Isolated domains of recombinant human apo-metallothionein 1A are folded at neutral pH: a denaturant and heat-induced unfolding study using ESI-MS

• Published in: Bioscience reports Vol. 38; no. 4
• Main Authors: Irvine, Gordon W, Korkola, Natalie, Stillman, Martin J
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 31.08.2018
• Subjects: metal-induced folding; ESI-MS; protein conformation; metallothionein; denaturant
• ISSN: 0144-8463; 1573-4935
• DOI: 10.1042/BSR20180592

Metallothioneins (MTs) are characterized by their high metal loading capacity, small molecular weight, and abundant cysteine residues. It has long been thought that metal-free, or apo-MT peptides were unstructured and only adopted as a distinct conformation upon forming the metal clusters, described as metal-induced folding. More recent studies have suggested that the presence of a globular, yet loosely defined structure actually exists that can be disrupted or unfolded. Residue modification and ion-mobility ESI (IM-ESI)-MS have been used to examine this unusual unfolding process. The structure of apo-MT plays a critical role as the starting point in the flexible metalation pathways that can accommodate numerous soft metals. ESI-MS measurements of the product species formed following the cysteine alkylation of the isolated domain fragments of recombinant human apo-MT 1A with -ethylmaleimide (NEM) were used in the present study to monitor the denaturant- and heat-induced unfolding at physiological pH. The results indicate that these apo-MT fragments adopt distinct structures at neutral pH that react co-operatively with NEM when folded and non-cooperatively when heated or exposed to high concentrations of the denaturant guanidinium chloride (GdmCl). From these studies, we can conclude that at neutral pH, the domain fragments are folded into globular structures where some of the free cysteine residues are buried within the core and are stabilized by hydrogen bonds. Metalation therefore, must take place from the folded conformation.