Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex.
Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex.
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DOI：
10.1007/s13361-017-1882-x
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发表时间：
2018-04
影响因子：
3.2
通讯作者：
Paša-Tolić L
中科院分区：
文献类型：
--
作者：
Zhou M;Yan J;Romano CA;Tebo BM;Wysocki VH;Paša-Tolić L
锰的氧化是一个重要的生物地球化学过程，主要受细菌通过酶反应调节。然而，由于分离和表征这些未知酶的挑战，对详细的机制知之甚少。来自芽孢杆菌PL-12的锰氧化酶Mnx已成功地以活性形式过量表达为分子量为211 kDa的蛋白质复合物。我们最近使用表面诱导解离（SID）和离子迁移-质谱（IM-MS）来释放和检测折叠的亚复合物，以确定亚基连接和四级结构。从本地质谱实验的数据导致了一个合理的结构模型，这种多铜氧化酶一直难以研究传统的结构生物学方法。由于每个Mnx亚基结合可变数量的铜离子，由于系统的异质性和有限的质量分辨率，分配一些观察到的峰的模糊性仍然是充分理解金属和潜在未知配体在Mnx中的作用的障碍。在本研究中，我们在一个改良的傅立叶变换-离子回旋共振质谱仪上进行了电离层分离。FTICR提供的高质量准确度和分辨率揭示了对蛋白质的意想不到的人工修饰，这些蛋白质先前被认为是基于较低分辨率光谱的铁结合物质。此外，释放的亚复合物的同位素分辨光谱揭示了在不同的结构水平的金属结合的化学计量。这种方法具有很大的潜力，深入表征金属蛋白质和蛋白质配体复合物。
Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility - mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase which has been difficult to study by conventional structural biology methods. Because each Mnx subunit binds a variable number of copper ions, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx due to the heterogeneity of the system and limited mass resolution. In this study, we performed SID in a modified Fourier transform – ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes.
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DOI：
10.1007/s13361-013-0664-3
发表时间：
2013-11
影响因子：
3.2
作者：
Flick, Tawnya G.;Merenbloom, Samuel I.;Williams, Evan R.
通讯作者：
Williams, Evan R.
DOI：
10.1007/s13361-017-1781-1
发表时间：
2017-12
影响因子：
3.2
作者：
Belov AM;Viner R;Santos MR;Horn DM;Bern M;Karger BL;Ivanov AR
通讯作者：
Ivanov AR
影响因子：
48
作者：
Heck, Albert J. R.
通讯作者：
Heck, Albert J. R.
影响因子：
48
作者：
Gault J;Donlan JA;Liko I;Hopper JT;Gupta K;Housden NG;Struwe WB;Marty MT;Mize T;Bechara C;Zhu Y;Wu B;Kleanthous C;Belov M;Damoc E;Makarov A;Robinson CV
通讯作者：
Robinson CV
影响因子：
5.7
作者：
Baldwin, Andrew J.;Lioe, Hadi;Benesch, Justin L. P.
通讯作者：
Benesch, Justin L. P.