Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

✍ Scribed by Masayuki Nara; Fumiaki Yumoto; Koji Nagata; Masaru Tanokura; Hiroyuki Kagi; Takao Ojima; Kiyoyoshi Nishita

• Publisher: Wiley (John Wiley & Sons)
• Year: 2004
• Tongue: English
• Weight: 87 KB
• Volume: 74
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.20048

✦ Synopsis

Abstract

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 m__M__). 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+^. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004