In-cell Structural Biology using Electron Paramagnetic Resonance Spectroscopy

这项研究的目的是了解蛋白质组内部发生的相互作用，从而了解生命所需的相互作用。要做到这一点，我们首先需要了解所涉及的蛋白质的结构和作用模式。该项目使用电子顺磁共振（EPR）光谱来确定蛋白质如何组合在一起，以及这种结构如何使它们发挥功能。EPR允许我们测量电子自旋的能量，从而确定它们彼此之间的相互作用。相互作用的变化依赖于自旋之间的距离，因此我们可以使用EPR来推断自旋分离。通过在特定位置将自旋连接到蛋白质上，我们可以确定蛋白质之间的物理相互作用。EPR最适合测量纳米距离，因此它是x射线晶体学、电子显微镜和核磁共振等更熟悉的方法的补充工具。迄今为止，大多数使用EPR的蛋白质结构研究都是在细胞环境外观察蛋白质。这给了我们一个很好的功能指示，但没有考虑到相互作用和变化的蛋白质行为作为一个更大的，受调节的系统的一部分。EPR用于细胞内部的测量，从而在自然环境中获得结构信息的范围很大。这项研究蛋白质的生物分子作用的动机是用定量证据确定蛋白质如何在细胞中发挥作用，以及这种作用模式的变化如何引起疾病。我们将使用各种不同的细胞系、蛋白质和自旋标签来测试我们方法的适用性以及它可以应用的程度。

The aim of this research is to understand the interactions that occur within the proteome and as a consequence, the interactions required for life. To achieve this, we first need to understand the structures and modes of action of the proteins involved. This project uses electron paramagnetic resonance (EPR) spectroscopy to determine how proteins fit together and in turn how this structure enables their function. EPR allows us to measure the energies of electron spins, and so determine the interactions they have with each other. The interactions change dependent on the distance between spins, and so we can use EPR to infer spin separation. By attaching spins to proteins at specific positions we can determine how proteins physically interact with one another. EPR is best suited to measuring nanometre distances, and so is a tool that is complementary to the more familiar methods of X-ray crystallography, electron microscopy and NMR. To-date most structural studies of proteins using EPR have looked at the proteins outside the cellular environment. This gives us a good indication of the function, but doesn't take into account interactions and changes in protein behaviour as part of a larger, regulated system. There is great scope to use EPR for measurements inside cells and thus obtain structural information in a native environment. The motivation for this research into the biomolecular action of proteins is to determine with quantitative evidence how proteins perform their roles in the cell and how changes to this mode of action can give rise to disease. We will be working with a variety of different cell lines, proteins and spin labels to test the applicability of our methodology as well as the extent to which it can be applied.