CORDIS - EU research results

Ultrahigh resolution NMR tools for chemistry and structural biology

Final Report Summary - RTACQ4PSNMR (Ultrahigh resolution NMR tools for chemistry and structural biology)

Nuclear magnetic resonance (NMR) spectroscopy is an invaluable tool in a wide range of scientific disciplines, in particular chemistry and structural biology. The power of NMR arises from its structural specificity, which allows the chemical environments of individual atoms to be probed, and from its versatility, which enables the experimenter to tailor the experimental methods used so as to extract the maximum useful information from a given sample. Modern NMR instrumentation allows the experimenter great freedom to vary the way in which radiofrequency pulses and other perturbations are used to tailor the excitation and observation of the signals of the nuclear spins, making it very easy for users to implement novel techniques. The challenge is to design new types of experiment to attack the problems fundamental to all types of spectroscopy: sensitivity, the ability to detect signals of interest against a background of electronic noise; and resolution, the ability to distinguish between different signals in crowded spectra. Recent advances in instrumentation have greatly improved the sensitivity of NMR experiments; this project concerned developments in and application of a new class of NMR experiments, real-time pure shift NMR, that can offer greatly improved resolution and are of potential use in determining the structures of very complex molecules.

The research project focused on the development of heteronuclear real-time pure shift NMR experiments, which allow information about two different stable nuclear isotopes, for example hydrogen-1 and carbon-13 or hydrogen-1 and nitrogen-15, to be correlated in a single experiment. This makes it possible to distinguish thousands of distinct atomic sites in a complex molecule such as a protein, far more than could be distinguished using a single isotope. The prototype of this class of experiments is known as heteronuclear single quantum correlation, or HSQC, and correlates the signals of the nuclei of atoms that are directly bonded to one another. The single most important result of the project has been the development, analysis and application of a real-time pure shift HSQC experiment suitable for use with small and medium-sized proteins. The pulse sequence code for this experiment, which makes it straightforward for potential users to implement the new method on the great majority of commercial NMR spectrometers, has been made freely available via the NMR methodology group website; this page has been accessed over 70000 times.

Example parameter sets and pulse sequence programs have also been provided for the major NMR spectrometer hardware manufacturers (Bruker and Varian/Agilent). Applications of the new pure shift HSQC technique included investigation of the L80C mutant of the N-terminal domain of PGK (phosphoglycerate kinase) and two mutants of a small antifungal protein called PAF (Penicillium chrysogenum Antifugal Protein). The methodology development work was done using the globular protein ubiquitin, which is readily available with and without isotopic labelling; the other protein samples were expressed and prepared in the laboratories of our collaboration partners Prof. Jon Waltho and Prof. Katalin Kövér, respectively. Full details, including detailed comparisons between different types of real-time pure shift method, have been published as J. Biomol. NMR 62, 43-52 (2015), DOI: 10.1007/s10858-015-9913-z. Parallel developments in other pure shift methods have been published as RSC Advs. 5, 52902-52906 (2015), DOI 10.1039/C5RA10192A and Chem. Commun. 51, 15410-15413 (2015), DOI 10.1039/c5cc06293d.

Real-time pure shift versions of more complex experiments for protein backbone assignment (including the three-dimensional HNCO and HNCA experiments, and the transverse relaxation optimised experiment TROSY) have also been implemented. The implementation of real time pure shift HSQC with non-uniform data sampling (NUS) has allowed us to reduce the experiment time needed to collect multidimensional spectra with high resolution. This has the potential to allow pure shift NMR methods to be incorporated into routine protocols for the determination of biomolecular structure. Such methods are both crucial in improving our understanding of the structures and functions of proteins, and key tools in developing new drugs.

The research project provided the context for an extensive training program for the Fellow, Dr Peter Kiraly. Formal training included seven workshops and three online training courses organized by the University of Manchester, and a 5 day residential course delivered by the NMR spectrometer manufacturer Bruker. PK participated in the weekly meetings of the NMR methodology group throughout the two years, leading nine meetings. Informal training was provided by the scientist in charge, Professor Gareth Morris, and by other members of the Manchester NMR methodology group, in NMR theory; analytical and numerical simulation of NMR experiments; statistical analysis of the results of NMR experiments; VnmrJ programming; NMR electronics; control and correction of systematic errors; Bruker programming and hardware; and protein and peptide NMR.

Further information may obtained from the Fellow, Dr Peter Kiraly ( or the scientist in charge, Professor Gareth Morris (