Jorge Christine, Marques Bryan S, Valentine Kathleen G, Wand A Joshua
Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Methods Enzymol. 2019;615:77-101. doi: 10.1016/bs.mie.2018.09.040. Epub 2018 Dec 4.
Protein hydration is a critical aspect of protein stability, folding, and function and yet remains difficult to characterize experimentally. Solution NMR offers a route to a site-resolved view of the dynamics of protein-water interactions through the nuclear Overhauser effects between hydration water and the protein in the laboratory (NOE) and rotating (ROE) frames of reference. However, several artifacts and limitations including contaminating contributions from bulk water potentially plague this general approach and the corruption of measured NOEs and ROEs by hydrogen exchange-relayed magnetization. Fortunately, encapsulation of single protein molecules within the water core of a reverse micelle overcomes these limitations. The main advantages are the suppression hydrogen exchange and elimination of bulk water. Here we detail guidelines for the preparation solutions of encapsulated proteins that are suitable for characterization by NOE and ROE spectroscopy. Emphasis is placed on understanding the contribution of detected NOE intensity arising from magnetization relayed by hydrogen exchange. Various aspects of fitting obtained NOE, selectively decoupled NOE, and ROE time courses are illustrated.
蛋白质水合作用是蛋白质稳定性、折叠和功能的关键方面，但仍难以通过实验进行表征。溶液核磁共振（NMR）提供了一条途径，通过水合水与蛋白质在实验室（NOE）和旋转（ROE）参考系中的核Overhauser效应，实现对蛋白质-水相互作用动力学的位点分辨观察。然而，包括大量水的污染贡献在内的一些假象和局限性可能困扰这种通用方法，以及氢交换中继磁化对测量的NOE和ROE的破坏。幸运的是，将单个蛋白质分子封装在反胶束的水核中克服了这些局限性。主要优点是抑制氢交换和消除大量水。在这里，我们详细介绍了适用于通过NOE和ROE光谱表征的封装蛋白质溶液的制备指南。重点在于理解由氢交换中继的磁化所产生的检测到的NOE强度的贡献。说明了拟合获得的NOE、选择性去耦NOE和ROE时间进程的各个方面。
