Understanding the Mechanism of Membrane Protein Insertion

了解膜蛋白插入的机制

• 批准号: BB/M003604/1
• 负责人: Ian Collinson
• 金额: $ 44.56万
• 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: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM003604%2F1
• 关键词: Understanding; Mechanism; Membrane; Protein; Insertion

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in and separating different biochemical reactions. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know very little about how membranes are formed. In particular, the fundamental process through which proteins are inserted into membranes is poorly understood. Our proposal aims to address this outstanding problem. The process is facilitated by a number of different protein translocation systems (or translocons), including the ubiquitous Sec-machinery responsible for both protein secretion and membrane protein insertion. We aim to learn more about how this particular system works by studying an example from the common gut bacterium Escherichia coli. This is much more experimentally tractable than the human counterpart, but nonetheless should tell us a lot about how similar systems work in our own bodies. A collaborative project between the Collinson (Bristol) and Schaffitzel (Grenoble) Labs has for the first time succeeded in producing and assembling the complete bacterial membrane protein insertion machinery - aka the holo-translocon (HTL), composed of 7 individual subunits. The availability of this active machinery provides a unique opportunity to study the mechanism of membrane protein insertion. The molecular structure of the complex has been investigated, revealing a partially enclosed internal cavity that we have strong reasons to believe is composed of phospholipids. This lipid pool may provide a protected environment into which individual membrane-spanning segments of protein are inserted prior to their folding and release into the bilayer. This is an attractive hypothesis because it mirrors the way soluble (non-membrane) proteins are folded within a water-filled interior of large chaperone complexes.The proposal aims to build on these exciting developments to characterise the activity of HTL and explore the progression of an inserting membrane protein through the complex. An important first step will be to exploit our ability to reconstitute the insertion process from purified components and conduct a comprehensive analysis of basic biochemical rules and requirements of the machinery. The work will also employ new synthetic biology methods to overcome the limitations of the classical biochemical and biophysical approaches employed so far. Collinson and Jones (Cardiff) will combine forces to apply genetic reprogramming to introduce non-natural amino acids into proteins that allow the introduction of novel properties into target proteins. This technology will provide the tools to report on the environment of a protein during its passage into the membrane, as well as on the corresponding architecture of the HTL. Combined with the structure of the active complex, this information will challenge and develop the hypothesis involving the encapsulated insertion of membrane proteins. The results of the project will be important because they relate to an essential and fundamental biological concept, which may then lead to new ideas about its disruption for the development of anti-bacterial drugs. Moreover, the ideas and principles implemented and developed will be accessible to the analysis of other complex membrane protein systems.

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。细胞膜扮演着分子“皮肤”的角色，将细胞内部保持在一起，并将不同的生化反应分开。需要以有控制的方式打破这一障碍，以允许营养物质、废物的运输以及与外部世界的沟通；这是通过广泛的膜插入蛋白质实现的。我们对膜蛋白所具有的运输、呼吸、光合作用等多种生物学功能有了很深的了解。然而，我们对膜是如何形成的知之甚少。特别是，人们对蛋白质插入膜的基本过程知之甚少。我们的建议旨在解决这一悬而未决的问题。这一过程由许多不同的蛋白质转位系统(或转运子)推动，包括无处不在的负责蛋白质分泌和膜蛋白插入的SEC机制。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，更多地了解这一特殊系统是如何工作的。这在实验上比人类的同类要容易得多，但尽管如此，它应该会告诉我们许多类似的系统在我们自己的身体中是如何工作的。柯林森(布里斯托尔)实验室和沙菲策尔(格勒诺布尔)实验室的合作项目首次成功地生产和组装了完整的细菌膜蛋白插入机械--也就是全息转运子(HTL)，由7个单独的亚基组成。这种主动机制的出现为研究膜蛋白插入的机制提供了一个独特的机会。对该络合物的分子结构进行了研究，揭示了一个部分封闭的内腔，我们有充分的理由相信它是由磷脂组成的。该脂质池可提供保护环境，其中在折叠之前插入单个跨膜蛋白质片段并将其释放到双层中。这是一个有吸引力的假说，因为它反映了可溶(非膜)蛋白质在大型伴侣复合体充满水的内部折叠的方式。该提案旨在这些令人兴奋的发展基础上，表征HTL的活性，并探索插入膜蛋白通过复合体的进展。重要的第一步将是开发我们的能力，从纯化的组件重新构建插入过程，并对机器的基本生化规则和要求进行全面分析。这项工作还将采用新的合成生物学方法，以克服迄今采用的经典生化和生物物理方法的局限性。柯林森和琼斯(卡迪夫)将联合起来，应用基因重新编程将非天然氨基酸引入蛋白质，从而使目标蛋白质具有新的特性。这项技术将提供工具来报告蛋白质进入膜过程中的环境，以及HTL的相应结构。结合活性复合体的结构，这些信息将挑战和发展涉及膜蛋白囊化插入的假说。该项目的结果将是重要的，因为它们与一个基本的生物学概念有关，这可能会导致关于它对抗菌药物开发的破坏的新想法。此外，实施和发展的思想和原理也将适用于其他复杂的膜蛋白系统的分析。

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

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

• DOI: 10.7554/elife.60669
• 发表时间: 2020-11-04
• 期刊: eLife
• 影响因子: 7.7
• 作者: Alvira S;Watkins DW;Troman L;Allen WJ;Lorriman JS;Degliesposti G;Cohen EJ;Beeby M;Daum B;Gold VA;Skehel JM;Collinson I
• 通讯作者: Collinson I

Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon.

大肠杆菌中的多蛋白复合物生产：SecYEG-SecDFYajC-YidC Holotranslocon。

• DOI: 10.1007/978-1-4939-6887-9_18
• 发表时间: 2017
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Berger I

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

SecA-a New Twist in the Tale.

• DOI: 10.1128/jb.00736-16
• 发表时间: 2017-01-15
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Collinson I