Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling
- Journal Article
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-[C-13,H-1]-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. Show more
Journal / seriesAngewandte Chemie. International Edition
Pages / Article No.
Subjectdelayed decoupling; isotopic labeling; NMR spectroscopy; Proteins; XL-ALSOFAST
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