Ultrafast nuclear magnetic resonance spectroscopy using parallel detection and DNP enhancement for studies of molecular dynamics

Nuclear Magnetic Resonance (NMR) is a spectroscopy technique which makes it possible to measure inter-nuclear distances in conjunction with chemical selectivity. The design of sophisticated techniques which can be used to probe also the connectivity of nuclei in large biomolecules has made NMR the method of choice in studies of biomolecular structure. However, NMR studies of molecular dynamics such as changes of protein conformation, protein folding or ligand-protein binding are limited to relatively slow time scales of the order of seconds. This problem arises because a delay of approximately one seconds is required after an excitation to allow the nuclear spin system to return to equilibrium before another NMR experiment can be carried out. Another problem in NMR is the relatively low sensitivity in comparison with other spectroscopy techniques. Frequently signal averaging is required to improve the signal-to-noise of an experiment thus creating another factor which limits the possible time resolution. In this research programme we propose to combine several technologies to improve the time resolution of NMR spectroscopy experiments substantially. We will use parallel detection to monitor the NMR signal at six different sample locations in a purpose designed probehead. By starting six experiments separately with short time delays we will be able to monitor fast molecular dynamics within the sample. This novel NMR probehead will be interfaced to a control console with six independent channels. We will develop novel programmes which will take maximal advantage of the multi-channel configuration to achieve the best possible time resolution. To maximise the signal-to-noise in the proposed NMR experiments we will use dynamic nuclear polarisation (DNP). This technology makes it possible to generate nuclear spin systems which have a substantially higher polarisation compared to the thermal polarisation obtained when exposing the spin systems to a strong magnetic field. DNP relies on the interactions between unpaired electrons of a radical and the surrounding nuclear spins.The nuclear polarisation using DNP can be enhanced up to four orders of magnitude if the sample temperature is also changed quickly between -271 degree C and 20 degree C. We will integrate our novel parallel detection strategy in a DNP-NMR prototype system that has been designed to perform this temperature jump in about 1 seconds. Our novel technology will be extremely useful in studies of protein-ligand interactions. Frequently, the first events in such interactions are not complete understood due to a lack of suitable experimental method. With an successful implementation of our novel technology we aim to shed light on such fast molecular dynamic processes.

NMR spectroscopy has currently a time resolution in the order of seconds. This limits its application to only relatively slow processes of molecular dynamics. Frequently, the observation of fast processes such as first events in ligand binding is currently not possible using NMR spectroscopy. To improve the time resolution in an NMR experiment substantially we have designed a novel strategy which involves parallel detection of the NMR signal in a purpose built NMR probehead at six different sample locations. The excitation and detection will be controlled by six independent channels thus making it possible to trigger the six acquistions consecutively with short delays between their starting times. We will combine this novel detection scheme with hyperpolarisation technologies to improve the signal-to-noise of the NMR experiment. Ligand molecules with high spin polarisation will be generated by low temperature dynamic nuclear polarisation in conjunction with a fast temperature jump generated by a dissolution step. The ligand solution will be combined with a protein solution in a mixing device before injection into the novel NMR probehead. The parallel detection scheme will be implemented in conjunction with two different DNP strategies. The first strategy involves pneumatic shuttling of the ligand solution between a small home built polariser and the main NMR magnet; the second strategy is based on a novel DNP-NMR spectrometer which is built around an unique two-centre magnet. The novel parallel detection scheme will make it possible to acquire either 1D spectra or fast gradient-assisted 2D spectra. We will demonstrate the potential of our novel NMR strategy using model ligand - protein systems.