Development of Surface-Induced Dissociation Ion Mobility MS, a Structrl Biol Tool

 描述(由申请人提供)：描述蛋白质复合体的整体拓扑和亚基间接触，以及它们的组装/拆解和展开途径，这是至关重要的，因为这些复合体调节关键的生物过程，包括故障导致疾病的过程。虽然许多蛋白质复合体可以用现有的工具来表征，包括X射线结晶学和核磁共振，但其他的不适合这些方法。该领域这一差距的一个解决方案是在结构生物学方法套件中增加本地质谱学(MS)工具。虽然天然MS对蛋白质复合体提供了许多有用的见解，但最常见的激活方法是碰撞诱导解离(CID，与气体靶的多次低能碰撞以诱导碎片)，通常会诱导一条展开途径，产生高电荷的未折叠单体和互补的(n-1)-MERS，。这些信息不足以直接表征络合物的拓扑结构和亚基间的接触。相比之下，PI的实验室已经证明，表面诱导解离(SID)可以直接解离到亚复合体，也可以有效地探测从CID谱中不明显的微妙结构变化(例如，离子源中的预展开)。这项提议的总体目标是开发这种新颖的、可供选择的MS激活方法-表面诱导解离与离子迁移率(SID/IM)，作为表征多聚体蛋白质复合体结构特征的工具，从而使这些特征能够与功能联系在一起。目的I表征日益复杂的五辛配体结构，包括金属离子与小分子配体和蛋白质配体与C反应蛋白和PTX3的结合。目的II将确定SID/IM是否能够确定ROP(引物抑制物)二聚体突变体和ROP突变体：RNA复合体的结构特征和展开倾向。目的III将确定SID/IM是否能够确定组蛋白的离散修饰是否影响核小体(组蛋白：DNA)的断裂/拆解，其方式与预期的失稳有关。目的IV将利用SID/IM来表征从洗涤剂(例如，DDM)、两性极体或纳米盘喷洒的脂质：蛋白质复合体(CRP：DMPC)、膜受体：蛋白质复合体(CTB：GM1)和膜蛋白(视紫红质、水通道蛋白0、YIDC、YIDC：PF3涂层)。多个合作者，特定蛋白质复合体的生产/纯化/功能行为专家，但需要获得更好的表征工具，已同意提供不同复杂性的蛋白质(Bortazzi(人类临床研究所；PTX3)；Magliery(OSU；ROP突变体)；Poirier和Ottesen(OSU；修饰核小体)；Brown(UA；视紫红质)；Schey(Vanderbilt；AQP-0)；Dalbey(OSU；YIDC，Pf3 Coat)。对创新的SID/IM方法的表征、与CID/IM的直接比较以及样本类型知识的发展，其中SID方法对蛋白质复杂结构的确定至关重要，对于天然MS作为一种强大的结构生物学工具的持续发展至关重要。

 DESCRIPTION (provided by applicant): Characterization of the overall topology and inter-subunit contacts of protein complexes, and their assembly/disassembly and unfolding pathways, is critical because these complexes regulate key biological processes, including processes where malfunction leads to disease. Although many protein complexes can be characterized by existing tools, including X-ray crystallography and NMR, others are not amenable to these approaches. One solution to this gap in the field is addition of native mass spectrometry (MS) tools to the suite of structural biology approaches. While native MS has provided many useful insights on protein complexes, the most common activation method, collision-induced dissociation (CID, multiple low-energy collisions with gaseous targets to induce fragmentation), often induces an unfolding pathway that produces highly charged unfolded monomers and complementary (n-1)-mers,. This information is insufficient to directly characterize the topology and intersubunit contacts of the complexes. Surface-induced dissociation (SID), in contrast, has been shown by the PI's lab to induce direct dissociation to subcomplexes and to also effectively probe subtle structural changes that are not evident from CID spectra (e.g. pre-unfolding in the ion source). The overarching goal of this proposal is to develop this novel, alternative MS activation method, surface-induced dissociation coupled to ion mobility (SID/IM), as a tool for characterization of structural features of multimeric protein complexes so that these features can be tied to function. Aim I is to characterize pentraxin-ligand structures of increasingly highe complexity, including binding of metal ions plus small molecular ligands and protein ligands to C-reactive protein and PTX3. Aim II will determine whether SID/IM can define the structural features and unfolding propensity of ROP (repressor of primer) dimer mutants and ROP mutant:RNA complexes. Aim III will determine whether SID/IM can define whether discrete modifications of histone proteins influences nucleosome (histone:DNA) fragmentation/disassembly in ways related to their expected destabilization. Aim IV will utilize SID/IM to characterize a lipid:protein complex (CRP:DMPC), membrane receptor:protein complex (CTB:GM1) and membrane proteins (rhodopsin, aquaporin0, YidC, YidC:Pf3 coat) sprayed from detergent (e.g., DDM), amphipols, or nanodiscs. Multiple collaborators, experts in the production/purification/functional behavior of particular protein complexes but who need access to better characterization tools, have agreed to provide proteins of varying complexity (Bortazzi (Instituto Clinico Humanitas; PTX3); Magliery (OSU; ROP mutants); Poirier and Ottesen (OSU; modified nucleosomes); Brown (UA; rhodopsin); Schey (Vanderbilt; AQP-0); Dalbey (OSU; YidC, Pf3 Coat). Characterization of the innovative SID/IM approach, direct comparison with CID/IM, and development of knowledge of sample types where the SID approach is vital to protein complex structure determination, is critical for the continued development of native MS as a strong structural biology tool.