New strategies for spin labelling cysteine-rich proteins.

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.

Knowledge of the structure of proteins and their complexes (i.e. conglomerations of proteins or proteins with nucleic acids etc) throughout their functional cycle is crucial to understanding how proteins work which is necessary for mimicking their properties for nanotechnology or for truly targeted and rational drug design. There are many physical techniques for exploring the structure of proteins but each requires certain conditions to be affected and can only reveal a partial view of the overall protein. The technique we will develop is complementary to other methods: it enables solution-state studies of the structure, on the nanometre-lengthscale, and dynamics of a protein uses electron paramagnetic resonance (EPR) spectroscopy.

Although EPR is not a new method, advances in recent years both for the technique and for sample preparation has meant that it is now emerging from being a niche method used to study, for example, the oxidation states of metalloproteins, to being an important and generally applicable biophysical tool, that can be used for accurate structural studies. The technique relies on the use of a spin label (i.e. stable radical) that is normally bound to cysteines and which may be engineered into the site or sites of interest. Many successful structural studies have now been carried out with EPR. However, this proposal describes work that if successful would overcome the problem that many proteins that could be investigated by EPR methodologies cannot currently be spin labelled since they are rich in cysteines. The completion of the investigations outlined in this proposal will significantly impact on the range of proteins that can be investigated with EPR. This will lead to greater knowledge and understanding of a potentially vast number of proteins, from P450s and nitric oxide synthases to proteins involved in the regulation of transcription and beyond. In turn this will have long-term benefits in medicine and biotechnology.

The systematic investigation that will be carried out into spin labelling unnatural amino acids, tethering the label to a natural amino acid, and ultimately labelling pairs of natural amino acids in the presence of single copies of identical amino acids will not only impact the specific applications outlined in this project, but also on other technologies which require, or would benefit from, the site-specific labelling of cysteine-rich proteins. For example, methodologies requiring fluorescence labelling. In fact, even the first objective of attempting several types of chemical reaction to label unnatural amino acids in aqueous, protein tolerant, conditions will aid cellular and in vivo labelling technologies and will help spear-head the use of precision EPR measurements in these environments.

The PDRA will have a one year "masterclass" in interdisciplinary research through exposure to a wide range of chemical and chemical biology techniques from solid phase peptide synthesis and wide ranging methods in organic synthesis to manipulating proteins to selectively incorporate the spin labels. They will also have the opportunity to learn EPR at one of the best-equipped laboratories in the world. Combining this skills-base with the experience of presenting their results at national/international conferences will lead to a very qualified person ready to work in a variety of chemistry-related research activities (industrial or academic).

Economic benefits will arise directly from the training of the PDRA and commercial and academic research in the fields of structural, chemical, molecular and synthetic biology as well as further developing the applications of EPR spectroscopy.