Characterising structure, interactions and dynamics of large molecular machines and intrinsically disordered proteins using novel carbon-detected NMR

In nuclear magnetic resonance (NMR) spectroscopy nuclear magnetisations, such as proton, carbon and nitrogen, are excited using specifically designed radio waves (pulse sequences) and the resulting radio wave transmitted from the sample provides the researcher with information about the local environment of the nucleus in question and its motional properties. NMR spectroscopy is therefore often the preferred tool to elucidate molecular structure, flexibility, and interactions since NMR provides descriptions of the molecular dynamics and interactions at atomic resolution. The main aim of the proposed research is to explore and develop a relative avenue in biomolecular NMR spectroscopy, that it, carbon-detected NMR spectroscopy. Our new applications will allow us to, among others, visualise the motions and mechanisms of macromolecular machines.

Within the research areas of protein complexes, macromolecular machines and intrinsically disordered proteins the main body of NMR spectroscopic applications rely on detection of proton magnetisations. This is because the magnetic strength of the proton is four times stronger than that of carbon and ten times stronger than that of nitrogen, thereby giving raise to stronger NMR signals that are easier to detect. However, the spin-physics and the nuclear interactions that govern the linewidth of the NMR signals also depend on the magnetic strength of the interacting nuclei and researchers have therefore been diluting the protons out in molecular machines to sharpen the NMR signals, avoid overlaps of signals, and thereby facilitate new characterisations.

Others and the applicants of this proposal have recently shown the potential in using carbon-detected NMR spectroscopy for the investigation of large proteins and also intrinsically disordered proteins. Although the carbon-detected NMR signals are weaker than the proton detected signals there are many benefits that we are planning on exploiting and developing further with the new equipment that we are applying for. Firstly, carbon signals are in general sharper than the corresponding proton signals - because of their lower magnetic strength. Secondly, the average distance between carbon signals are much higher than that of proton signals, thereby leading to significantly less overlap of signals and facilitating analyses of the underlying parameters. In general terms, this means that we will be able to monitor motions and interactions of proteins during the formations of their structure (protein folding), macromolecular machines such as the cells protein producing machinery (Ribosome) and DNA remodelling enzymes (histone deacetylases). We all have excellent track-records in applying and developing new methodologies for NMR spectroscopy also within the field of carbon-detected NMR.

The research will be carried out at the Institute of Structural and Molecular Biology (ISMB) at University College London (UCL). A state-of-the-art and stimulating research environment with dedicated NMR machines that are optimised for detection of carbon nuclei, and the highly collaborative environment and world-class expertise open up the possibility for many fruitful collaborations across disciplines and many novel applications to come.

This is an exciting time for NMR spectroscopy, as it is gradually becoming possible to probe large macromolecular machines - even within a cellular environment, follow how proteins fold, and elucidate how disordered parts of macromolecules regulate functions. Such studies have mainly become possible because of the developments of new hardware by the spectrometer manufacturers, developments in production of isotope labelled proteins, and developments of new applications to provide high enough sensitivities in order to address the full range of biological questions.

The very recent introduction of a new cryogenic NMR probe that is optimised for carbon-detection now paves the way for exciting new NMR applications to be exploited. Most of the previously used NMR methods are based on detection of proton magnetisation because its higher magnetic moment relative to the carbon and nitrogen nuclei. However, now with the introduction of the carbon-optimised cryogenic probes, carbon-detected NMR spectroscopy of, for example, macromolecular machines has become possible.

Our objective it to exploit carbon-detected NMR spectroscopy to gain new insights into a plethora of interdisciplinary projects, including large macromolecular machines, intrinsically disordered protein and dynamic regions within enzymes. For example: We will employ and develop new carbon-detected NMR applications to characterise the side-chains of large molecular machines, oncogenes and kinases, and protein folding intermediates in order to understand the underlying interactions, regulations and dynamics properties.

We believe that carbon-detected NMR spectroscopy is a very promising technique that, when new applications are coming up over the next decade, will open up for new exciting avenues due to the existence of slow-relaxing carbon coherences (sharp and intense signals) and the large dispersion of carbon chemical shifts (less overlap).

We strongly believe that the outcome of the proposed research, to be made possible with the acquisition of the new carbon-optimised cryogenic NMR probe, will be beneficial for both the academic sector (new applications, new protocols, new science), the private sector (commercial drug discovery, spectrometer manufactures), the public sector, and in general beneficial for enhancing the knowledge of society by attracting highly skilled people to the UK.

Some of the applicants, in particular DFH, JC and FLG, have parts of their research program aimed at employing and developing new applications for drug-discovery. This includes combining NMR with organic chemistry in a fragment-based approach (DFH), development of modulators of intrinsically disordered proteins (JC), and pushing the boundaries of allosteric kinase inhibitors by combining multi-scale molecular dynamics simulations and NMR spectroscopy (FLG). Thus, the drug-discoveries themselves can be used as lead and trail compounds by the pharmaceutical companies or as modulators in biochemical assays, however even more important is the methodologies and frameworks that are being established that could impact future drug-discovery strategies.

A major objective of the proposed research is to pave the way for new applications of direct carbon-detected NMR spectroscopy to probe the regulation, interactions, dynamics and structure of macromolecular machines and intrinsically disordered proteins. Since carbon-detected NMR is a relative new field within biomolecular NMR, we expect that these new developed protocols and frameworks will have a great impact on the NMR field. This impact could for example be that other researchers will start using our protocols and software and also that the manufactures (see support letter from Bruker) will tailor their new hardware to benefit our new applications. As mentioned above, new breakthroughs that impact a field often come as a consequence of simultaneous developments on many fronts - this could for example be the development of new NMR pulse sequences concomitantly with technological developments of the NMR hardware (new probe technology, new spectrometers, etc.).

Students and postdoctoral fellows will, as a part of their overall training, be trained in employing the new equipment for carbon-detected NMR spectroscopy and developing new applications. Thus, when these students and postdoctoral fellows leave UCL to continue their academic or professional careers they will have gained cutting-edge knowledge and expertise that they will be able to use and impact the research in their new environment. Several of our past students and postdoctoral fellows have now moved on to careers supported by prestigious grants and fellowships and using and developing NMR spectroscopy.

The scientific results and new methodologies will be made available by publications in internationally high-quality peer-reviewed journals and by presentations at national and international conferences. The software and protocols that will be developed during the project will be made available online, or via email as we have done previously. Moreover, and importantly, the experiences that we gain and knowledge that we acquire while using the new equipment will be shared with the manufacturer, such that they can potentially benefit from our applications and thereby tailor and optimise the next generation of NMR machines.