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Nuclear magnetic resonance studies of calcyclin and annexin XI.

钙环蛋白和膜联蛋白 XI 的核磁共振研究。

基本信息

批准号：
06044105
负责人：
HIDAKA Hiroyoshi
金额：
$ 7.04万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

Ca^<2+>-binding proteins is classified into major 3 groups based on structure of the Ca^<2+>-binding domain, 1) EF-hand proteins, 2) annexins, 3) C2-domain proteins. This study was mainly aimed at the elucidation of three-dimensional structure of calcyclin, a member of S100 family with 2 EF-hands and annexin XI which is a ligand of calcyclin with NMR spectroscopy. The three dimensional structure of calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among Ca^<2+>-binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of Ca^<2+>-binding proteins.The subcellular localization of annexin XI is, unique among the family, mainly in a uncleus of cells such … More as COS-7 and is altered by phosphorylation. In this study, it was revealed that annexin XI localized in a nucleus by the regulatory domain as nuclear localization signal, and the localization is regulated depending on developmental and growth stages, and cell types, and annexin XI isoforms generated by alternative splicing in the N-terminal regulatory domain differed in subcellular localization.We indentified the calcyclin binding domain on annexin XI with annexin XI-GST fusion proteins. This results indicated that calcyclin bound in the region from Gln49 to Thr62. Chou-Fasman analysis of the binding domain suggest a highly probability for a-helical formation with hydrophobic residues aligning onto one side of a theoretical amphipathic helix. It was suggested that the annexin XI hydrophobic face was the binding site for calcyclin. We synthesized the peptide containing the calcyclin binding site of annexin XI,which could bind to calcyclin. We have been analyzed the three-dimensional structure of the complex of calcyclin and the calcyclin binding peptide of annexin XI with NMR spectroscopy. These results obtained in this study contribute to the studies of not only the structure analysis of Ca^<2+>-binding proteins but also the protein-protein interaction in other field. Less

根据Ca^2+结合结构域的结构，Ca^2+结合蛋白主要分为3类：1）EF-手蛋白，2）膜联蛋白，3）C2-结构域蛋白。本研究的主要目的是利用核磁共振波谱技术对S100家族中具有两个EF-手的钙周期素及其配体annexin XI的三维结构进行解析。钙周期蛋白的三维结构已被确定在溶液中的载脂蛋白状态的NMR光谱和计算策略，包括一个系统的对接协议。这种结构揭示了一种对称的同源二聚体折叠，这在Ca^2+结合蛋白中是独一无二的。二聚化是由几个高度保守的残基的疏水性接触介导的，这表明钙周期蛋白鉴定的二聚体折叠将作为Ca^<2+>结合蛋白S100亚家族的结构范例。 ...更多信息与COS-7一样，并通过磷酸化改变。本研究发现，annexin XI通过调节结构域作为核定位信号定位于细胞核内，其定位受发育和生长阶段以及细胞类型的调节，并且通过N端调节结构域的选择性剪接产生的annexin XI亚型在亚细胞定位上存在差异，我们用annexin XI-GST融合蛋白鉴定了annexin XI上的钙周期蛋白结合结构域。该结果表明，钙周期蛋白结合在Gln 49至Thr 62的区域。结合结构域的Chou-Fasman分析表明，疏水残基排列在理论两亲性螺旋的一侧上的α-螺旋形成的可能性很高。这表明膜联蛋白XI的疏水表面是钙周期蛋白的结合位点。我们合成了含有膜联蛋白XI的钙周期蛋白结合位点的多肽，它可以与钙周期蛋白结合。我们用核磁共振波谱分析了钙周期蛋白和钙周期蛋白结合肽膜联蛋白XI的复合物的三维结构。这些结果不仅为Ca^2+结合蛋白的结构分析提供了理论依据，而且为其它领域的蛋白质相互作用研究提供了理论依据。少