Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA; Biophysics and Chemistry Department, The University of Michigan, Ann Arbor, MI 48109-1055, USA; Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA; Biomedical Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA.
Biochim Biophys Acta Biomembr. 2020 Sep 1;1862(9):183332. doi: 10.1016/j.bbamem.2020.183332. Epub 2020 May 1.
Lipid-nanodiscs have been shown to be an exciting innovation as a membrane-mimicking system for studies on membrane proteins by a variety of biophysical techniques, including NMR spectroscopy. Although NMR spectroscopy is unique in enabling the atomic-resolution investigation of dynamic structures of membrane-associated molecules, it, unfortunately, suffers from intrinsically low sensitivity. The long data acquisition often used to enhance the sensitivity is not desirable for sensitive membrane proteins. Instead, paramagnetic relaxation enhancement (PRE) has been used to reduce NMR data acquisition time or to reduce the amount of sample required to acquire an NMR spectra. However, the PRE approach involves the introduction of external paramagnetic probes in the system, which can induce undesired changes in the sample and on the observed NMR spectra. For example, the addition of paramagnetic ions, as frequently used, can denature the protein via direct interaction and also through sample heating. In this study, we show how the introduction of paramagnetic tags on the outer belt of polymer-nanodiscs can be used to speed-up data acquisition by significantly reducing the spin-lattice relaxation (T) times with minimum-to-no alteration of the spectral quality. Our results also demonstrate the feasibility of using different types of paramagnetic ions (Eu, Gd, Dy, Er, Yb) for NMR studies on lipid-nanodiscs. Experimental results characterizing the formation of lipid-nanodiscs by the metal-chelated polymer, and their increased tolerance toward metal ions are also reported.
脂质纳米盘已被证明是一种令人兴奋的创新，作为一种模拟膜系统的方法，可用于多种生物物理技术研究，包括 NMR 光谱学。尽管 NMR 光谱学在原子分辨率研究膜相关分子的动态结构方面具有独特的优势，但不幸的是，它的灵敏度本质上较低。为了提高灵敏度，通常使用长时间的数据采集，但这对于敏感的膜蛋白来说并不理想。相反，顺磁弛豫增强（PRE）已被用于减少 NMR 数据采集时间或减少获取 NMR 光谱所需的样品量。然而，PRE 方法涉及在系统中引入外部顺磁探针，这可能会导致样品和观察到的 NMR 光谱发生不期望的变化。例如，如经常使用的添加顺磁离子，可以通过直接相互作用和样品加热使蛋白质变性。在这项研究中，我们展示了如何在外聚合物纳米盘的外带上引入顺磁标签，通过显著减少自旋晶格弛豫（T）时间来加速数据采集，同时最小化对光谱质量的影响。我们的结果还证明了使用不同类型的顺磁离子（Eu、Gd、Dy、Er、Yb）进行脂质纳米盘 NMR 研究的可行性。还报告了表征金属螯合聚合物形成脂质纳米盘的实验结果及其对金属离子的更高耐受性。
