Effect of cysteine replacements at positions 13 and 50 on metallothionein structure.
Recombinant wild-type and mutant Chinese hamster metallothioneins, purified from the yeast Saccharomyces cerevisiae, were analyzed for their chemical and spectroscopic properties. The mutant proteins contain cysteine to tyrosine replacements at positions 13 and 50. Wild-type and mutant metallothioneins, in their cadmium-bound forms, all showed characteristic ultraviolet absorption spectra with shoulders at 245-250 nm due to cadmium-thiolate charge transfer. Upon acidification, these absorption shoulders were abolished. In all cases, two distinct titrations were seen, presumably corresponding to two independent cadmium binding domains in each of the proteins. Analysis of domain structures was performed both with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) and with the protease subtilisin. These studies indicated that both mutations affected domain structure by disrupting the normally tight protein clusters. Circular dichroism spectra obtained for wild-type and mutant metallothioneins showed unique structural rearrangements in mutants containing a cysteine-50 to tyrosine alteration. These data, along with previously obtained 113Cd NMR data, were incorporated into a model which can account for the in vivo and in vitro properties of these mutant proteins.
Cismowski
M J
MJ
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Huang
P C
PC
D000818:Animals; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D003545:Cysteine