Enabling ultra-high resolution hydrogen/deuterium exchange for challenging biomedical systems

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Whilst protein structure and conformation have been traditionally accomplished by traditional X-ray crystallography, NMR, and Cryo-EM, these structural techniques are often extremely labour intensive and have significant technical limitations for many challenging protein systems. Hydrogen/deuterium exchange (HDX) has emerged as a complement, valuable and rapid technique for probing protein structure, functional flexibility, conformational variability, allosteric changes, as well as for monitoring and characterising protein interactions with other macromolecules, small metabolites and drugs. Moreover, HDX applications also have focused on monitoring protein stability in response of post-translational modifications and covalent ligand/drug binding as well as protein engineering. Despite the significant progress in HDX method developments, all current HDX techniques are limited by either the system size and complexity or resolution, with which HDX data can be obtained. As a result, the traditional high-resolution (HR) HDX techniques have only restricted (if any) applicability for many challenging biological systems, such as multidomain proteins, transient and multicomponent protein-protein complexes, large intrinsically disordered proteins/regions, and membrane proteins. We propose to develop a time- and cost-efficient HDX 3D NMR approach, applicable for the high-resolution characterisation of large challenging proteins. The proposed project will significantly enhance the current HDX capabilities, improving its effectiveness, efficiency and applicability to the biological and biomedical systems, currently inaccessible via the other HR HDX techniques. It will allow the collection and analysis of high resolution HDX data, obtained with the use of physiological protein concentrations (as low as sub-uM) and physiological buffers (without limitations on salt concentration and buffer composition). We will performed read-out 3D NMR experiments in aprotic organic solvent DMSO that preserves HDX patterns obtained under physiological conditions, but drastically improves the quality of NMR spectra by unfolding the protein of interest. We will exploit the most recent developments in ultra-resolution fast multidimensional NMR to allow time-optimised, semi-automatic data acquisitions, NMR assignments and analysis of HDX read-out 3D NMR spectra. We will vigorously test and optimise the proposed approach using several model systems, which represent the most common types of challenging protein systems, including multidomain proteins, intrinsically disordered proteins, transient protein complexes, and membrane proteins. Next, we will examine the accuracy and applicability of the HDX 3D NMR approach for drug developments, particularly, for monitoring conformational perturbations induced by small molecule binding. As model systems we will use molecular chaperones BIP and Hsp90 that undergo subtle, but functionally important conformational changes upon binding to small molecule inhibitors. This project will provide a long-awaited high-resolution method for structural and dynamic characterisation of challenging and complex biological systems in the near-native context. In a long term, it will provide new opportunities for elucidation of molecular mechanisms of action for large, multicomponent biological machines and facilitate future drug developments for such systems.