Amide protection in an early folding intermediate of cytochrome c.
Amide protection in an early folding intermediate of cytochrome c.
复制标题
细胞色素 c 早期折叠中间体中的酰胺保护。
DOI：
10.1016/s1359-0278(98)00040-6
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Roder,H
中科院分区：
文献类型：
--
作者：
Sauder,JM;Roder,H
背景：对于许多蛋白质来说，在天然结构形成的限速步骤之前很久就出现了紧密状态。一个关键的问题是，无论是随机的或更具体的疏水interactions.Results：氢交换标记结合NMR的链的初始崩溃被用来监测形成稳定的氢键和溶剂排除结构在马细胞色素（cytc）。使用氢交换/折叠竞争方案在可变pH和短竞争时间（2 ms）下测量保护。在cytc的所有三个α螺旋中观察到3倍至8倍的保护因子，而其他区域没有显示出显著的保护作用。这表明存在的致密态包含边缘稳定的氢键结构的片段。当中间体不稳定时，只有来自Cys 14、Ala 15和His 18的酰胺质子表现出显着的保护作用，这表明未折叠蛋白质中共价结合的血红素基团附近存在持久的残留结构区域。荧光检测的停流研究表明，在早期中间体的最大保护因子是一致的，其展开平衡constant.Conclusions：连同以前的荧光和CD结果，所观察到的酰胺保护模式是一致的，与早期形成的α-螺旋核心结构域在一个合奏的紧凑状态，表明有效的折叠是促进逐步收购的天然结构元素。这些特定的早期相互作用建立在亚毫秒的时间尺度上，在折叠的限速步骤之前。
Background:For many proteins, compact states appear long before the rate-limiting step in the formation of the native structure. A key issue is whether the initial collapse of the chain is driven by random or more specific hydrophobic interactions.Results:Hydrogen-exchange labeling coupled with NMR was used to monitor the formation of stable hydrogen-bonded and solvent-excluded structure in horse cytochromec(cytc). Protection was measured using a hydrogen exchange/folding competition protocol at variable pH and short competition time (2 ms). Protection factors of threefold to eightfold were observed in all threeαhelices of cytc, whereas other regions showed no significant protection. This suggests that the compact states that are present contain segments of marginally stable hydrogen-bonded structure. When the intermediate(s) are destabilized, only amide protons from Cys14, Ala15 and His18 show significant protection, indicating a region of persistent residual structure near the covalently bound heme group in the unfolded protein. Fluorescence-detected stopped-flow studies showed that the maximum protection factor in the early intermediate is consistent with its unfolding equilibrium constant.Conclusions:Together with previous fluorescence and CD results, the observed pattern of amide protection is consistent with the early formation of anα-helical core domain in an ensemble of compact states, indicating that efficient folding is facilitated by stepwise acquisition of native structural elements. These specific early interactions are established on the sub-millisecond time scale, prior to the rate-limiting step for folding.