Ensemble and single molecule analysis of protein translocation

所有的细胞都被膜包围，膜由一层叫做磷脂的脂肪分子组成。它们充当了理想的“皮肤”，将细胞的内部保持在里面！在缺乏其他成分的情况下，它们将起到屏障的作用，阻止营养物质和废物以及蛋白质等较大分子在环境和细胞内部之间的必要快速交换。这样的通道是许多蛋白质发挥其生物学功能所必需的--例如，血液中丰富的蛋白质白蛋白必须从其在肝细胞中的合成部位跨膜分泌。为了克服这一潜在问题，生物膜包含许多易位系统，使蛋白质和其他有用物质(底物)能够通过磷脂屏障。就蛋白质底物而言，这些转位系统通过嵌入构建它们的氨基酸序列中的信号识别要转位的特定蛋白质。我们的目标是通过研究常见肠道细菌大肠杆菌的一个例子来更多地了解这种易位系统是如何工作的。在实验上，这种细菌比人类细胞更容易使用，但尽管如此，它应该会告诉我们很多类似的系统在我们自己的身体里是如何工作的。就像我们自己的一样，细菌易位系统(转位系统)负责将蛋白质从细胞内部分泌到外部。它由两个成分组成--一个名为SecYEG的三蛋白质复合体，它形成了一个穿过细胞膜的通道，以及一个名为SecA的马达蛋白质，它驱动蛋白质通过这个通道，能量由ATP提供，也就是所谓的细胞“能量货币”。我们知道，当马达蛋白SecA将ATP分解成两个更小的分子--ADP和磷酸时，蛋白质转运的能量就会释放出来。我们不了解的是，这个过程实际上是如何驱动转运蛋白的运动的。然而，很明显，很可能会涉及赛卡和赛吉格形状变化的循环，称为构象变化，就像内燃机中涉及活塞和凸轮的运动一样。正是这些构象变化将在拟议的项目中进行探索。为了做到这一点，我们将使用重组DNA技术将氨基酸半胱氨酸引入蛋白质底物和我们怀疑在易位过程中移动的易位子中的位置。这种特殊类型的氨基酸具有化学反应活性，这意味着我们可以选择性地连接荧光或磁性探针，利用它们我们可以在蛋白质转运和ATP分解的不同阶段监测每个地方的环境。特别是，探针对之间的距离可以通过分别称为Förster共振能量转移(FRET)和电子自旋共振(ESR)的物理技术来测量。我们还将检查半胱氨酸对是否足够接近，以便通过定义长度的交联分子化学连接在一起，如果是的话，我们将看到这种连接在一起对易位机制的功能有什么影响。这些类型的实验在试管中进行，一次对数百万个转座子进行，在所谓的“整体”条件下，应该非常清楚地揭示了这种机制。然而，在这样的群体中，要使移位的机器同步是非常困难的，这样当我们观察它们时，它们都同时处于机械周期的同一阶段。因此，为了补充这一方法，我们还将利用非常灵敏的显微技术的发展，这将使我们能够跟踪单个转位的构象变化，以及相关的蛋白质转位。综上所述，集合和单分子方法应该可以让我们了解所有细胞中必不可少的分子机器的内部工作原理。

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. These act as an ideal 'skin', keeping the cell's insides in! In the absence of other components they would act as barriers, preventing the necessary rapid exchange of nutrients and waste products, and of larger molecules like proteins, between the environment and the cell interior. Such passage is required for many proteins to perform their biological functions - for example the abundant protein albumin of the blood has to be secreted across the membrane from its site of synthesis in liver cells. To overcome this potential problem, biological membranes contain a number of translocation systems that enable proteins and other useful substances ('substrates') to pass across the phospholipid barrier. In the case of protein substrates, these translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed. We aim to learn more about how such translocation systems work by studying an example from the common gut bacterium Escherichia coli, which is experimentally easier to work with than human cells, but nonetheless should tell us a lot about how similar systems work in our own bodies. Like our own, the bacterial translocation system (the 'translocon') serves to secrete proteins from the interior of the cell to the outside. It comprises two components - a three-protein complex named SecYEG that forms a channel through the membrane, and a motor protein named SecA that drives the passage of proteins through the channel, fuelled by energy provided by ATP, the so-called 'energy currency' of the cell. We know that the energy for protein translocation is released when the motor protein SecA breaks down ATP into two smaller molecules, ADP and phosphate. What we don't understand is how this process actually drives movement of the translocating protein. However, it is clear that a cycle of changes in the shapes of SecA and SecYEG, termed conformational changes, are likely to be involved, much as the movements of pistons and cams are involved in internal combustion engines. It is these conformational changes that will be explored in the proposed project. To do this, we will use recombinant DNA techniques to introduce the amino acid cysteine into the protein substrate and at places in the translocon that we suspect move during the translocation process. This particular type of amino acid is chemically reactive, meaning that we can selectively attach fluorescent or magnetic probes with which we can monitor the environment at each place during different stages of protein translocation and ATP breakdown. In particular, the distances between pairs of probes can be measured by physical techniques known as Förster resonance energy transfer (FRET) and electron spin resonance (ESR) respectively. We will also examine whether pairs of cysteines are sufficiently close to each other to be chemically linked together by cross-linking molecules of defined length, and if so, we will see what effect this tethering together has on the function of the translocation machinery. These types of experiments, conducted in the test tube on millions of translocons at a time under so-called 'ensemble' conditions, should be very revealing of the mechanism. However, in such ensembles it is very difficult to synchronise the translocation 'machines' so that they are all simultaneously at the same stage of their mechanical cycles when we observe them. To complement this approach we will therefore also take advantage of the development of very sensitive microscopy techniques, which will allow us to follow the conformational changes of a single translocon, and the associated translocation of protein, at a time. Taken together, the ensemble and single molecule approaches should allow us to understand the inner workings of a molecular machine essential in all cells.