title: Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+.
abstract: Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing experiments were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, respectively. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Co(n)-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric analysis of the titration suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titration of free sulfhydryl groups (S) in Co(n)-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Co(n)-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.
authors.affiliation: Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ejnik
authors.forename: James
authors.initials: J
authors.lastname: Robinson
authors.forename: Jianyu
authors.initials: J
authors.lastname: Zhu
authors.forename: Holger
authors.initials: H
authors.lastname: Försterling
authors.forename: C Frank
authors.initials: CF
authors.lastname: Shaw
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000478:Alkylation; D000818:Animals; D002104:Cadmium / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*
chemical_list: D002104:Cadmium; D003035:Cobalt; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D015032:Zinc