Investigations of interactions in dynamic protein complexes by mass spectrometry

项目摘要 拟议的研究是集中在蛋白质构象动力学和相互作用的运输和交付的同源配体和治疗剂的细胞的至关重要性。分子治疗学非常强调必须将活性成分精确地递送到其靶位点的候选药物。实现这一目标的一个潜在方法是利用现有的细胞机制，特别是那些将分子转运到细胞中的系统。本论文主要研究转铁蛋白/转铁蛋白受体系统的动力学过程。 本实验室采用的一种用于研究蛋白质高级结构和动力学的强大而灵敏的技术是质谱法。我们将开发新的实验策略，将溶液中的氢/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构象和动力学。新的HDX MS方法将能够以接近单个氨基酸残基水平的分辨率表征蛋白质的高阶结构，以确定对功能至关重要的蛋白质的动态区域。最近开发的几种片段化技术的应用将提高当前的能力，以包括否则逃避这种表征的蛋白质（如富含二硫键的蛋白质），并且还能够选择和表征特定的构象异构体。 我们将使用这些方法来解决与向细胞递送治疗剂相关的紧迫的生物医学问题。具体来说，我们将应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及其由金属和缀合治疗剂的调节。金属结合和游离形式的转铁蛋白的蛋白质动力学的变化，以及其在细胞外环境与内体中的行为的比较对于理解这种运输过程至关重要。该体外模型将通过将各种转铁蛋白-细胞毒素缀合物的构象和受体结合性质与其在体内穿过血脑屏障并在恶性细胞中积累的能力相关联来验证。

Project Summary 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.