VideoAFM of 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.