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Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

  • Rößler P Institute of Molecular Biology and Biophysics, Department of Biology and Biomolecular NMR Spectroscopy Platform, Department of Biology, ETH Zürich, Otto-Stern-Weg 5, 8093, Zürich, Switzerland.
  • Mathieu D Bruker BioSpin GmbH, Silberstreifen 4, 76287, Rheinstetten, Germany.
  • Gossert AD Institute of Molecular Biology and Biophysics, Department of Biology and Biomolecular NMR Spectroscopy Platform, Department of Biology, ETH Zürich, Otto-Stern-Weg 5, 8093, Zürich, Switzerland.
  • 2020-08-04
Published in:
  • Angewandte Chemie (International ed. in English). - 2020
NMR spectroscopy
XL-ALSOFAST
delayed decoupling
isotopic labeling
proteins
English Current biological research increasingly focusses on large human proteins and their complexes. Such proteins are difficult to study by NMR spectroscopy because they often can only be produced in higher eukaryotic expression systems, where deuteration is hardly feasible. Here, we present the XL-ALSOFAST-[13 C,1 H]-HMQC experiment with much improved sensitivity for fully protonated high molecular weight proteins. For the tested systems ranging from 100 to 240 kDa in size, 3-fold higher sensitivity was obtained on average for fast relaxing signals compared to current state-of-the-art experiments. In the XL-ALSOFAST approach, non-observed magnetisation is optimally exploited and transverse relaxation is minimized by the newly introduced concept of delayed decoupling. The combination of high sensitivity and superior artefact suppression makes it ideal for studying inherently unstable membrane proteins or for analysing therapeutic antibodies at natural 13 C abundance. The XL-ALSOFAST and delayed decoupling will therefore expand the range of biomolecular systems accessible to NMR spectroscopy.
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  • English
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hybrid
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https://sonar.rero.ch/global/documents/113763
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