Development of structural biology methods for solid-state NMR utilizing sensitivity enhancement via dynamic nuclear polarization (DNP) and their application to drug-binding of membrane proteins

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Solid state NMR spectroscopy (ssNMR) is a powerful tool of structural biology. In contrast to crystallography it does not require molecular long-range order. This facilitates sample preparation and allows for more physiological sample conditions. SsNMR finds an application to structural studies of membrane proteins and their interaction with drugs.One membrane protein of major interest is M2 of influenza A. It has been the target of the antiviral drugs rimantadine and amantadine. Due to mutations of M2 these drugs lost their efficacy completely. To facilitate the development of alternative drugs major efforts have been invested to unravel the structural details of the M2-drug interaction. Unfortunately the contradictory data does not lead to a conclusive picture. One of the reasons seem to be structural artefacts introduced by the used membrane mimetics.SsNMR allows for structure determination of membrane proteins in native and physiological lipid bilayers; while the intrinsically low sensitivity of NMR requires large sample amounts, expensive isotope labelling and long measurement times.Dynamic nuclear polarization (DNP) can be used to transfer the large electronic polarization of stable radicals to nuclear spins to amplify the NMR signal by two orders of magnitude. Depending on the sample under investigation the resonance line width under cryogenic DNP conditions can be increased, possibly complicating resonance assignment.Novel methods for sensitivity and resolution enhancement shall be developed and implemented in DNP spectrometers of up to 800 MHz proton frequency.These techniques shall allow for the development of novel high-dimensional NMR spectra to facilitate resonance assignment and internuclear distance determination and will find application in the structure determination of M2 in complex with its drugs.