© 2005 The Japanese Biochemical Society
Regular Paper |
NMR Analysis of the Mg2+-Binding Properties of Aequorin, a Ca2+-Binding Photoprotein
1 RIKEN Genomic Sciences Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045; and 2 Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa, Yokohama 236-8605
* To whom correspondence should be addressed. Tel: +81-45-503-9211, Fax: +81-45-503-9210, E-mail: hirota{at}gsc.riken.jp
Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.