EARLY HYDROGEN-BONDING EVENTS IN THE FOLDING REACTION OF UBIQUITIN
EARLY HYDROGEN-BONDING EVENTS IN THE FOLDING REACTION OF UBIQUITIN
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DOI：
10.1073/pnas.89.6.2017
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发表时间：
1992-03-15
影响因子：
11.1
通讯作者：
RODER, H
中科院分区：
文献类型：
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作者：
BRIGGS, MS;RODER, H
泛素是一种小的细胞质蛋白，具有延伸的β -薄片和围绕着明显疏水核心的α -螺旋结构，在折叠反应中氢键结构的形成已经通过氢-氘交换标记结合快速混合方法和二维核磁共振分析进行了研究。测定了26个主酰胺质子在自然条件下可形成稳定的氢键并进行缓慢的交换。β -sheet和α -helix中的酰胺质子，以及参与螺旋/sheet界面氢键的质子，在最初的8-ms折叠阶段得到80%的保护，表明这两个二级结构元素在一个共同的协同折叠事件中形成和结合。残基59、61和69的保护率稍慢，为表面环的后续稳定提供了证据。大多数探针还表现出两个时间常数约为100 ms和10 s的小相位。在观察到的残基中，只有Gln-41和Arg-42表现出明显的慢折叠相，幅度分别为37%和22%，这可能是由于含有Pro-37和/或Pro-38的顺式肽键的天然折叠中间体。与脉冲标记研究的细胞色素c、核糖核酸酶、藤蔓酶等其他蛋白质相比，泛素中氢键结构的初始形成速度更快，慢折叠物种不太突出。
The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 backbone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.