Investigations of interactions in dynamic protein complexes by mass spectrometry

描述（由申请人提供）：拟议的研究重点是蛋白质构象动力学和相互作用对同源配体和治疗剂向细胞的运输和递送的关键重要性。分子疗法非常强调候选药物必须精确地将活性成分传递到其目标部位。实现这一目标的一个潜在方法是利用现有的细胞机制，特别是那些将分子运送到细胞内的系统。本研究主要关注转铁蛋白/转铁蛋白受体系统的动态过程。本实验室用于研究蛋白质高阶结构和动力学的一种强大而灵敏的技术是质谱分析。我们将开发新的实验策略，将溶液中氢/氘交换（HDX）与电喷雾电离质谱（ESI MS）检测相结合，使用一系列气相离子碎片技术以前所未有的细节水平表征蛋白质的结构和动力学。新的HDX质谱方法将能够在接近单个氨基酸残基水平的分辨率下表征蛋白质的高阶结构，以便精确定位对功能至关重要的蛋白质动态区域。最近开发的几种碎片化技术的应用将提高目前的能力，以包括那些否则无法进行这种表征的蛋白质（例如富含二硫键的蛋白质），并使特定构象的选择和表征成为可能。我们将使用这些方法来解决与向细胞输送治疗剂有关的紧迫的生物医学问题。具体来说，我们将运用我们的方法来破译转铁蛋白-转铁蛋白受体系统中蛋白质-蛋白质相互作用的详细分子机制，以及金属和偶联治疗剂对其的调节。铁蛋白在金属结合和自由形式下的蛋白质动力学变化，以及其在细胞外环境和核内体中的行为的比较，对于理解这一转运过程至关重要。通过将各种转铁蛋白-细胞毒素偶联物的构象和受体结合特性与它们在体内穿过血脑屏障并在恶性细胞中积累的能力相关联，将验证这一体外模型。

DESCRIPTION (provided by applicant): The proposed research is focused on the critical importance of protein conformational dynamics and interactions for the transport and delivery of both cognate ligands and therapeutic agents to cells. Molecular therapeutics places a great emphasis on drug candidates that must precisely deliver the active ingredients to their target sites. One potential method to achieve this aim is to harness the existing cellular machinery, particularly those systems that transport molecules into the cell. This research focuses on the dynamic processes in the transferrin/transferrin receptor system. A powerful and sensitive technique employed in this laboratory for the study of protein higher order structure and dynamics is mass spectrometry. We will develop new experimental strategies combining hydrogen/deuterium exchange in solution (HDX) with electrospray ionization mass spectrometry (ESI MS) detection that use an array of gas phase ion fragmentation techniques to characterize protein conformation and dynamics at an unprecedented level of detail. The new HDX MS methods will enable characterization of protein higher order structure at resolution close to the single amino-acid residue level, in order to pinpoint dynamic regions of proteins critical for function. Application of several recently developed fragmentation techniques will advance current capabilities to include proteins that have otherwise eluded such characterization (such as those rich in disulfide bonds), and also enable selection and characterization of specific conformers. We will use these methods to address pressing biomedical questions related to delivery of therapeutic agents to cells. Specifically we will apply our methodologies to decipher the detailed molecular mechanism of protein-protein interaction in the transferrin-transferrin receptor system and its modulation by metals and conjugated therapeutic agents. The changes in protein dynamics of transferrin in the metal-bound and free form, and comparison of its behavior in the extracellular environment versus the endosome are critical to understanding this transport process. This in vitro model will be verified by correlating the conformational and receptor-binding properties of various transferrin-cytotoxin conjugates with their ability to traverse the blood-brain barrier in vivo and accumulate in malignant cells.