31P-edited pulse sequences for protein NMR

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying molecular structure. NMR relies on the interaction of nuclei with strong magnetic fields, and our ability to perturb these interactions using pulses of radiofrequency radiation. The particular sequence of radiofrequency pulses determines the information provided by the resulting spectrum.

Specialised NMR techniques can be applied to large biological molecules such as proteins. We can build complex pulse sequences that allow multidimensional NMR spectra to be acquired, which can be used to determine protein structures and to investigate their interactions with other molecules, both of which are vital to properly understand a protein. However, traditional methods are limited to smaller proteins, as larger proteins give too many signals in their spectra to allow individual signals to be resolved, and also lose signal intensity faster than smaller proteins. This loss of signal intensity prevents the use of longer, more complex pulse sequences that might otherwise be used to reduce the crowding of signals in the spectrum. One method that allows us to overcome both problems is the use of spectral editing and filtering. Spectral editing and filtering allow us to select signals for atoms bound to particular other atoms. For example, 13C editing selects only signals from atoms bound to 13C.

While proteins do not contain phosphorus, many other molecules that bind to proteins do. Recent advances in NMR hardware have for the first time allowed us to observe hydrogen, carbon, nitrogen and phosphorus in a single NMR experiment. However, pulse sequences that take advantage of this ability have not yet been developed. The work proposed here will develop such pulse sequences. This would allow a huge leap forward in the types of experiments that we can perform on proteins that bind phosphorus-containing molecules, including DNA, RNA and a wide range of vitamins and their derivatives. These proteins are often important in cellular processes, and several have been implicated in diseases, including cancer. Therefore, pulse sequences that allow new types of experiments to be performed on these proteins will help to advance our understanding of the processes underlying disease.

Over half of all known proteins bind to phosphate-containing molecules such as DNA, RNA and various cofactors and phosphorylated metabolites. Such proteins often have important cellular functions and many have been implicated in disease processes. The development of novel tools to study such proteins is therefore of great interest and broad applicability.

The proposed work will develop 31P-edited NOESY experiments for use in protein NMR, by incorporation of 31P half-filter elements or other editing techniques into two- and three-dimensional NOESY pulse sequences. The resulting experiments will be validated and tested using the model protein dihydrofolate reductase. The use of gradient pulses to select desired magnetisation transfer pathways, phase cycling to reduce spectral artefacts, and additional modifications to the basic pulse sequences will be fully investigated in order to maximise their usefulness. The optimised pulse sequences will subsequently be tested on more challenging proteins to demonstrate their wider utility.

The incorporation of 31P editing will dramatically simplify the resulting spectra. Because only a small number of signals in the protein will be selected by 31P editing, the resulting spectra will contain only a small number of signals, even in a large protein where individual resonances may be impossible to resolve using traditional methods. As the spectra obtained will be focused on the active site of the protein, they will provide meaningful information of direct relevance to the protein's function, particularly in combination with selective isotopic labeling NMR methods, X-ray crystallography or homology modeling to provide additional structural information.

The novel NMR pulse sequences developed here will provide new methods for the study of proteins that bind phosphate-containing molecules. They will therefore facilitate research in a range of fields within BBSRC's remit.

The development of 31P-edited NOESY pulse sequences for protein NMR will enable a wide range of novel research across a range of fields. As such it will have considerable academic impact in the short term, and has great potential for broader impact in the longer term though the application of the resulting techniques.

In order that the pulse sequences can be fully exploited by the wider scientific community, dissemination and communication is vital. The sequences will be disseminated through the NMR manufacturer Bruker as part of their standard pulse sequence library, and will also be made directly available to other NMR spectroscopists. The work will be published in internationally leading, peer-reviewed journals, and will be presented at conferences and NMR user meetings. Where appropriate, this will be extended to the popular press due to the potential for longer-term societal benefits outside the scope of this proposal. It is worthy of note that the applicant has significant experience in science communication.

The pulse sequences developed here will enable novel experiments in protein NMR, facilitating research in a range of fields. In the first instance, the research will be of greatest interest to academics (see 'academic beneficiaries'). However, there is great potential for industrial collaboration in the longer term, as many of the systems that this research can be applied to are involved in disease processes. The true impact of this work will not be felt for some years after the completion of the project.

It is not anticipated that intellectual property will arise from this work. However, progress and findings will periodically be discussed with the Research, Innovation and Enterprise Services (RIES, formally RACD) at Cardiff University to assess any outcomes in this area. RIES is well equipped to protect intellectual property, set up license arrangements and handle all aspects of commercial exploitation in support of this project.

Finally, the research technician working on the project will gain excellent training, providing scientific knowledge and skills as well as wider transferable skills suitable for future employment in a range of sectors or for future postgraduate study.