Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q).
Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).
Nara
Masayuki
M
Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Chiba 272-0827, Japan. nara.las@tmd.ac.jp
Yumoto
Fumiaki
F
Nagata
Koji
K
Tanokura
Masaru
M
Kagi
Hiroyuki
H
Ojima
Takao
T
Nishita
Kiyoyoshi
K
D000818:Animals; D001669:Biochemical Phenomena; D001671:Biochemistry; D002942:Circular Dichroism; D008274:Magnesium / Q000737:chemistry*; D008974:Mollusca; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D019209:Troponin C / Q000235:genetics* / Q000378:metabolism
D019209:Troponin C; D008274:Magnesium