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首页> 外文会议>American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics >Extending the Cross-Linking/MS Strategy: Investigation of Nidogen-1 Complexes by Incorporated Photo-Amino Acids and Photo-Cross-Linking
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Extending the Cross-Linking/MS Strategy: Investigation of Nidogen-1 Complexes by Incorporated Photo-Amino Acids and Photo-Cross-Linking

机译：扩展交联/ MS策略：通过掺入的光氨基酸和光交联的纳米酮-1个配合物研究

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摘要

The interactions of nidogen-1 were investigated using a combination of chemical cross-linking with an amine-reactive cross-linker [4] and photo-cross-linking [5] with high-resolution mass spectrometry. The diazirine-containing amino acids photo-methionine and photoleucine were incorporated into nidogen-1. The C-terminal globular G3 domain of nidogen-1 is known to interact with the epidermal growth factor-like modules LE 3-5 (novel nomeclature: LEb2-4) in laminin γ1 and this complex has been crystallized (pdb entry 1NPE) [3]. Therefore, we chose the complex between nidogen-1 and laminin γ1 LE 3-5 as a system to validate our photo-cross-linking approach. Photo-amino acid incorporation rates into nidogen-1 were much higher for photo-methionine than for photo-leucine, but despite the relatively low incorporation rate of photo-leucine, several cross-linked products were identified. Cross-linking data with the aminereactive cross-linker BS~2G and photo-cross-linking were complementary. Our results indicate that laminin γ1 LE 3-5 does not exclusively interact with the G3 domain of nidogen-1, but also with additional N-terminal regions of nidogen-1. Moreover, the analysis of intramolecular cross-links within nidogen-1 yielded a number of cross-links between N- and C-terminal regions suggesting a globular structure of nidogen-1 rather than a linear domain arrangement. Based on the obtained distance constraints, a 3D-structural model was created of the nidogen-1/ laminin γ1 LE 3-5 complex.
机译：使用与胺 - 反应性交联剂[4]和化学交联的组合巢蛋白-1的相互作用进行了调查光交联[5]使用高分辨率质谱。氨基酸照片甲硫氨酸和photoleucine含吖丙因-分别并入巢蛋白-1。巢蛋白-1的C末端球形G3域是已知的相互作用与表皮生长因子样模块LE 3-5：层粘连蛋白γ1（新颖nomeclature LEb2-4）和该复合物已结晶（PDB条目1NPE） 3]。因此，我们选择巢蛋白-1和层粘连蛋白γ1LE 3-5之间的复杂的系统来验证我们的光交联方法。照片氨基酸掺入率成巢蛋白-1高得多的照片蛋氨酸比照片亮氨酸，但尽管照片亮氨酸的相对较低的掺入率，分别确定了几个交联产物。交联与胺反应的交联剂BS〜2G和光交联的数据是互补的。我们的研究结果表明，层粘连蛋白γ1LE 3-5不完全互动与巢蛋白-1的G3域名，但也与巢蛋白-1的额外的N末端区域。此外，巢蛋白-1内的分子内交联的分析产生了一些N-和C-末端区域之间的交联表明巢蛋白-1，而不是线性域排列的球状结构的。基于所获得的距离的限制，在3D-结构模型创建巢蛋白-1 /层粘连蛋白γ1LE 3-5络合物。

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