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Protein dynamics in Escherichia coli

大肠杆菌中的蛋白质动力学

基本信息

批准号：
BB/E009751/1
负责人：
Conrad Mullineaux
金额：
$ 40.17万
依托单位：
Queen Mary University of London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE009751%2F1
关键词：
Protein dynamics Escherichia coli

项目摘要

The interior of a living cell is a complex environment which is densely packed with many different kinds of molecules. These include numerous proteins - biological macromolecules which carry out many essential functions of the cell. There are two traditional approaches to understanding the processes occurring in the cell. The biochemical approach involves isolating specific cell components, studying their behaviour in vitro, and then inferring their behaviour within the intact cell, usually on the assumption that the cell interior is a rather simple, fluid 'bag' of molecules. A second approach involves microscopic structural studies on the organisation of the cell interior and specific cell components. Neither approach gives a complete picture of the function of the cell interior. The biochemical approach neglects the complex, structured, and crowded nature of the cell interior, while the conventional microscopic approach shows cell structure but gives little information on the movement of molecules in the cell. However, there are number of ways to study the movement of proteins within living cells. A key technique is Fluorescence Recovery after Photobleaching (FRAP). For FRAP, the protein of interest must be somehow be labelled with a fluorescent tag. When the cell is observed in a fluorescence microscope, the protein can then be detected. Usually, individual protein molecules are not observed - instead the fluorescence micrograph shows the distribution of hundreds or thousands of protein molecules within the cell. The diffusion of the protein can be measured by using a highly-focussed laser beam to rapidly 'bleach' fluorescence in a small area of the cell. The bleached area appears as a dark area in the fluorescence micrograph. If the protein population is mobile, the bleached area spreads and fills in a characteristic way. The rate of diffusion of the fluorescent protein can then be estimated. There have been numerous FRAP studies on individual proteins in different kinds of cells, but surprisingly there has to date been no systematic study of the factors that control the rates of protein diffusion. We will carry out such a systematic study, using as a model organism the well-known gut bacterium Escherichia coli. E. coli is arguably the organism we understand best. For our study, it has the advantage of a relatively simple cell structure. Furthermore, it is very easy to genetically manipulate. A perceived disadvantage of E. coli for FRAP studies is that it has relatively small cells, which can make studies based on optical microscopy harder. However, we have developed ways to overcome this problem, and shown that it is possible to use FRAP to make accurate measurements of protein diffusion in E. coli cells. We will use genetic methods to add fluorescent tags to proteins of different sizes and properties, which will be synthesised inside the cell. Depending on the properties of the proteins, they will either remain in the cytoplasm, be associated with the inner membrane that surrounds the cytoplasm, or be exported into the periplasm, the compartment between the inner and outer membranes of the cell. The diffusion rates of the various proteins will provide an incisive probe of the physical properties of these environments in the cell. We will use the data to construct physical models for these environments. There have been several attempts to construct mathematical models for processes involving protein diffusion in E. coli, but these have not been based on accurate experimental measurements of the rates of protein diffusion. We will use our data to construct more realistic models for cell processes, including the way that chemical messages travelling within the cell control the direction in which it swims, and the way the cell is able to divide at its midpoint. The project will help to us to understand how a cell functions as a dynamic physical system.

活细胞的内部是一个复杂的环境，其中密集地挤满了许多不同种类的分子。这些包括许多蛋白质-生物大分子，它们执行细胞的许多基本功能。有两种传统的方法来理解细胞中发生的过程。生物化学方法包括分离特定的细胞成分，研究它们在体外的行为，然后推断它们在完整细胞内的行为，通常假设细胞内部是一个相当简单的流体“袋”。第二种方法涉及对细胞内部组织和特定细胞成分的显微结构研究。这两种方法都不能给出细胞内部功能的全貌。生物化学方法忽略了细胞内部复杂、结构化和拥挤的性质，而传统的显微镜方法显示了细胞结构，但几乎没有提供细胞中分子运动的信息。然而，有许多方法可以研究活细胞内蛋白质的运动。其中一项关键技术是光漂白后的荧光恢复（FRAP）。对于FRAP，感兴趣的蛋白质必须以某种方式用荧光标签标记。当在荧光显微镜下观察细胞时，可以检测到蛋白质。通常，单个蛋白质分子不会被观察到-相反，荧光显微照片显示了细胞内数百或数千个蛋白质分子的分布。蛋白质的扩散可以通过使用高度聚焦的激光束在细胞的小区域中快速“漂白”荧光来测量。漂白区域在荧光显微照片中显示为暗区。如果蛋白质群体是移动的，则漂白区域以特征性方式扩散和填充。然后可以估计荧光蛋白的扩散速率。已经有许多FRAP研究个别蛋白质在不同种类的细胞，但令人惊讶的是，迄今为止还没有系统的研究控制蛋白质扩散速率的因素。我们将进行这样一个系统的研究，作为一个模式生物，众所周知的肠道细菌大肠杆菌。E.大肠杆菌可以说是我们最了解的有机体。对于我们的研究，它具有相对简单的细胞结构的优点。此外，它很容易进行基因操纵。一个明显的缺点是E。FRAP研究的另一个优势是它的细胞相对较小，这使得基于光学显微镜的研究更加困难。然而，我们已经开发了克服这个问题的方法，并表明使用FRAP来精确测量E.大肠杆菌细胞。我们将使用遗传方法将荧光标记添加到不同大小和特性的蛋白质上，这些蛋白质将在细胞内合成。根据蛋白质的性质，它们将保留在细胞质中，与包围细胞质的内膜结合，或者被输出到周质中，周质是细胞内外膜之间的隔室。各种蛋白质的扩散速率将为细胞中这些环境的物理性质提供一个敏锐的探针。我们将使用这些数据来构建这些环境的物理模型。已经有几次尝试构建数学模型的过程，涉及蛋白质扩散在大肠杆菌。大肠杆菌，但这些都没有基于蛋白质扩散速率的准确实验测量。我们将利用我们的数据来构建更真实的细胞过程模型，包括细胞内传播的化学信息控制其游动方向的方式，以及细胞在中点分裂的方式。该项目将帮助我们了解细胞作为一个动态物理系统的功能。