Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura , Nishikyo-ku, Kyoto 615-8510, Japan.
Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho , Sakyo-ku, Kyoto 606-8501, Japan.
Anal Chem. 2017 Jul 18;89(14):7286-7290. doi: 10.1021/acs.analchem.7b01816. Epub 2017 Jul 5.
Shear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.
切应力可引起蛋白质的结构变形，从而导致聚集物的形成。流变核磁共振（Rheo-NMR）光谱学具有在原子水平下监测切应力下蛋白质结构变化的潜力；然而，现有的 Rheo-NMR 方法学对于探测蛋白质结构和动力学的灵敏度不足。在这里，我们提出了一种简单而通用的 Rheo-NMR 方法，该方法通过使用配备低温探头的光谱仪来最大限度地提高灵敏度。因此，该仪器的灵敏度在迄今为止报道的 Rheo-NMR 光谱仪中位居首位。我们证明，新开发的 Rheo-NMR 仪器可在切应力下获取蛋白质的高质量弛豫数据，并可实时跟踪蛋白质在纤维形成过程中的结构变化。所描述的方法将促进蛋白质结构变形的流变学研究，从而有助于从物理角度理解剪切诱导的淀粉样纤维形成。
