NEW CHEMICAL PROBES ENABLE MASS SPECTROMETRY-BASED FOOTPRINTING OF HUMAN PROTEIN STRUCTURE IN LIPID

现代质谱仪的灵敏度、分辨能力和速度现在提供了 有机会开发自下而上的足迹方法，能够解决重大的结构性问题， 以及膜蛋白的动力学问题。这种自下而上的方法从根本上说 自上而下的质谱（MS）研究的有力替代方案， 仅限于细菌膜蛋白。我们关注人类蛋白质，因为它们参与了 几乎所有的生理过程，并代表超过60%的药物靶点。然而， 代表了传统高分辨率结构方法最具挑战性的目标。 迄今为止，已知约100种蛋白质的结构，留下了很大的空白 MS填充。我们的长期目标是开发全面的足迹MS方法， 活细胞和体外脂质中膜蛋白的结构和动力学的独特方法 双层。我们的目标是合成新的化学探针， 覆盖面，以揭示铁转运蛋白的配体相互作用和动态运输运动，一个模型， 代表最大的膜转运蛋白超家族并维持铁 人体内的稳态我们的假设是：（1）互补化学可以最大限度地提高 覆盖足迹，从而提高其空间分辨率。此外，调整 标记试剂的物理性质将允许进入疏水区域， 膜蛋白(2)光活化快速足迹可以揭示动态运输运动 发生在毫秒内，这超出了目前膜结构生物学的范围。 (3)生物正交不可逆标记法可以优化，以揭示细胞的结构状态， 膜蛋白，一种晶体学或冷冻电镜难以捕捉的结构。使用这些 常规方法需要纯化的蛋白质，但大多数膜蛋白不足以 稳定以承受苛刻的净化。活细胞足迹完全避开了这个巨大的 困难我们的假设是建立在我们实验室产生的大量初步数据之上的。 具体来说，我们继续展示我们探索新化学和合成的能力。 新试剂我们正在进行的研究证明了MS足迹法可以揭示配体的原理- 结合相互作用的人膜蛋白在脂质双层，并可以报告其天然的 结构状态和运动。为了实现我们的目标，我们将努力实现三个具体目标。 目的：（1）开发新的化学探针，以提供高足迹覆盖膜 蛋白质;（2）在脂质膜系统中实现新的探针以研究配体相互作用 和ferroportin的毫秒运动;和（3）证明新探针与活的 通过检测细胞运动和膜铁转运蛋白的配体相互作用的细胞足迹。我们 创新的足迹法结合自下而上的MS蛋白质组学分析将建立有效的， 在活细胞和脂质膜中广泛的足迹。建议的意义 这种方法将得到扩展，因为基于MS的足迹法可以广泛应用于结构化 蛋白质组学研究人员对生物医学上重要的人类膜蛋白。

The sensitivity, resolving power, and speed of modern mass spectrometers now afford the opportunity to develop bottom-up footprinting methods capable of resolving significant structural and dynamics questions of membrane proteins. This bottom-up approach is a fundamentally more powerful alternative to the top-down mass spectrometry (MS) studies that have been mainly limited to bacterial membrane proteins. We focus on human proteins because they participate in almost all physiological processes and represent more than 60% of drug targets. They, however, represent the most challenging targets for traditional high-resolution structural methods. Structures of about 100 of these proteins are known to date, leaving a large gap for footprinting MS to fill. Our long-term goal is to develop comprehensive footprinting MS methods that offer a unique approach to structure and dynamics of membrane proteins in live cells and in vitro lipid bilayers. Our objective here is to synthesize new chemical probes that provide high footprinting coverage to reveal the ligand interaction and dynamic transport motion of ferroportin, a model protein representing the largest superfamily of membrane transporters and maintaining iron homeostasis in humans. Our hypotheses are: (1) Complementary chemistry can maximize the coverage of footprinting and thereby improve its spatial resolution. Furthermore, tuning the physical properties of the labeling reagents will allow access to the hydrophobic region of membrane proteins. (2) Photoactivated fast footprinting can reveal dynamic transporter motions taking place within milliseconds, which is beyond the current scope of membrane structure biology. (3) Bio-orthogonal irreversible labeling can be optimized to reveal the cellular structure state of membrane proteins, a structure that is elusive by crystallography or cryo-EM. Use of these conventional methods requires purified proteins, but most membrane proteins are insufficiently stable to withstand demanding purification. Live-cell footprinting completely avoids this giant difficulty. Our hypotheses are built on extensive preliminary data produced in our laboratories. Specifically, we continue to demonstrate our capability to explore new chemistry and synthesize new reagents. Our ongoing studies prove the principle that MS footprinting can reveal ligand- binding interaction of human membrane proteins in lipid bilayer, and can report on their native structural state and motion in live cells. To accomplish our goals, we will pursue three specific aims: (1) develop new chemical probes to provide high footprinting coverage of membrane proteins; (2) implement the new probes in lipid membrane systems to study the ligand interaction and millisecond motion of ferroportin; and (3) demonstrate the new probes’ compatibility with live- cell footprinting by the detection of cellular motions and ligand interactions of ferroportin. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish effective, broad-based footprinting in live cells and lipid membranes. The significance of the proposed approach will expand because MS-based footprinting can be broadly applied by structural proteomics researchers to biomedically important human membrane proteins.