title: Expression and characterization of EF-hand I loop mutants of aequorin replaced with other loop sequences of Ca2+-binding proteins: an approach to studying the EF-hand motif of proteins.
abstract: The binding properties of Ca(2+) to EF-hand I of aequorin (AQ) were characterized by replacing the loop sequence of EF-hand I (AQ[I]) with other known loop sequences of Ca(2+)-binding proteins, including photoproteins (aequorin, clytin-I, clytin-II and mitrocomin), Renilla luciferin-binding protein (RLBP) and calmodulin (CaM). For evaluation of the binding affinity of Ca(2+) to AQ[I] mutants, the half-decay time of the maximum intensity in the luminescence reaction triggered by Ca(2+) was used as an indicator and 22 kinds of AQ[I] mutants were expressed in Escherichia coli cells. AQ[I] mutants replaced with the EF-hand I and EF-hand III from photoproteins showed sufficient luminescence activity, but it was not shown by other EF-hands from RLBP and CaM. An AQ[I] mutant with a lysine or arginine residue at the second position of the non-conserved amino acid residue showed a slow-decay pattern of luminescence, indicating that the Ca(2+)-binding affinity to aequorin was reduced by a positive charge at the second position of the loop sequence. The specific loop sequence of the EF-hand I motif in aequorin caused the specific Ca(2+)-triggered luminescence pattern.
authors.affiliation: Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan sinouye@jnc-corp.co.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inouye
authors.affiliation: Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan.
authors.forename: Yuiko
authors.initials: Y
authors.lastname: Sahara-Miura
mesh_terms: D000331:Aequorin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020816:Amino Acid Motifs; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D000331:Aequorin
keywords: EF-hand structure; calmodulin; coelenterazine; helix–loop–helix; photoprotein