Applications of NMR Spectroscopy to Study Structure, Dynamics and Small Molecule Interactions Related to Protein Folding and Misfolding

NMR spectroscopy is a powerful technique to study the structure, dynamics and interactions of biomacromolecules in their natural environment in solution, including proteins the cellular milieu, and in the solid-state, including biological membranes, biomaterials and membrane-proteins. This methodology allows researchers to gain detailed atomic resolution pictures of these components and to also study the strength and frequency of the interactions incurred by these biomolecules, giving a comprehensive picture of the systems of interest. A unique feature of the solution biomolecular NMR, in particular recently developed TXO cryoprobes, is the ability to characterise residual structure and transient interactions by proteins which lack distinct three dimensional structures and which are referred to as intrinsically disordered. Intrinsic disorder is a primary characteristic of ~30% of the proteins encoded in the eukaryotic genome and these proteins are central to a number of cellular processes as well as aberrant human diseases including cancer, neurodegenerative disorders, dilated cardiomyopathies. Another key research area in biomolecular NMR is within the solid-state; where samples are in more rigid conformations due to self-assembly or interactions with biological membranes or protein networks. Biomolecular ssNMR has been driving the characterisation of protein amyloid fibrils and membrane proteins, and is an extremely accurate method to probe the structural properties of biomolecular assemblies, i.e.biomaterials, large protein complexes and hydrogels.

The NMR facility in the Department of Chemistry has been serving a large community of molecular scientists in the Cambridge area, including the Departments of Chemistry, the Centre for Misfolding Disease (CMD) and a number of companies and other Departments. Recent research developments in the research labs of the applicants have enabled significant discoveries in the field of protein aggregation and misfolding diseases, for which the CMD has been funded to boost the transitional research into finding cures for neurodegenerative disorders include Alzheimer's and Parkinson's diseases. The activities of the CMD, as well as of the other research labs accessing our NMR facility, would greatly benefit from the availability of high-throughput screening of small molecules on the precursor proteins linked with these diseases. This methodology cannot, currently, be applied with efficiency, despite our flagship 700 MHz instrument being equipped with a cryoprobe TXO and sample exchanger, and we need a more modern spectrometer console to boost sensitivity and apply fast methods of data acquisitions. The change of console is part of our bigger plan to rejuvenate our 700 MHz spectrometer, including maintenance to the magnet to ensure more cost effective management of the instrument and avoid future breakage.

In addition to our expertise in solution NMR, we have gained internationally relevant expertise in studying the aggregated species that are associated with Alzheimer's and Parkinson's disorders. In this context, we would like to equip our NMR facility with bio-solid NMR capability for protein investigations, which is surprisingly missing in the Cambridge area. This equipment will allow also, the drug-screening of selected molecules to specific protein aggregates associated with Alzheimer's and Parkinson's.

In addition to the great benefits that this application would have on the CMD activities associated with the study of intrinsically disordered proteins and protein aggregation in neurodegenerative disorders, the new NMR capability will serve the larger community of scientists at the University of Cambridge and the collaborating institutions that frequently access this facility, including industrial collaborators, faciliting and impacting on research encompassing various aspects of organic chemistry,material sciences and chemical biology.

This application will enhance the capability of the Department of Chemistry's 700 MHz NMR spectrometer to improve sensitivity and allow usage of the latest techniques of acquisition, enabling us to exploit, in full, the capability of our TXO cryoprobe. In addition the spectrometer will be equiped with bio-solid MAS ssNMR capabilities, including 3.2 mm and 1.3 mm MAS probes, filling a gap in the accessibility to biomolecular ssNMR in the Cambridge area. We will also rejuvenate the magnet of the spectrometer to enable continued access to the instrument, which serves an increasing number of users from academia and industry. Collectively these improvements will enable us to perform state of the art biomolecular NMR experiments in both solution and solid-state, and will add to the current capability provided by the TXO cryoprobe, which is unique in the region. In particular: 1) Rejuvenation of our 700 MHz magnet will ensure extended sustainability of this instrument and its components, including the existing TXO cryoprobe and sample exchanger, to offer cost-effective service to the research community in the region. 2) Upgrading the console will exploit, in full, the potential of the TXO cryprobe and sample exchanger by increasing sensitivity of solution NMR measurements and enabling acquisition techniques, i.e. NUS sampling, enabling a fast approach to molecular screening and boosting high-throughput drug discovery. These enhancements will increase service to a broader community of NMR spectroscopists in the region. 3) The biomolecular solid-state NMR will fill a gap in the access to biomolecular ssNMR in the Cambridge area. The two probes, 3.2 mm Efree and 1.3 mm ultrafast spinning probes, will collectively provide excellent biomolecular ssNMR versatility for a large variety of applications in homonuclear and heteronuclear 13C and 15N experiments as well as in 1H detected experiments in deuterated samples; including protein amyloid, hydrogels & bionanomaterials.

The impact of this research grant will be in both the academic and industrial sectors as well as towards both basic and applied sciences. The NMR instrumentation requested will provide a a key tool to generating knowledge towards the identification of new therapies to combat cancer, tuberculosis, and neurodegernerative diseases such as Alzheimer's and Parkinson's diseases. Moreover, the general characterisation of amyloid fibrils will have an impact on the research into novel biomaterials their growing application in material science. This will enhance dramatically our ability to study key molecular processes that are relevant to both academia and industry and extend the applicability of current spectroscopic methods to more complex processes.

INDUSTRIAL BENEFICIARIES: The equipment requested is likely to provide a significant impact to the pharma industry by advancing the use of NMR spectroscopy to understand complex molecular processes in neurodegenerative, and other diseases. Direct beneficiaries include those pharmaceutical companies working at the definition of therapeutic approaches to combat amyloid diseases such as Alzheimer's and Parkinson's. An additional area for applied sciences is the exploitation of new biomaterials based on amyloid fibrils. More broadly, the interdisciplinary NMR experiments that will be enabled by the requested equipment will enhance our ability to characterise the mechanisms of heterogeneous biological processes, with key impact on many bio-industrial areas.

MEDICAL CHALLENGES IN NEURODEGENERATIVE DISORDERS: Understanding the molecular bases of neurodegenerative disorders is one of the top current biomedical challenges. By producing structural data on protein aggregates and their interaction with small molecules, the requested equipment will set the scenario for drug discovery toward the identification of effective therapeutic molecules to target toxic species at the onset and development of Alzheimer's, Parkinson's and other diseases.

DISSEMINATION: We will make every effort to ensure that research produced in our NMR facility is disseminated widely to the research community by the Open Access publication in high-impact journals, presentations at international research meetings and the development of new collaborations. We are highly committed to make our findings available to the wider public, as shown by our track-record in outreach. Presentations explaining how NMR spectroscopy assists in the drug development, Alzheimer's and Parkinson's research and scientific discovery will be made at School-oriented presentations and Schools Open Days. The press office of the Cambridge University will assist in disseminating discoveries via the popular science press and other media formats.

TRAINING: The proposed grant will enable to develop and apply state-of-the-art methods of NMR spectroscopy to contribute in the wide area of structural biology and molecular medicine. This provides an excellent platform to enhance training of postdoctoral researchers, postgraduate students, as well as undergraduate students at the interface of biology, spectroscopy, medicine and chemical engineering.