Sharpening up protein NMR: ultra-high resolution experiments

Proteins are key targets and tools in both medicine (e.g. biopharmaceuticals) and industry (e.g. enzymic biotransformations). NMR is a vital method for the study of such proteins in solution, providing comprehensive site-specific information on structure, dynamics and interactions - but only if individual signals are resolved in the spectra. A range of multidimensional (nD) NMR techniques has therefore been developed. Spreading the signal in several dimensions both significantly increases resolution, and encodes structural information; however, nD NMR can be very costly in experiment time and the spectra produced can be difficult to visualise. Existing alternative methods sometimes use direct detection of 13C to improve signal dispersion, however these approaches are very insensitive and require both 13C labelling and special NMR probes. In this project we will develop new NMR experiments, based on so-called pure shift techniques, that are able both to improve resolution and to reduce experiment time. Pure shift NMR increases spectral resolution by a factor of 2 to 10, by collapsing multiplet structure. In small molecules this typically comes at a significant cost in sensitivity, but in protein NMR we use selective methods that can increase both resolution and sensitivity1.

New pure shift methods will be of most importance to two classes of polypeptides in which fast local motion provides the relatively slow T2 relaxation needed for pure shift methods: small (< 8 kDa) proteins and peptides, and intrinsically disordered regions within larger proteins. The primary class of experiments targeted is that of HSQC-based experiments1,2 such as HNCO, where the pure shift approach is particularly favourable for 15N-labelled proteins with natural abundance 13C. If the anticipated gains are realised, these new experiments will allow 1H-15N correlation planes to be recorded at significantly enhanced resolution, and at more than an order of magnitude lower in concentration than is currently possible with direct 13C acquisition.

Initial development work will use 15N-labelled ubiquitin as a test sample, with prototype pulse sequences then applied to ParG,3 a small protein involved in plasmid segregation which has both folded and functional unfolded regions, and on a larger protein REF2-1,4 implicated in recognition of cellular mRNA, as well as viral proteins which are under active investigation in our lab. The work will be divided between the School of Chemistry, which has 12 medium field superconducting spectrometers, and the Manchester Institute of Biotechnology, which houses three higher field (up to 800 MHz) spectrometers equipped with the triple-resonance cryoprobes needed for practical protein applications.

The supervisors are world experts in NMR, with a recent focus on pure shift methods1,2,5 (largely developed in Manchester), and on NMR studies of proteins in biology and biotechnology. This is a challenging and important project that spans the full range from physics to biology. The student will be trained in the practicalities of NMR (GAM, MN) and of structural biology in solution (AG), the basic theory of NMR (GAM), the use of computer-controlled instrumentation to perform novel experiments (GAM, MN), and advanced data analysis methods (AG, MN). The three initial 8-week rotations will provide training in the practical operation of NMR spectrometers, at the level needed for development of new experiments; the computer programming skills needed for pulse sequence development and data analysis, including graphical user interfaces and macro programming; and the production of isotopically-labelled proteins.