Coordination structures of Mg2+ and Ca2+ in three types of tobacco calmodulins in solution: Fourier‐transform infrared spectroscopic studies of side‐chain COO− groups

Calmodulin (CaM) is a Ca²⁺‐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 Mg²⁺ and Ca²⁺ 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 β‐strand bands and the weak 1661 cm^?1 band. Coordination structures of Mg²⁺ of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca²⁺‐binding manner was similar among the three CaMs. The amplitude differences of the band at 1554–1550 cm^?1 obtained by second‐derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca²⁺] increases under low [Ca²⁺] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca²⁺]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg²⁺‐binding manner and needs higher [Ca²⁺] for bidentate Ca²⁺ coordination of 12th Glu in EF‐hand motifs. The Ca²⁺‐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.