Understanding the Mechanism of Membrane Protein Insertion

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。细胞膜就像一层分子“皮肤”，将细胞内部保持在里面，并将不同的生化反应分开。屏障需要以受控的方式被突破，以允许营养物质，废物的运输以及与外界的交流;这是通过各种膜插入蛋白质来实现的。我们对膜蛋白赋予的各种生物功能有了很大的了解，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，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。这个过程是由许多不同的蛋白质易位系统（或translocons），包括无处不在的Sec机制负责蛋白质分泌和膜蛋白插入促进。我们的目标是通过研究来自常见肠道细菌大肠杆菌的一个例子来更多地了解这个特定系统是如何工作的。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）和Schaffitzel（格勒诺布尔）实验室之间的一个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制-即由7个单独亚基组成的全转位子（HTL）。这种活性机器的可用性提供了一个独特的机会来研究膜蛋白插入的机制。复合物的分子结构已被研究，揭示了一个部分封闭的内部空腔，我们有充分的理由相信是由磷脂。该脂质池可以提供受保护的环境，其中蛋白质的各个跨膜区段在其折叠和释放到双层中之前被插入。这是一个很有吸引力的假设，因为它反映了可溶性（非膜）蛋白质在大分子伴侣复合物充满水的内部折叠的方式。该提案旨在建立在这些令人兴奋的发展基础上，以验证HTL的活性，并探索插入膜蛋白通过复合物的进展。重要的第一步将是利用我们的能力，从纯化的成分中重建插入过程，并对基本的生物化学规则和机器的要求进行全面分析。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）将联合收割机应用遗传重编程将非天然氨基酸引入蛋白质，从而允许将新特性引入靶蛋白。这项技术将提供工具来报告蛋白质在进入膜期间的环境，以及HTL的相应结构。结合活性复合物的结构，这些信息将挑战和发展涉及膜蛋白的封装插入的假设。该项目的结果将是重要的，因为它们涉及到一个重要的和基本的生物学概念，这可能会导致新的想法，其中断的抗菌药物的发展。此外，实施和开发的思想和原则将可用于其他复杂膜蛋白系统的分析。

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.