Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes
Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes
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DOI：
10.1007/978-1-4939-3341-9_9
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发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
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作者：
Loessl, Philip;Sinz, Andrea
在过去的15年里，化学交联和高分辨率质谱学的结合已经成熟为分析蛋白质和蛋白质复合体三维结构的另一种方法。使用由交联链施加的距离限制，可以创建被研究的蛋白质或蛋白质复合体的模型。目前大多数的交联剂研究都是用同双官能团胺反应型交联剂进行的。我们通过添加补充的光交叉链接数据来扩展这一“传统的”交叉链接/MS策略。为此，含有二氮杂环的非天然氨基酸光亮氨酸和光蛋氨酸被结合到蛋白质中，并通过UV-A辐射诱导交联物的形成。光交联策略的优点是它不限于赖氨酸残基，并且可以针对蛋白质中的疏水区域进行定位，这有利于膜蛋白的研究。我们认为，将交联剂与正交反应性和距离相结合的策略非常适合于最大限度地增加从交联实验中获得的结构信息量。
During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.