Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR
Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR
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氢/氘交换 2D NMR 显示未折叠泛素的残留结构
DOI：
10.1016/j.bpj.2020.10.003
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发表时间：
2020
影响因子：
3.4
通讯作者：
Kuwajima Kunihiro
中科院分区：
文献类型：
--
作者：
Yagi-Utsumi Maho;Chandak Mahesh S.;Yanaka Saeko;Hiranyakorn Methanee;Nakamura Takashi;Kato Koichi;Kuwajima Kunihiro
未折叠蛋白质在浓变性溶液中残留结构的表征是目前蛋白质折叠研究中的一个重要问题，因为处于未折叠状态的残基结构可能形成折叠起始点并指导后续的折叠反应。在这里，我们研究了未折叠的人泛素在6M氯化胍中的氢/氢(H/D)交换行为。我们使用了二甲基亚砜(DMSO)猝灭的H/D交换核磁共振技术和旋转脱盐柱，这使得我们能够快速地从6M氯化胍到猝灭的DMSO溶液进行介质交换。基于泛素在DMSO溶液中的主链共振归属，我们成功地研究了泛素序列中60个肽酰胺基团的H/D交换动力学。虽然大多数酰胺基团没有受到保护，但在中间螺旋(残基23-34)和N-末端β-发夹(残基2-16)中的某些酰胺基团得到了显著的保护，保护系数为2.1-4.2，表明未折叠的泛素中存在残基结构，这些酰胺基团在残基结构中有52%以上的氢键。我们发现，即使在6M的氯化胍中，α-螺旋和β-发夹中也形成了氢键残基结构，这表明这些残基结构可能作为折叠起始位置来指导后续的泛素折叠反应。
The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23–34) and an N-terminalβ-hairpin (residues 2–16) were significantly protected with a protection factor of 2.1–4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in theα-helix and theβ-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.
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