Dynamic allostery of Sec machinery in protein transport and folding

所有的细胞都被油膜包围，保持细胞内部，不需要的物质和寄生虫。因此，选择性孔隙已经发展为营养物的供应和废物的去除。如果没有这些“转运蛋白”，细胞膜就会成为一个不可渗透的屏障，特别是对于蛋白质等较大的分子。许多蛋白质需要这样的通道来执行它们的生物功能，例如通过免疫细胞将抗体分泌到血流中。因此，生物膜也包含了一些易位系统，通过嵌入复合氨基酸序列中的信号识别特定的蛋白质进行易位。我们的目标是更多地了解大肠杆菌中的一个这样的易位系统是如何工作的：“translocon”，它从内部分泌蛋白质穿过膜并进入膜;甚至对于基本生存也是必不可少的。这种机制由两部分组成--一部分是嵌入细胞膜的蛋白通道，另一部分是一种名为SecA的马达蛋白，它通过ATP提供的能量驱动蛋白质通过通道，ATP是细胞的“能量货币”。当SecA将ATP分解成两个更小的分子时，蛋白质移位的能量就被释放出来，ADP和磷酸盐，这个过程与SecA和通道形状变化的循环中的易位蛋白的运动偶联，称为构象变化。由于有两个伙伴，我们特别感兴趣的是如何协调它们的行动。我们的目标是了解这种“舞蹈”是如何在时间和空间中编排的，即拍摄一部这对分子的分子电影。我们已经开发了工具，通过每次照亮其中一个伙伴，可以以高时间分辨率一次询问一个组件。这是通过快速交替的激光脉冲来实现的，同时通过将非常灵敏和快速的探测器（或摄像机）对准舞池来记录他们的行为。要做到这一点，我们必须使用遗传和生物化学技术，将光学报告分子（荧光探针）引入蛋白质，在易位过程中移动的地方，或者跳舞（如果你喜欢我们丰富多彩的比喻！）。这些荧光探针报告在蛋白质易位的不同阶段和ATP分解期间的特定运动。在过去的几年里，我们已经取得了相当大的成功，在这种方法观察分子回旋的个别合作伙伴。令人惊讶的是，我们发现易位子的舞蹈速度比它提供能量的运动伙伴快十倍。现在，我们想弄清楚这是怎么可能的，以及这种奇怪的舞蹈是如何协调的。为了做到这一点，我们将同时观察两个伙伴的电影，这需要相当多的分子生物学技巧，以便在正确的地方引入正确的记者。我们还需要更精细、更快的激光系统来照亮舞池，当然还有快速滚动的摄像机和其他探测器。在电影被记录下来之后，我们将从编码在检测到的信号中的单个电影中梳理出关于舞蹈的有用信息。为了做到这一点，我们开发了计算算法，将极其快速的信号转换为有意义的信息。这些发现将揭示构象舞蹈的细节，并从根本上解释马达和通道的协调和矛盾的不连贯行为如何导致蛋白质穿过并进入细胞膜。这些信息也可以告诉我们，在生物学的其他地方，即使有不同的舞蹈动作，也会有非常不同的舞者。因此，我们可以更多地了解ATP是如何帮助细胞周围不同类型的货物移动的，以及蛋白质的质量控制。此外，这些知识可以用于开发针对这些元素的化合物（舞者！）其是细菌所特有，潜在地用于高度期望的新型抗生素。

All cells are surrounded by oily membranes keeping the cell insides in, and unwanted substances and parasites out. Thus, selective pores have evolved for the supply of nutrients and removal of waste products. Without these 'transporters' the membrane presents an impermeable barrier, particularly for larger molecules like proteins. Such passage is required for many proteins to perform their biological functions, such as secretion of antibodies into the blood stream by immune cells. Therefore, biological membranes also contain a number of translocation systems that recognise the specific proteins to be translocated via signals embedded in the sequence of composite amino acids.We aim to learn more about how one such translocation system works in bacterium Escherichia coli: the "translocon", which secretes proteins from the interior across the membrane and also into it; essential even for basic survival. The machinery comprises two components -a protein-channel embedded within 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.The energy for protein translocation is released when SecA breaks down ATP into two smaller molecules, ADP and phosphate and this process is coupled to the movement of the translocating protein in a cycle of changes to the shape of SecA and the channel, termed conformational changes. Since there are two partners, we are particularly interested how their movement is coordinated. Our objective is to understand how this "dance" is choreographed in time and space, i.e. take a molecular movie of the pair.We have developed tools, which allows us to interrogate one component at a time with a high temporal resolution by illuminating one of the partners at a time. This is achieved with rapidly alternating laser pulses, while recording their behaviour by aiming very sensitive and fast detectors (or cameras) onto the dance floor. To do so, we have to use genetic and biochemical techniques to introduce optical reporters (fluorescence probes) onto the proteins, in places that move during the translocation process, or dance (if you prefer our colourful metaphor!).These fluorescent probes report on the specific motions during different stages of protein translocation and during ATP breakdown. In the past few years, we have had considerable success in this approach observing molecular gyrations of individual partners. Surprisingly, we found the translocon dances about ten times faster than its energy providing motor partner. Now, we want to figure out how this is possible, and how such a strange dance is coordinated. To do so we will observe movies of both partners simultaneously, which requires considerable molecular biology craft for the introduction of the right reporters in the right places. We also need more elaborate and faster laser systems to illuminate the dance floor and of course fast rolling cameras and other detectors. After the movie is recorded, we will tease out useful information about the dance from individual movies that are encoded in the detected signals. To do this we have developed computational algorithms to convert the extremely rapid signals into meaningful information. The findings will uncover details of the conformational dance and explain fundamentally how the coordinated, and paradoxically incoherent, behaviour of the motor and channel result in the passage of proteins across and into the membranes of cells. This information could also inform us about how very different dancers move elsewhere in biology, even with different routines. So, we could learn more how ATP is also used to, help move different types of cargo around the cell, and for protein quality control. Moreover, this knowledge could be exploited in the development of compounds that target those elements (dancers!) that are unique to bacteria, potentially for highly desirable novel antibiotic.