Topographical analysis of As-induced folding of α-MT1a.

Metallothionein binds multiple metals into two clustered domains. While the structure of the fully metalated protein is well known for the Cd- and Zn-containing protein, there is little known about the structures of the metal-free protein (apo-metallothionein) and even less about the partially metalated forms. However, the partially-metalated species are vitally important intermediates in the passage of the protein from translational synthesis to its homeostatic buffer or metal chaperone roles. Because multiple metals bind to metallothioneins, the partially-metalated species span a wide range depending on the metal bound. Up to 3 As(3+) bind stepwise to the α-domain fragment in a manner that allows measurement of each of the 4 species simultaneously with the number of free cysteines diminishing by 3 for every As(3+) bound: apo- (11 Cys), As1- (8 Cys), As2- (5 Cys) and As3-α-MT (2 Cys). The cysteine modifier benzoquinone (Bq), was used to determine the relative accessibility of the free cysteines in the α-MT fragment as a function of the number of As(3+) bound. The effect of each As(3+) was to induce folding in the protein. The ESI-MS results show that the whole protein folds significantly even when just one of the three As(3+) has bound. The profile of the Bq reacting with the unbound cysteines shows effects of steric hindrance in slowing down the reaction. By freezing the reaction midway to the endpoint, the mass spectral data show the 'mid-flight' concentrations of all the key species, 27 in all. Analysis of this mid-flight reaction profile gives insight into the topology of the partially metalated MT from the differential access to the unbound cysteinyl thiols by the Bq. Significantly, the metal-free, apo-α-MT also adopts a folded structure in the presence of the As(3+) even though there is no As(3+) bound. This can only happen if the apo-protein wraps around other metalated proteins in solution via protein-protein interactions.