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.
Bruker BioSpin GmbH, Silberstreifen 4, 76287, Rheinstetten, Germany.
Angew Chem Int Ed Engl. 2020 Oct 19;59(43):19329-19337. doi: 10.1002/anie.202007715. Epub 2020 Aug 26.
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, 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 C abundance. The XL-ALSOFAST and delayed decoupling will therefore expand the range of biomolecular systems accessible to NMR spectroscopy.
目前的生物研究越来越关注大型人类蛋白质及其复合物。由于这些蛋白质通常只能在高等真核表达系统中产生，而在该系统中氘化几乎是不可行的，因此用 NMR 光谱法研究此类蛋白质具有一定难度。本文提出了 XL-ALSOFAST-[C, H]-HMQC 实验，该实验对完全质子化的高分子量蛋白质具有更高的灵敏度。在我们所测试的大小在 100 到 240 kDa 之间的系统中，与当前最先进的实验相比，快速弛豫信号的灵敏度平均提高了 3 倍。在 XL-ALSOFAST 方法中，通过新引入的延迟去耦概念，最优地利用了未观察到的磁化强度并最小化了横向弛豫。高灵敏度和出色的伪影抑制相结合，使其非常适合研究固有不稳定的膜蛋白或在天然 13C 丰度下分析治疗性抗体。因此，XL-ALSOFAST 和延迟去耦将扩大可用于 NMR 光谱学的生物分子系统的范围。
