Effects of amino acid replacements on cadmium binding of metallothionein alpha-fragment.
To evaluate whether only 20 cysteine residues at invariant positions are needed to bind and coordinate the metals in metallothioneins (MTs), and whether changing the positions of cysteine residues in the sequence affects the metal-binding capacity and the coordination of MTs, we examined the cadmium-binding affinities of seven mutant MT alpha s using an Escherichia coli expression system. Five mutant MT alpha s in which the constitutive amino acid residues other than cysteines of the alpha-fragment were replaced with glycine residues, and the remaining two mutant MT alpha s in which the invariant positions of the cysteine residues of the alpha-fragment were shifted, were analysed for their ability to be expressed as cadmium-binding forms and for their biochemical properties. The results showed that extreme alteration of the constitutive amino acid residues other than cysteines in the MT alpha-fragment leads to disruption of their cadmium-binding abilities and of their structure. However, mutant MT alpha s containing changes of the invariant positions of the cysteine residues were expressed in a cadmium-binding form in Escherichia coli, although the invariant positions of 20 cysteine residues in the MTs are thought to be important for their metal-binding abilities. These results suggest that the position of cysteine residues and the chemical nature of the other amino acids in the amino acid sequence of MTs are less critical than expected.
Yamasaki
F
F
Department of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan. futoshi@eesbio.hokudai.ac.jp
Kurasaki
M
M
Oikawa
S
S
Emoto
T
T
Okabe
M
M
Kojima
Y
Y
D000595:Amino Acid Sequence; D002104:Cadmium / Q000378:metabolism*; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
C491053:MT2A protein, human; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D003545:Cysteine