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.

所有细胞都被膜包围，由称为磷脂的双层脂肪分子组成。细胞膜充当分子“皮肤”，使细胞的内部内部并分离不同的生化反应。需要以受控的方式破坏障碍，以允许营养物质，废物产品运输以及与外界的沟通；这是通过各种膜插入蛋白来实现的。我们对膜蛋白赋予的多种生物学功能（例如运输，呼吸，光合作用）的多种生物学功能了解很多。但是，我们对膜的形成方式知之甚少。特别是，插入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个杰出的问题。该过程由许多不同的蛋白质易位系统（或转运系统）促进，包括负责蛋白质分泌和膜蛋白插入的无处不在的SEC机械。我们的目标是通过研究常见的肠道细菌大肠杆菌的示例来了解有关该特定系统如何工作的更多信息。这在实验上比人类对应物更具实验性，但是尽管如此，应该告诉我们很多有关类似系统如何在我们自己的身体中起作用的信息。 Collinson（Bristol）和Schaffitzel（Grenoble）实验室之间的合作项目首次成功生产和组装完整的细菌膜蛋白插入机械-AKA aka Holo -Translocon（HTL），由7个单独的亚基组成。这种主动机械的可用性为研究膜蛋白插入机理提供了独特的机会。已经研究了该复合物的分子结构，揭示了我们有强烈的理由相信由磷脂组成的部分封闭的内部空腔。该脂质池可以提供一个受保护的环境，在该环境中，在蛋白质折叠之前插入并释放到双层之前，将蛋白质的单个跨膜段插入其中。这是一个有吸引力的假设，因为它反映了可溶性（非膜）蛋白的方式折叠在大型伴侣复合物的水内部内部。该提案旨在建立在这些令人兴奋的开发基础上，以表征HTL的活性并探索通过该复合物插入膜蛋白的进展。重要的第一步是利用我们从纯化的组件重新插入插入过程的能力，并对机械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来克服迄今为止使用的经典生化和生物物理方法的局限性。 Collinson和Jones（Cardiff）将结合力以应用遗传重编程将非天然氨基酸引入蛋白质中，从而使新特性将新特性引入靶蛋白。这项技术将为蛋白质进入膜以及HTL的相应体系结构时提供有关蛋白质环境的工具。结合活性复合物的结构，该信息将挑战和发展涉及膜蛋白插入的假设。该项目的结果将很重要，因为它们与一个基本和基本的生物学概念有关，这可能会导致有关其对抗菌药物开发的破坏的新想法。此外，分析其他复杂的膜蛋白系统的分析将可以访问和开发的思想和原理。

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: 10.1101/589077
• 发表时间: 2020-01-01
• 期刊: bioRxiv
• 作者: Alvira, S.;Watkins, DW.;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

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

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