Ensemble and single molecule analysis of protein translocation

蛋白质易位的整体和单分子分析

• 批准号: BB/I008675/1
• 负责人: Ian Collinson
• 金额: $ 58.29万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI008675%2F1
• 关键词: Ensemble; single; molecule; analysis; protein

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.

所有的细胞都被膜所包围，膜由称为磷脂的双层脂肪分子组成。这些作为一个理想的“皮肤”，保持细胞的内部！在没有其他成分的情况下，它们将充当屏障，阻止环境和细胞内部之间营养物质和废物以及蛋白质等较大分子的必要快速交换。许多蛋白质需要这样的通道来执行它们的生物学功能-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位点跨膜分泌。为了克服这个潜在的问题，生物膜含有许多易位系统，使蛋白质和其他有用的物质（“底物”）通过磷脂屏障。在蛋白质底物的情况下，这些易位系统通过嵌入在构建它们的氨基酸序列中的信号识别待易位的特定蛋白质。我们的目标是通过研究来自普通肠道细菌大肠杆菌的例子来更多地了解这种易位系统是如何工作的，大肠杆菌在实验上比人类细胞更容易使用，但仍然应该告诉我们很多关于类似系统如何在我们自己的身体中工作的信息。像我们自己的一样，细菌易位系统（“translocon”）用于将蛋白质从细胞内部分泌到外部。它包括两个组成部分-一个名为SecYEG的三蛋白复合物，它形成了一个穿过膜的通道，一个名为SecA的马达蛋白，它驱动蛋白质通过通道，由ATP提供能量，即所谓的细胞“能量货币”。我们知道，当运动蛋白SecA将ATP分解为两个更小的分子ADP和磷酸盐时，蛋白质转运的能量被释放。我们不明白的是这个过程实际上如何驱动移位蛋白质的运动。然而，很明显，可能涉及SecA和SecYEG形状变化的循环，称为构象变化，就像内燃机中涉及的活塞和凸轮的运动一样。这些构象变化将在拟议的项目中进行探索。为此，我们将使用重组DNA技术将氨基酸半胱氨酸引入到蛋白质底物中，并在我们怀疑在易位过程中移动的易位子中。这种特殊类型的氨基酸具有化学反应性，这意味着我们可以选择性地附着荧光或磁性探针，在蛋白质易位和ATP分解的不同阶段，我们可以监测每个地方的环境。特别地，探针对之间的距离可以分别通过称为福斯特共振能量转移（FRET）和电子自旋共振（ESR）的物理技术来测量。我们还将检查成对的半胱氨酸是否足够接近，以通过确定长度的交联分子化学连接在一起，如果是这样，我们将看到这种束缚在一起对易位机制的功能有什么影响。这些类型的实验，在所谓的“集合”条件下，在试管中一次对数百万个translocons进行，应该非常揭示机制。然而，在这样的集合体中，很难使易位“机器”同步，以便当我们观察它们时，它们都同时处于它们的机械周期的同一阶段。为了补充这种方法，我们也将因此利用非常敏感的显微镜技术的发展，这将使我们能够遵循一个单一的translocon的构象变化，以及相关的蛋白质的易位，在同一时间。综合考虑，整体和单分子方法应该使我们能够理解所有细胞中必不可少的分子机器的内部运作。

项目成果

Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion.

• DOI: 10.7554/elife.77586
• 发表时间: 2022-04-29
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Allen, William J.;Corey, Robin A.;Watkins, Daniel W.;Oliveira, A. Sofia F.;Hards, Kiel;Cook, Gregory M.;Collinson, Ian
• 通讯作者: Collinson, Ian

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

• DOI: 10.1038/srep38399
• 发表时间: 2016-12-07
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Botte M;Zaccai NR;Nijeholt JL;Martin R;Knoops K;Papai G;Zou J;Deniaud A;Karuppasamy M;Jiang Q;Roy AS;Schulten K;Schultz P;Rappsilber J;Zaccai G;Berger I;Collinson I;Schaffitzel C
• 通讯作者: Schaffitzel C

Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion

质子动力刺激蛋白质分泌需要特定的心磷脂-SecY 相互作用

• DOI: 10.1101/202184
• 发表时间: 2017
• 作者: Corey R

Channel crossing: how are proteins shipped across the bacterial plasma membrane?

• DOI: 10.1098/rstb.2015.0025
• 发表时间: 2015-10-05
• 期刊: Philosophical transactions of the Royal Society of London. Series B, Biological sciences
• 作者: Collinson I;Corey RA;Allen WJ
• 通讯作者: Allen WJ

Composition and Activity of the Non-canonical Gram-positive SecY2 Complex.

• DOI: 10.1074/jbc.m116.729806
• 发表时间: 2016-10-07
• 期刊: The Journal of biological chemistry
• 作者: Bandara M;Corey RA;Martin R;Skehel JM;Blocker AJ;Jenkinson HF;Collinson I
• 通讯作者: Collinson I