Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry
Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry
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DOI：
10.1007/s13361-013-0734-6
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发表时间：
2013-12-01
影响因子：
3.2
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
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作者：
Pettelkau, Jens;Thondorf, Iris;Sinz, Andrea
通过化学交联和高分辨质谱研究了GCAP-2同源二聚体的拓扑结构。补充进行的尺寸排阻色谱和分析超离心研究表明，GCAP-2形成一个同源二聚体在不存在和存在的Ca 2+。交联产物的深入MS和MS/MS分析由（15）N-标记的GCAP-2辅助。使用同位素标记的蛋白质提供了可靠的结构信息的GCAP-2同源二聚体，使一个单体（分子内）或两个亚基（分子间）之间的交联之间的明确区分。在Ca 2+结合状态下获得的有限数量的交联使我们能够通过对接和分子动力学方法推导出定义的同源二聚体GCAP-2结构。在无Ca 2+状态下，GCAP-2更灵活，如由更高数量的交联所指示的。我们认为稳定的同位素标记是不可或缺的，从多亚基蛋白质组装体的化学交联数据中获得可靠的结构信息。
The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by (15) N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.