Atomic-level visualization of signalling protein activity in living mammalian cells using In-Cell NMR spectroscopy

每个活细胞都含有遗传信息，编码一系列指令，以产生它们的定义成分蛋白质。细胞蛋白质仅由20个氨基酸组成，有多种形状(或折叠)和大小。蛋白质独特的3D形状，甚至是暴露在给定蛋白质表面的单一氨基酸，赋予了特定的能力，使蛋白质能够控制所有的细胞过程，包括生长、分化、新陈代谢、运动、动态平衡和运输。当然，蛋白质并不是独立起作用的，而是作为细胞内不断变化的、完整的齿轮组成的庞大网络的一部分。因此，为了了解细胞如何运动或生长，我们必须了解蛋白质在正常网络中的行为。这是一项极具挑战性的工作，因为我们缺乏在原子水平上观察蛋白质驻留在细胞中的技术。因此，研究人员不得不通过将单个蛋白质从其自然环境中分离出来，放入纯缓冲液甚至水中，对其进行详细研究。虽然这为蛋白质在原子水平上的功能甚至它们在网络中的关系提供了巨大的洞察力，但未来的主要任务是在活细胞中获取这些相同的数据。我的研究团队使用一种名为核磁共振光谱学的先进技术来观察蛋白质的原子3D形状和动态性质。这种方法需要用15-氮等同位素标记蛋白质。虽然这种标记对蛋白质的功能没有影响，但它为我们提供了一个巨大的优势，因为我们添加到蛋白质中的任何未标记的东西都不会被核磁共振检测到。因此，我们可以观察蛋白质在复杂混合物中的表现，即与其他蛋白质、代谢物、药物，甚至是完整的、破碎的细胞的混合。这提供了令人兴奋的新数据，详细说明了重要的细胞蛋白质是如何发挥作用的，我们在此提议进一步，开始研究活细胞内的蛋白质。这项技术被称为细胞内核磁共振波谱，或IC-核磁共振，它提供了巨大的潜力来真正了解蛋白质如何整合和控制细胞命运，作为其自然网络的组成部分。我们正在提高将标记的蛋白质带入活细胞并进行核磁共振实验的能力，现在建议观察一些非常关键的细胞蛋白质GTP酶和激酶的活性。这些类型的数据对于促进我们对细胞蛋白质、网络以及最终细胞生物学的理解至关重要。

Every living cell contains genetic information encoding a set of instructions to produce their defining component proteins. Generated from a set of just 20 amino acids, cellular proteins come in many shapes (or folds') and sizes. The distinct 3D shape of a protein, and even singular amino acids exposed on the surface of a given protein, bestow specific capabilities that allow proteins to govern all cellular process, including growth, differentiation, metabolism, motility, homeostasis and transport. Of course, proteins do not act independently but instead as part of a vast network of constantly changing, integrated cogs inside of cells. Thus, to understand how cells move or grow, we must understand how proteins behave while part of their normal network. This is an extremely challenging endeavor, as we lack the technology to observe proteins at an atomic level while they reside in cells. Researchers have therefore, by necessity, studied individual proteins in detail by isolating them from their native environment into pure buffers or even water. While this has provided tremendous insight into how proteins function at an atomic level and even their relationships within networks, a major task for the future is to acquire these same data inside a living cell. My research team uses an advanced technique called NMR spectroscopy to observe' the atomic 3D shape and dynamic properties of proteins. This approach requires proteins to be labelled with isotopes such as 15-Nitrogen. While this labelling has no effect on a protein's function, it provides us a massive advantage in that anything we add to our protein that is unlabelled remains invisible' by NMR. Thus, we can observe how proteins behave in complex mixtures i.e. with other proteins, metabolites, drugs, or even entire, broken up cells. This has provided exciting new data detailing how essential cellular proteins function, and we here propose to take this one step further and begin to study our proteins inside of living cells. This technique is called In-Cell NMR spectroscopy, or IC-NMR, and offers tremendous potential to truly understand how proteins integrate and control cell fate as components of their natural networks. We are improving our ability to get labelled proteins into live cells and conduct NMR experiments, and now propose to observe the activity of some very key cellular proteins called GTPases and kinases. These types of data are crucial to advance our understanding of cellular proteins, networks, and ultimately the biology of cells.