VideoAFM of membrane proteins

膜蛋白是细胞功能的核心，因此对生命至关重要，但我们对它们的结构信息知之甚少。造成这种情况的一个主要原因是很难生长出足够有序的用于电子显微镜的二维晶体或用于x射线晶体学的三维晶体。对于膜蛋白在其天然膜环境或生长晶体中的排列，以及稳定这些联系的力量，人们知之甚少。最近，原子力显微镜（AFM）已被证明是研究膜蛋白结构和组织的有力工具，特别是光捕获复合物，并且AFM在温度和ph的生理条件下具有图像采集的优势。然而，AFM技术的广泛应用受到时间长度（通常为几分钟）的阻碍，它需要获得单张图像。VideoAFM的最新发展，以及博士后移动应用研究人员Vasilev博士对其的改进，使AFM的成像速率提高了1000倍，允许在亚秒时间尺度上进行处理，同时保持纳米空间分辨率。该项目的目的是将Vasilev博士在这项新技术上的技能转移到谢菲尔德大学的分子生物学和生物技术部门，并将其应用于光捕获膜蛋白复合物的结晶。这些配合物为VideoAFM研究结晶过程的适用性提供了理想的测试。这项技术的图像扩展能力，高度有组织的光收集复合物阵列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对晶体的大面积进行分子分辨率成像，提供第一个具有统计学意义的膜晶体生长延时信息。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础的认识。最后，将探讨使用VideoAFM跟踪结晶过程的可行性，现场和实时。如果成功，这将为生长晶体的动力学和组织提供宝贵的信息，适用于这些和许多其他膜蛋白复合物的结构研究。

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.