Investigations of interactions in dynamic protein complexes by mass spectrometry

项目摘要 拟议的研究集中在蛋白质构象动力学和相互作用对于同源配体和治疗剂向细胞的运输和递送的关键重要性。分子治疗学非常重视必须将活性成分准确地输送到目标部位的候选药物。实现这一目标的一个潜在方法是利用现有的细胞机械，特别是那些将分子输送到细胞内的系统。本研究主要研究转铁蛋白/转铁蛋白受体系统中的动态过程。 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学。我们将开发新的实验策略，结合溶液中氢/氢交换(HDX)和电喷雾电离质谱仪(ESI MS)检测，使用一系列气相离子碎裂技术来以前所未有的详细程度表征蛋白质的构象和动力学。新的HDX MS方法将能够以接近单一氨基酸残基水平的分辨率表征蛋白质的高阶结构，以精确定位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技术的应用将提高目前的能力，包括原本无法进行这种表征的蛋白质(例如那些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相关的紧迫的生物医学问题。具体地说，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，以及金属和结合治疗药物对其调节作用。转铁蛋白在金属结合和游离状态下的蛋白质动力学变化，以及它在细胞外环境和内体中的行为比较，对于理解这一转运过程至关重要。通过将各种转铁蛋白-细胞毒素结合物的构象和受体结合特性与它们在体内穿越血脑屏障并在恶性肿瘤细胞中蓄积的能力相关联，这一体外模型将得到验证。

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.