title: Roles of the C-terminal residues of calmodulin in structure and function.
abstract: Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+induced conformational change and to form a proper Ca2+-saturated conformation.
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, N10 W8, Sapporo 060-0810, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kitagawa
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, N10 W8, Sapporo 060-0810, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Nakatomi
authors.affiliation: Center for Nano Materials and Technology (CNMT), Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Dasol
authors.initials: D
authors.lastname: Hwang
authors.affiliation: Center for Nano Materials and Technology (CNMT), Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Issey
authors.initials: I
authors.lastname: Osaka
authors.affiliation: Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamatecho, Suita, Osaka 564-8680, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Fujimori
authors.affiliation: Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamatecho, Suita, Osaka 564-8680, Japan.
authors.forename: Hideya
authors.initials: H
authors.lastname: Kawasaki
authors.affiliation: Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamatecho, Suita, Osaka 564-8680, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Arakawa
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, N10 W8, Sapporo 060-0810, Japan.
authors.forename: Yota
authors.initials: Y
authors.lastname: Murakami
authors.affiliation: Center for Nano Materials and Technology (CNMT), Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohki
keywords: CD; ESI-MS; NMR; activity; truncation