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Structural characterization of protein-protein interaction sites by FT-ICR MS

通过 FT-ICR MS 表征蛋白质-蛋白质相互作用位点的结构

基本信息

批准号：
11680619
负责人：
AKASHI Satoko
金额：
$ 1.28万
依托单位：
RIKEN (The Institute of Physical and Chemical Research)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680619/
关键词：
protein-protein interaction H D exchange mass spectrometry cystatin lipid-micelle melittin

项目摘要

The interaction between cystatin and papain was investigated by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision induced dissociation (CID) in a hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain examining the mass of each fragment ion produced by hexapole-CID.In the absence of papain, amide hydrogens in short amino-terminal fragments were highly deuterated within 1min. In the case of cystatin-papain complex, significant drops in initial deuterium contents were recognized throughout the sequence of cystatin. Remarkable reduction in deuterium content in the region of residues l-10 was recognized for hours, suggesting that the flexible N-terminal region should have been tightly fixed in the binding pocket with hydrogen bonds. These resu … More lts were consistent with the previous studies on the structure and inhibition mechanism of cystatin. It was demonstrated that protein-protein interaction can be characterized by H/D exchange in combination with CID using ESI-FTICR MS within a short time using a small amount of sample.ESIMS was applied to the complex of papain and cystatin with partly lagged N-terminus. When cystatin was mixed with equimolar quantity of papain, the relative intensity of the free full-length cystatin dramatically decreased. It might be caused by the higher binding affinity of the intact cystatin for papain than those of the truncated forms. These results were consistent with those of the H/D exchange of cystatin-papain complex.The structural change of a peptide, melittin, caused by the interaction with lipid-micelles, dodecylphosphocholine, was investigated using the same method. It was confirmed that melittin is in a stable conformation in the presence of the lipid, while its structure is very flexible in the absence of the lipid. Less

采用氢-氘（H/D）交换和碰撞诱导解离（CID）连续分析，结合电喷雾电离-傅里叶变换离子回旋共振质谱（ESI-FTICR MS），研究了胱抑素和木瓜蛋白酶的相互作用。在存在或不存在木瓜蛋白酶的情况下，分析了不同时间点胱抑素主胺氢中氘的掺入情况，并检测了六极- cid产生的每个片段离子的质量。在没有木瓜蛋白酶的情况下，氨基末端短片段中的酰胺氢在1min内被高度氘化。在胱抑素-木瓜蛋白酶复合物的情况下，在整个胱抑素序列中都可以识别到初始氘含量的显著下降。残基l-10区域的氘含量在数小时内显著降低，这表明柔性n端区域应该被氢键紧密地固定在结合口袋中。这些结果与以往关于胱抑素结构和抑制机制的研究结果一致。结果表明，采用ESI-FTICR质谱法，在少量样品的情况下，可以在短时间内通过H/D交换和CID来表征蛋白质-蛋白质相互作用。ESIMS应用于部分n端滞后的木瓜蛋白酶和胱抑素复合物。当胱抑素与等摩尔量的木瓜蛋白酶混合时，游离全长胱抑素的相对强度显著降低。这可能是由于完整的胱抑素比截断的胱抑素对木瓜蛋白酶具有更高的结合亲和力。这些结果与胱抑素-木瓜蛋白酶复合物H/D交换结果一致。用同样的方法研究了肽蜂毒素与脂质胶束十二烷基磷脂胆碱相互作用引起的结构变化。证实了蜂毒蛋白在有脂质存在时结构稳定，而在没有脂质的情况下结构非常灵活。少