Integrated Methods for Structural Elucidation of Proteins and Macromolecular Comp

描述(申请人提供)：大多数细胞功能是由大分子组装完成的，关于这些高阶复合体的结构和功能的信息可以在分子水平上更好地理解许多生物过程，包括疾病是如何发展的。这项研究的目标是开发一种综合的方法来确定蛋白质和大分子组件的高阶结构的详细信息，该方法将高分辨率离子迁移率光谱(IMS)与溶液相化学和氢氚交换(H/DX)与增压串联质谱学相结合，并应用这些方法来确定发生在重要的大分子络合物中的结构和结构转变的详细信息，在这些信息中，传统的高分辨率结构方法只能获得有限的信息。将建造一个低压漂移管装置，并与QTOF质谱仪对接，以获得大分子络合物的绝对碰撞截面。这些横截面可以提供有关复合体的结构/功能关系的信息，也可以为使用商业上可买到的行波IMS仪器的其他人提供参考测量，这些仪器只提供相对横截面测量。IMS的这种能力将被用来开发新的H/DX方法，这种方法使用增压试剂在毫秒或亚毫秒的电喷离子形成时间范围内对组成分子进行展开、解离和高度充电，从而消除了传统酸淬法可能发生的任何反向交换。目标是使用电子转移或电子捕获解离串联质谱仪获得接近单个氨基酸分辨率的主链酰胺交换率，这已被证明可以将气相H/D扰乱和随之而来的交换率信息的损失降至最低。将调查使用这些试剂获得的增加电荷对可能发生的任何气相H/D扰乱的影响。将采用蛋白质分解‘划痕’策略来显著增加这种自上而下方法可以使用的分子大小范围。将开发一种新的方法来测量短时间内的组装动力学，并用于寻找有潜力开发成治疗炭疽感染的有效药物和正在开发的癌症治疗蛋白酶体抑制剂的分子骨架。这些集成的方法可能会提高获得高保真结构信息的速度、灵敏度和分子大小范围，并将提供其他结构方法的补充信息，包括电子显微镜、核磁共振、结晶学和分子建模。这些方法将被用于研究几个信息有限的复合体中发生的结构和结构转变，包括炭疽毒素复合体和26S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.