title: Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification.
abstract: Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the bound oxygen. After light emission, aequorin may be regenerated by incubating the protein with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol. To understand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine. Six of the seven modified aequorins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin. Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type aequorin. The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity.
authors.affiliation: Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kurose
authors.forename: S
authors.initials: S
authors.lastname: Inouye
authors.forename: Y
authors.initials: Y
authors.lastname: Sakaki
authors.forename: F I
authors.initials: FI
authors.lastname: Tsuji
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D007700:Kinetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids
chemical_list: D008164:Luminescent Proteins; D000331:Aequorin; D003545:Cysteine; D002118:Calcium