VideoAFM of membrane proteins

膜蛋白的视频AFM

• 批准号: EP/F027591/1
• 负责人: Christopher Hunter
• 金额: $ 10.43万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FF027591%2F1
• 关键词: VideoAFM; membrane; proteins

Membrane proteins are central to the function of cells, and hence to life, but we have structural information for remarkably few of them. One of the main reasons for this is the difficulty in growing sufficiently well ordered two dimensional crystals for electron microscopy or three dimensional crystals for X-ray crystallography. Little is known about the arrangement of membrane proteins in either their native membrane environment or in growing crystals, or of the forces that stabilise these associations. Recently atomic force microscopy (AFM) has proved to be a powerful tool for studying the structure and organisation of membrane proteins, particularly light harvesting complexes, and AFM has the advantage of image acquisition under physiological conditions of temperature and pH. However, the wider application of the AFM technique has been hampered by the length of time, typically minutes, that it takes to obtain a single image. The recent development of VideoAFM, and its enhancement by Dr Vasilev who is the researcher on this post-doctoral mobility application, gives a thousand-fold increase in the imaging rate of AFM, allowing access to processes that occur over sub-second timescales while maintaining nanometre spatial resolution. The aim of this project is to transfer Dr Vasilev's skills in this new technique, established in the Department of Physics and Astronomy, to the Department Molecular Biology and Biotechnology at the University of Sheffield and to apply them to the crystallization of light harvesting membrane protein complexes. These complexes provide an ideal test of applicability of VideoAFM to study the crystallisation process. The capability of this technique to image expanding, highly organised arrays of light harvesting complexes will be explored and suitable protocols developed. The very rapid scan rates of VideoAFM will allow large areas of a crystal to be imaged with molecular resolution at reasonable speeds, providing the first statistically significant time-lapse information of membrane crystal growth. Obtaining such large data sets under different conditions will greatly enhance our knowledge of the fundamentals of membrane protein crystallisation. Finally, the feasibility of using VideoAFM to follow the crystallisation process, in-situ and in real-time, will be explored. If successful, this will provide invaluable information on the kinetics and organisation of growing crystals, applicable to structural studies of these and many other membrane protein complexes.

膜蛋白是细胞功能的核心，因此也是生命的核心，但我们掌握的结构信息非常少。其主要原因之一是难以生长足够有序的二维晶体用于电子显微镜或三维晶体用于X射线晶体学。关于膜蛋白在其天然膜环境中或在生长的晶体中的排列，或者稳定这些关联的力，我们知之甚少。最近，原子力显微镜（AFM）已被证明是一个强大的工具，用于研究膜蛋白的结构和组织，特别是捕光复合物，和AFM具有的优势，在生理条件下的温度和pH值的图像采集。然而，更广泛的应用的AFM技术已受到阻碍的时间长度，通常是分钟，它需要获得一个单一的图像。VideoAFM的最新发展，以及Vasilev博士对该博士后流动性应用的研究，使AFM的成像速率提高了数千倍，允许访问在亚秒时间尺度上发生的过程，同时保持纳米空间分辨率。该项目的目的是将Vasilev博士在物理和天文学系建立的这项新技术的技能转移到谢菲尔德大学的分子生物学和生物技术系，并将其应用于光捕获膜蛋白复合物的结晶。这些配合物提供了一个理想的测试VideoAFM的适用性，研究结晶过程。这项技术的能力，图像扩大，高度组织阵列的光捕获复合物将被探索和开发合适的协议。VideoAFM非常快的扫描速率将允许以合理的速度以分子分辨率对晶体的大面积进行成像，从而提供膜晶体生长的第一个统计上显著的时间推移信息。在不同条件下获得如此大的数据集将大大提高我们对膜蛋白结晶基本原理的认识。最后，使用VideoAFM跟踪结晶过程的可行性，原位和实时，将进行探索。如果成功的话，这将提供宝贵的信息的动力学和组织生长的晶体，适用于这些和许多其他膜蛋白复合物的结构研究。