New strategies for spin labelling cysteine-rich proteins.

点击翻译按钮获取中文摘要

The maxim that Structure Underlies Function is true at any scale. However, probing structure over nanometre lengths can be difficult and yet is necessary to understand the structure and therefore fully understand the function of proteins. One method for measuring nanometre distances on proteins is by using electron paramagnetic resonance (EPR) spectroscopy which can accurately measure the dipole-dipole interaction between pairs of molecules, in a similar way that the attraction between bar magnets feels stronger as the two get closer together. In order for this method to work, the molecules must contain magnetic species called radicals. However, these are not very common in proteins and so they must be added to the structure at particular, pre-determined, positions. Most commonly these sites are particularly reactive amino acids called cysteines. This process is called spin labelling. Proteins which function outside a cell usually contain very few cysteines which allows them to have this amino acid engineered in at points of interest, and so then be specifically spin-labelled.However, proteins that function within the cell may contain many cysteines and if the protein were isolated and mixed with spin label there would be lots of labels attached - a disadvantage for the EPR distance measurement technique which is most accurate in the simplest case of a pair of radicals. Additionally, it would be useful to be able to label proteins specifically within a living cell to enable measurements of their interactions there, however if cysteine reactive labels were injected they would label all cysteine-containing proteins - not just the one of interest. These principles and problems extend to all sorts of techniques that require specific labelling of isolated proteins or proteins within a living organism. The work proposed here seeks to systematically explore some of the options for spin labelling unnatural amino acids - these are amino acids that have been designed with specific reactivities and can be inserted into proteins as they are made in a living cell. Cutting-edge chemical reactions such those developed by 2001 Nobel prize winner B. K. Sharpless and 2010 Nobel prize winner A. Suzuki, shall be utilised. Since the accuracy of the EPR distance measurements is affected when the linker between the backbone of the protein and the label itself is long, and this may be necessary for efficient incorporation of the unnatural amino acid, we shall develop spin labels that can be coupled both to the unnatural amino acid and a natural amino acid adjacent to it. This will reduce the uncertainty in the position of the spin label. The lessons learned from this will be used to test whether it might be possible to specifically label pairs of natural amino acids - e.g. can pairs of adjacent cysteines be labelled specifically in a cysteine-rich protein? This one-year project will lay the groundwork for efficiently and site-specifically spin labelling proteins, regardless of whether they contain multiple cysteines or not. Further, the work will develop the more general technique of chemically modifying unnatural amino acids for any technology that requires labelling or tagging. This will have wide-reaching impact on a range of academic, commercial and medical techniques.