Quadruple‐Resonance Magic‐Angle Spinning NMR Spectroscopy of Deuterated Solid Proteins |
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Authors: | Dr Ümit Akbey Dr Andrew J Nieuwkoop Dr Sebastian Wegner Anja Voreck Dr Britta Kunert Dr Priyanga Bandara Dr Frank Engelke Prof?Dr Niels Chr Nielsen Prof?Dr Hartmut Oschkinat |
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Institution: | 1. Leibniz Institute for Molecular Pharmacology, Robert Roessle Str. 10, 13125 Berlin (Germany);2. Bruker BioSpin GmbH, Silberstreifen 4, 76287 Rheinstetten (Germany);3. Institute de Biologie et Chemie des Protéines, UMR 5086, CNRS/Université de Lyon?1, 7 passage du Vercors, 69367 Lyon (France);4. Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNano) and Department of Chemistry, Aarhus University (Denmark) |
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Abstract: | 1H‐detected magic‐angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back‐exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using 2H excitation instead of 1H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, “quadruple‐resonance NMR spectroscopy”, is presented which relies on an efficient 2H‐excitation and 2H‐13C cross‐polarization (CP) step, combined with 1H detection. We show that by using 2H‐excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties. |
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Keywords: | deuteration high sensitivity proteins quadruple‐resonance MAS NMR spectroscopy structure elucidation |
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