Understanding the Mechanism of Membrane Protein Insertion

了解膜蛋白插入的机制

基本信息

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

项目摘要

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，膜由双层脂肪分子（称为磷脂）组成。细胞膜充当分子“皮肤”，保持细胞内部并分隔不同的生化反应。需要以受控方式突破屏障，以允许营养物质、废物的运输以及与外界的沟通；这是通过多种膜插入蛋白实现的。我们非常了解膜蛋白赋予的多种生物功能，例如运输、呼吸、光合作用。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个突出问题。许多不同的蛋白质易位系统（或易位子）促进了这一过程，包括无处不在的负责蛋白质分泌和膜蛋白插入的 Sec 机器。我们的目标是通过研究常见肠道细菌大肠杆菌的例子来更多地了解这个特定系统的工作原理。这在实验上比人类的对应物更容易处理，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。 Collinson（布里斯托尔）和 Schaffitzel（格勒诺布尔）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器 - 又名全息转位子 (HTL)，由 7 个单独的亚基组成。这种活性机制的可用性为研究膜蛋白插入机制提供了独特的机会。对该复合物的分子结构进行了研究，揭示了一个部分封闭的内部空腔，我们有充分的理由相信它是由磷脂组成的。这种脂质池可以提供一个受保护的环境，在蛋白质的各个跨膜片段折叠并释放到双层中之前将其插入其中。这是一个有吸引力的假设，因为它反映了可溶性（非膜）蛋白质在大型伴侣复合物的充满水的内部折叠的方式。该提案旨在以这些令人兴奋的进展为基础，来表征 HTL 的活性并探索穿过复合物的插入膜蛋白。重要的第一步将是利用我们从纯化成分重建插入过程的能力，并对基本生化规则和机器要求进行全面分析。这项工作还将采用新的合成生物学方法来克服迄今为止采用的经典生物化学和生物物理方法的局限性。 Collinson 和 Jones（卡迪夫）将联手应用基因重编程，将非天然氨基酸引入蛋白质中，从而将新特性引入目标蛋白质中。该技术将提供工具来报告蛋白质在进入膜期间的环境以及相应的 HTL 结构。结合活性复合物的结构，这些信息将挑战并发展涉及膜蛋白封装插入的假设。该项目的结果非常重要，因为它们涉及一个重要且基本的生物学概念，这可能会带来关于其对抗菌药物开发的破坏的新想法。此外，所实施和开发的想法和原理将可用于其他复杂膜蛋白系统的分析。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
作者：
Alvira, S.;Watkins, DW.;Collinson, I.
通讯作者：
Collinson, 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

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。