Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.
Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.
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DOI：
10.1002/anie.201405362
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发表时间：
2014-10-20
期刊：
影响因子：
--
通讯作者：
Gopalan V
中科院分区：
文献类型：
--
作者：
Ma X;Lai LB;Lai SM;Tanimoto A;Foster MP;Wysocki VH;Gopalan V
我们证明了表面诱导解离（SID）结合离子迁移质谱（IM-MS）是确定Mg2+中组装的多亚基核糖核蛋白（RNP）复合物的化学计量学和四级结构的有力工具。我们在此研究了一种催化tRNA 5 '成熟的古细菌RNP——狂热焦球菌（Pfu） RNase P。先前，利用Pfu RNase P的催化RNA亚基和两个相互作用的蛋白质辅因子对（RPP21•RPP29和POP5•RPP30）对Pfu RNase P的Mg2+依赖性重构揭示了通往RNase P酶的功能性RNP中间体，但没有提供亚基化学计量学的信息。我们的原生质谱研究仅显示了RPP21•RPP29和（POP5•RPP30）2复合物，但当其中一个或两个蛋白复合物结合同源RNA时，所有亚基的组成为1:1。这些结果突出了SID和IM-MS在解决构象异质性和对RNP组装产生见解方面的效用。
We demonstrate that surface-induced dissociation (SID) coupled with ion mobility-mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry and quaternary structure of a multi-subunit ribonucleoprotein (RNP) complex assembled in Mg2+. We investigated here Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step-wise, Mg2+-dependent reconstitutions of Pfu RNase P using its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21•RPP29 and POP5•RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins alone showed RPP21•RPP29 and (POP5•RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.