NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein.
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.
Ohashi
Wakana
W
RIKEN Genomic Sciences Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045.
Inouye
Satoshi
S
Yamazaki
Toshio
T
Hirota
Hiroshi
H
D000331:Aequorin / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation
D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D000331:Aequorin; D008274:Magnesium