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

 描述（由申请人提供）：蛋白质复合物的总体拓扑结构和亚基间接触及其组装/拆卸和解折叠途径的表征至关重要，因为这些复合物调节关键生物过程，包括功能障碍导致疾病的过程。虽然许多蛋白质复合物可以通过现有的工具来表征，包括X射线晶体学和NMR，但其他蛋白质复合物不适合这些方法。该领域中的这一差距的一个解决方案是在结构生物学方法套件中添加天然质谱（MS）工具。虽然天然MS提供了许多有用的见解蛋白质复合物，最常见的活化方法，碰撞诱导解离（CID，多次低能量碰撞与气态目标诱导碎片），往往诱导解折叠途径，产生高度带电的未折叠单体和互补的（n-1）-聚体。这些信息不足以直接表征复合物的拓扑结构和亚基间接触。相反，PI的实验室已经证明表面诱导解离（SID）可以诱导亚复合物的直接解离，并且还可以有效地探测CID光谱中不明显的细微结构变化（例如，离子源中的预解折叠）。该提案的首要目标是开发这种新型的替代MS活化方法，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物结构特征的工具，以便这些特征可以与功能联系起来。目的一是表征越来越复杂的五聚体配体结构，包括金属离子加小分子配体和蛋白质配体与C-反应蛋白和PTX 3的结合。目的二是确定SID/IM是否可以定义ROP（引物阻遏物）二聚体突变体和ROP突变体：RNA复合物的结构特征和去折叠倾向。目的III将确定SID/IM是否可以定义组蛋白的离散修饰是否以与其预期的不稳定相关的方式影响核小体（组蛋白：DNA）片段化/解体。目的IV将利用SID/IM来表征从洗涤剂（例如，DDM）、Amphipol或纳米盘。多个合作者，特定蛋白质复合物的生产/纯化/功能行为方面的专家，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（Instituto Clinico Humanitas; PTX 3）; Magliery（OSU; ROP突变体）; Poirier和Ottesen（OSU;修饰的核小体）; Brown（UA;视紫红质）; Schey（范德比尔特; AQP-0）; Dalbey（OSU; YidC，Pf 3 Coat）。创新SID/IM方法的表征，与CID/IM的直接比较，以及SID方法对蛋白质复合物结构测定至关重要的样品类型知识的开发，对于继续开发天然MS作为强大的结构生物学工具至关重要。

 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.