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

现代质谱仪的灵敏度、分辨率和速度现在提供了 开发能够解决重大结构性问题的自下而上足迹方法的机会 以及膜蛋白的动力学问题。这种自下而上的方法基本上是一种 强大的替代自上而下的质谱学(MS)研究，主要是 仅限于细菌膜蛋白。我们关注人类蛋白质是因为它们参与了 几乎所有的生理过程，并代表了超过60%的药物靶点。然而，他们， 代表了传统高分辨率结构方法最具挑战性的目标。 到目前为止，大约有100个这样的蛋白质的结构是已知的，这为足迹留下了一个很大的缺口 毫秒以填补。我们的长期目标是开发全面的足迹MS方法，以提供 研究活细胞膜蛋白和体外脂质结构和动力学的独特途径 双层的。我们这里的目标是合成新的化学探针，提供高足迹 用覆盖率揭示配基相互作用和铁蛋白的动态转运运动 代表最大的膜转运蛋白超家族和维持铁的蛋白质 人类体内的动态平衡。我们的假设是：(1)补充化学可以最大化 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性区域 膜蛋白。(2)光激活快速足迹技术可以显示转运蛋白的动态运动 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可用于揭示细胞的结构状态。 膜蛋白，一种通过结晶学或冷冻-EM难以捉摸的结构。使用这些工具 传统的方法需要纯化的蛋白质，但大多数膜蛋白是不够的 稳定，经得起苛刻的净化。活细胞足迹完全避免了这个庞然大物 困难重重。我们的假设建立在实验室产生的大量初步数据的基础上。 特别是，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足迹可以揭示配体- 人膜蛋白在脂质双层中的结合作用及其天然报道 活细胞中的结构状态和运动。为了实现我们的目标，我们将追求三个具体目标 目标：(1)开发新的化学探针，以提供高足迹覆盖率的膜 (2)在脂膜体系中应用新的探针研究配体相互作用。 和毫秒级的运动；以及(3)证明了新的探针与活体动物的兼容性。 通过检测细胞运动和铁蛋白的配基相互作用来确定细胞足迹。我们的 创新的足迹和自下而上的MS蛋白质组学分析将建立有效的， 活细胞和脂膜中广泛存在的足迹。建议的重要意义 这种方法将得到扩展，因为基于MS的足迹可以通过Structure广泛应用 蛋白质组学研究人员发现了具有重要生物医学意义的人类膜蛋白。

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.