Integrated Methods for Structural Elucidation of Proteins and Macromolecular Comp

描述（由申请人提供）：大多数细胞功能由大分子组装体执行，关于这些高阶复合物的结构和功能的信息可以在分子水平上提高对许多生物过程的理解，包括疾病如何进展。拟议研究的目标是开发一种综合方法，以确定有关蛋白质和大分子组装体的高阶结构的详细信息，该方法将高分辨率离子迁移谱（IMS）与溶液相化学和氢氘交换（H/DX）相结合。并应用这些方法来确定关于重要的大分子复合物中发生的结构和结构转变的详细信息，其中使用常规方法只能获得有限的信息。高分辨率结构方法一个低压漂移管装置将被构建和接口到QTOF质谱仪，以获得大分子复合物的绝对碰撞截面。这些横截面可以提供关于复合物的结构/功能关系的信息，以及使用仅提供相对横截面测量的市售行波IMS仪器为其他人提供参考测量。这种IMS能力将用于开发新的H/DX方法，该方法使用增压试剂在通过电喷雾形成离子的ms或亚ms时间范围内展开、解离和高度充电组成分子，这消除了传统酸淬灭方法可能发生的任何反交换。我们的目标是使用电子转移或电子捕获解离串联质谱法获得接近单个氨基酸分辨率的骨架酰胺交换率，这已被证明可以最大限度地减少气相H/D干扰和随之而来的交换率信息损失。将研究用这些试剂获得的任何气相H/D扰乱的增加的充电的效果。将采用蛋白水解“切口”策略，以显著增加可使用这种自上而下方法的分子大小范围。一种新的方法来测量组装动力学在短时间内将被开发和用于寻找分子框架，有可能被开发成有效的药物，用于治疗炭疽感染和蛋白酶体抑制剂的发展癌症治疗。这些集成的方法可以潜在地提高速度，灵敏度和分子大小范围，可以高保真地获得结构信息，并将为其他结构方法提供补充信息，包括电子显微镜，NMR，晶体学和分子建模。这些方法将被应用于调查的结构和结构转换，发生在几个复杂的有限的信息已经获得，包括炭疽毒素复合物，和26 S蛋白酶体。

DESCRIPTION (provided by applicant): Most cellular functions are performed by large molecular assemblies and information about the structures and functions of these higher-order complexes can lead to an improved understanding of many biological processes, including how diseases progress, at the molecular level. The goal of the proposed research is to develop an integrated approach to determining detailed information about the higher-order structures of proteins and macromolecular assemblies that combines high-resolution ion mobility spectrometry (IMS) with solution-phase chemistry and hydrogen deuterium exchange (H/DX) with supercharging tandem mass spectrometry, and apply these methods to determine detailed information about the structures and structural transitions that occur in important macromolecular complexes where only limited information has been obtained using conventional high resolution structural methods. A low-pressure drift tube apparatus will be constructed and interfaced to a QTOF mass spectrometer to obtain absolute collision cross sections of large macromolecular complexes. These cross sections can provide information about structure/function relationships of the complex, as well as provide reference measurements for others using commercially available traveling wave IMS instruments that only provide relative cross section measurements. This IMS capability will be used to develop new H/DX methods that use supercharging reagents to unfold, dissociate, and highly charge the constituent molecules in the ms or sub-ms time frame of ion formation by electrospray, which eliminates any back exchange that can occur with conventional acid quenching methods. The goal is to obtain backbone amide exchange rates with close to individual amino acid resolution using electron transfer or electron capture dissociation tandem mass spectrometry, which have been shown to minimize gas-phase H/D scrambling and the concomitant loss of exchange rate information. The effect of increased charging obtained with these reagents on any gas-phase H/D scrambling that may occur will be investigated. A proteolytic 'nicking' strategy will be pursued to significantly increase the molecular size range where this top-down method can be used. A novel method to measure assembly kinetics at short times will be developed and used to find molecular frameworks that have the potential to be developed into effective drugs for treatment of anthrax infection and proteasome inhibitors in development for cancer therapy. These integrated approaches can potentially increase the speed, sensitivity, and molecular size range for which structural information can be obtained with high fidelity, and will provide complementary information to other structural methods, including electron microscopy, NMR, crystallography, and molecular modeling. These approaches will be applied to investigate the structures and structural transitions that occur in several complexes where limited information has been obtained, including Anthrax toxin complexes, and the 26S proteasome.