The crystal structure of the photoprotein aequorin at 2.3 Å resolution
The crystal structure of the photoprotein aequorin at 2.3 Å resolution
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DOI：
10.1038/35012659
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发表时间：
2000-05-18
期刊：
影响因子：
64.8
通讯作者：
Shimomura, O
中科院分区：
文献类型：
--
作者：
Head, JF;Inouye, S;Shimomura, O
Aequorin是一种钙敏感的光蛋白，最初从水母Aequorea Aequorea(1)中获得。由于Aequorin对钙离子高度敏感，且对生物无害，因此被广泛用作监测细胞内游离钙水平的探针。Aequorin分子包含四个螺旋-环-螺旋‘EF-Hand’结构域，其中三个能与钙结合(2)。该分子还含有作为发色配体的腔肠净(3)。当加入钙时，蛋白质复合体分解成载脂蛋白、腔肠酰胺和二氧化碳，并伴随着光的发射。在无钙的情况下，通过与土豆蔻嗪、氧气和硫醇试剂(5)孵育，可以将皂苷再生为活性皂苷。Aequorin互补DNA的克隆和表达最早是在1985年报道的(参考文献2，6)，并描述了重组蛋白晶体的生长(7)；然而，直到最近才发展出制备最高纯度的重组Aequorin的技术(8)，从而允许进行全面的结晶学研究。本文报道了用X-射线单晶衍射法测定重组马兜铃素的结构。Aequorin被发现是一种球状分子，含有一个疏水的核心空腔，该空腔容纳了配体coelenterazine-2-过氧化氢。该结构显示了稳定过氧化氢的蛋白质成分，并暗示了钙激活可能发生的机制。
Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea(1). Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix-loop-helix 'EF-hand' domains, of which three can bind calcium(2). The molecule also contains coelenterazine as its chromophoric ligand(3). When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light(4). Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent(5). Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described(7); however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity(8), permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.