DMSO 淬火 H/D 交换 2D NMR 光谱学及其在蛋白质科学中的应用。

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.

机构信息

Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Exploratory Research Center on Life and Living Systems and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Aichi, Japan.

出版信息

Molecules. 2022 Jun 10;27(12):3748. doi: 10.3390/molecules27123748.

DOI:10.3390/molecules27123748

PMID:35744871

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9230524/

Abstract

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.

摘要

氢/氘（H/D）交换结合二维（2D）NMR 光谱已广泛用于研究蛋白质的结构、稳定性和动力学。当我们应用 H/D 交换方法研究蛋白质的非天然状态，如平衡和动力学折叠中间体时，H/D 交换猝灭技术是必不可少的，因为交换反应通常太快，无法通过 2D NMR 进行跟踪。在本文中，我们将描述二甲亚砜（DMSO）猝灭 H/D 交换方法及其在蛋白质科学中的应用。在该方法中，用非质子性的 DMSO 溶液取代 H/D 交换缓冲液，从而猝灭交换反应。我们通过使用脱盐柱改进了 DMSO 猝灭方法，该方法用于将 H/D 交换缓冲液中的介质交换到 DMSO 溶液中。这种改进使我们能够在高浓度盐或变性剂下监测蛋白质的 H/D 交换。我们描述了改进的 DMSO 猝灭方法的方法细节，并通过使用改进的方法对 6 M 盐酸胍中展开的人泛素的 H/D 交换行为进行了案例研究。