Coordination Structures of Mg 21 and Ca 21 in Three Types of Tobacco Calmodulins in Solution: Fourier-Transform Infrared Spectroscopic Studies of Side-Chain COO 2 Groups Nanao Suzuki, 1,2 Lica Fabiana Imai, 2 Yusuke Kato, 2 Koji Nagata, 2 Yuko Ohashi, 3 Kazuyuki Kuchitsu, 4 Masaru Tanokura, 2 Akira Sakamoto, 5 Masayuki Nara, 6 Minoru Nakano, 1 Naoto Yonezawa 1 1 Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan 2 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan 3 Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan 4 Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan 5 Department of Chemistry, Faculty of Science, Saitama University, Shimo-okubo 255, Sakura-ku, Saitama 338-8570, Japan 6 Laboratory of Chemistry, College of Liberal and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan Received 3 October 2012; revised 25 December 2012; accepted 5 January 2013 Published online 19 January 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22203 This article was originally published online as an accepted preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com Coordination Structures of Mg 21 and Ca 21 in Three Types of Tobacco Calmodulins in Solution: Fourier-Transform Infrared Spectroscopic Studies of Side-Chain COO 2 Groups Correspondence to: Naoto Yonezawa; e-mail: nyoneza@faculty.chiba-u.jp ABSTRACT: Calmodulin (CaM) is a Ca 21 -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg 21 and Ca 21 among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak b-strand bands and the weak 1661 cm 21 band. Coordination structures of Mg 21 of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca 21 -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554–1550 cm 21 obtained by second- derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca 21 ] increases under low [Ca 21 ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca 21 ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg 21 -binding manner and needs higher [Ca 21 ] for bidentate Ca 21 coordination of 12th Glu in EF-hand motifs. The Ca 21 -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation- dependent conformational change in NtCaM13 is unique among the three NtCaMs. # 2013 Wiley Periodicals, Inc. Biopolymers 99: 472–483, 2013. Keywords: tobacco calmodulin; infrared spectroscopy; secondary structure; coordination structure Contract grant sponsors: Ministry of Education, Science, Sports and Culture of Japan; National Project on Protein Structural and Functional Analyses V V C 2013 Wiley Periodicals, Inc. 472 Biopolymers Volume 99 / Number 7