Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix
Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix
复制标题
具有非天然 α-螺旋的 β-乳球蛋白初始折叠中间体的高度塌陷构象
DOI：
10.1016/j.jmb.2015.07.018
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发表时间：
2015
影响因子：
5.6
通讯作者：
Satoshi Takahashi
中科院分区：
文献类型：
--
作者：
Tsuyoshi Konuma;Kazumasa Sakurai;Masanori Yagi;Yuji Goto;Teturo Fujisawa;Satoshi Takahashi
在β-乳球蛋白（β-lactoglobulin，βLG）的折叠过程中，一种主要由β折叠蛋白组成的蛋白质，在几毫秒内形成一种含有过量非天然α-螺旋的瞬时中间体。为了表征βLG的早期折叠动力学的二级结构内容和紧凑性，我们进行了亚毫秒分辨圆二色性（CD）和小角X射线散射（SAXS）测量。尿素快速稀释后的时间分辨CD显示在200 μs内形成非天然α-螺旋。时间分辨小角X射线散射（SAXS）显示，在300 μs时，中间体的回转半径（Rg）为23.3 ± 0.7 μ m，表明它从Rg为35.1 ± 7.1 μ m的未折叠状态发生了相当大的塌陷。在28 ± 11 ms的时间常数下，进一步压缩到21.2 ± 0.3 μ m。对分布函数表明，在300 μs时，中间体由一个单一的坍缩畴和一个小的起伏畴组成，第二次坍缩后，起伏畴变得更加紧凑。在2，2，2-三氟乙醇存在下的动力学测量表明，在几毫秒内，中间体具有增加的α-螺旋量，但相似的Rg为23.0 ± 0.8 Ω，表明中间体在不同溶剂中的形状相似。因此，初始塌缩发生全局的紧凑状态与一个小的波动域无关的非天然α-螺旋的内容。波动结构域的第二次塌陷发生与链A周围的非天然螺旋的稳定化一致。链A周围的非天然螺旋可能有助于形成βLG折叠所需的长程接触。
In the folding of β-lactoglobulin (βLG), a predominantly β-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of βLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200 μs. Time-resolved SAXS showed that the radius of gyration (Rg) of the intermediate at 300 μs was 23.3 ± 0.7 Å, indicating a considerable collapse from the unfolded state havingRgof 35.1 ± 7.1 Å. Further compaction toRgof 21.2 ± 0.3 Å occurred with a time constant of 28 ± 11 ms. Pair distribution functions showed that the intermediate at 300 μs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similarRgof 23.0 ± 0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of βLG.