The use of 13CF3 nuclear spin probes to reduce the size limit of NMR spectroscopy.

NMR spectroscopy is one of the only techniques that can determine the structure and dynamics of a protein in the solution state. However, when NMR is applied to large proteins, the resulting spectra suffer from a lack of signal intensity and resolution. This resolution problem has reduced the number of proteins that can be studied effectively by NMR, meaning that important information on their dynamics is being missed. Techniques such as protein deuteration as well as TROSY and methyl-TROSY NMR pulse sequences have been developed to increase the resolution in the NMR spectra of large proteins. While these developments have proved successful, they have not been able to solve the problem.
The purpose of this research project is to develop these techniques further to study even larger proteins. The current best technique, methyl-TROSY, works by labelling proteins with 13CH3 groups. Relaxation interference between different dipolar and chemical shift anisotropy (CSA) relaxation mechanisms within the 13CH3 group. This leads to each peak of the quartet in the NMR spectrum having a different linewidth and intensity, known as the TROSY effect. Phase cycling can then be used to plot only the sharpest, most intense peak in the spectrum, enhancing the resolution of the spectrum. Fluorine nuclei have a large asymmetric electron distribution around them, leading to a large CSA. Therefore, it is thought that 13CF3 nuclei will lead to greater relaxation interference enabling enhanced NMR signal intensity and resolution. James' research will focus on the modelling of NMR relaxation rates followed by expression 13CF3 labelled proteins to see if this labelling can improve the NMR spectrum resolution. Whilst previous attempts to measure the TROSY effect in 13CF3 labelled proteins have been unsuccessful, these have been in shorter peptide chains. In mathematical models, it has been predicted that the TROSY effect will exist for larger proteins, but this needs to be proved experimentally. If the TROSY effect is observed for 13CF3 labelled proteins and the effect is stronger than for 13CH3 labelled proteins, a new protocol to study large proteins via NMR will have been developed, significantly increasing signal intensity and resolution. Another benefit of using a 13CF3 probe is that 19F nuclei are rarely found in biology, meaning that the protein of interest could be studied in-vivo without the spectrum suffering from background noise. This protocol could then be applied to a range of large biologically relevant proteins whose NMR spectra currently suffer from poor signal intensity and resolution. Thereby, important structures and dynamics of such proteins can be determined.