulfide exchange, our method correctly predicts the experimentally observed regiospecificity and selectivity of the cysteine-rich protein. While redox reactivity has been examined primarily on the level of transition states and activation barriers, our results argue for accessibility of the disulfide by the attacking thiol given the highly dynamic and sterically demanding protein as a major bottleneck of thiol-disulfide exchange. This scenario may be similarly at play in other proteins with or without an evolutionarily designed active site.

SUBMITTER: Kolsek K

PROVIDER: S-EPMC5575259 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Accessibility explains preferred thiol-disulfide isomerization in a protein domain.

Kolšek Katra K Aponte-Santamaría Camilo C Gräter Frauke F

Scientific reports 20170829 1

Disulfide bonds are key stabilizing and yet potentially labile cross-links in proteins. While spontaneous disulfide rearrangement through thiol-disulfide exchange is increasingly recognized to play an important physiological role, its molecular determinants are still largely unknown. Here, we used a novel hybrid Monte Carlo and Molecular Dynamics scheme to elucidate the molecular principles of thiol-disulfide exchange in proteins, for a mutated immunoglobulin domain as a model system. Unexpected ...[more]

PMID: 28851879

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Project description:This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide Isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5-10nM, can utilize a range of thiol substrates (including 5µM dithiothreitol, 10µM reduced RNase thiols, and 5mM glutathione; GSH), and can operate from pH 6-9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-?-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1µM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

Dataset Information

Accessibility explains preferred thiol-disulfide isomerization in a protein domain.

Publications

Accessibility explains preferred thiol-disulfide isomerization in a protein domain.

Similar Datasets

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