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

每一个活细胞都含有编码一组指令的遗传信息，以产生它们的定义成分-蛋白质。 细胞蛋白质由20种氨基酸组成，有许多形状（或“折叠”）和大小。蛋白质独特的3D形状，甚至暴露在给定蛋白质表面的单一氨基酸，赋予蛋白质控制所有细胞过程的特定能力，包括生长，分化，代谢，运动，稳态和运输。当然，蛋白质并不是独立发挥作用的，而是作为细胞内不断变化的庞大网络的一部分。因此，要了解细胞如何移动或生长，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奋进，因为我们缺乏在原子水平上观察蛋白质的技术。因此，研究人员必须通过将它们从天然环境中分离到纯缓冲液甚至水中来详细研究单个蛋白质。虽然这为蛋白质如何在原子水平上发挥作用以及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内获取这些相同的数据。我的研究团队使用一种称为NMR光谱的先进技术来“观察”蛋白质的原子3D形状和动态特性。这种方法需要用同位素如15-氮标记蛋白质。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可以通过NMR“看不见”。因此，我们可以观察蛋白质在复杂混合物中的行为-即与其他蛋白质，代谢物，药物，甚至整个破碎的细胞。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如何发挥作用，我们在这里建议更进一步，开始研究活细胞内的蛋白质。这种技术被称为细胞内NMR光谱，或IC-NMR，并提供了巨大的潜力，真正了解蛋白质如何整合和控制细胞命运作为其天然网络的组成部分。我们正在提高将标记蛋白质导入活细胞并进行NMR实验的能力，现在我们计划观察一些非常关键的细胞蛋白质（称为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.