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

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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.