Confinement and Stabilization of Fyn SH3 Folding Intermediate Mimetics within the Cavity of the Chaperonin GroEL Demonstrated by Relaxation-Based NMR.
Confinement and Stabilization of Fyn SH3 Folding Intermediate Mimetics within the Cavity of the Chaperonin GroEL Demonstrated by Relaxation-Based NMR.
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DOI：
10.1021/acs.biochem.6b01237
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发表时间：
2017-02-21
期刊：
影响因子：
2.9
通讯作者：
Clore GM
中科院分区：
文献类型：
--
作者：
Libich DS;Tugarinov V;Ghirlando R;Clore GM
利用基于15N弛豫的核磁共振波谱（寿命谱线展宽、暗态交换饱和转移和弛豫色散）研究了模型蛋白底物Fyn SH3的两种折叠中间模拟物与超分子折叠酶/展开酶机器伴侣蛋白GroEL的相互作用。这两种模拟物包括野生型和三突变体（A39V/N53P/V55L） Fyn SH3的c端截断，其中SH3结构域的c端链展开，同时保留其余结构域结构。数据的定量分析表明，可以检测到SH3结构域在GroEL腔内的移动状态，可能是通过与无序的富含蛋氨酸的c端尾部相互作用，并且折叠中间模拟物的天然状态通过在载脂蛋白GroEL内的限制和与载脂蛋白GroEL的结合而稳定。这些数据为理解GroEL作为折叠酶/展开酶的被动活性提供了基础：在没有疏水GroEL结合一致序列的情况下，未展开状态在空腔内不稳定，因为与折叠中间体相比，其旋转半径更大，而折叠中间体相对于天然状态稳定，因为暴露于有利于GroEL结合的疏水斑块。
The interaction of two folding intermediate mimetics of the model protein substrate Fyn SH3 with the chaperonin GroEL, a supramolecular foldase/unfoldase machine, has been investigated by 15N relaxation-based nuclear magnetic resonance spectroscopy (lifetime line broadening, dark state exchange saturation transfer, and relaxation dispersion). The two mimetics comprise C-terminal truncations of wild-type and triple-mutant (A39V/N53P/V55L) Fyn SH3 in which the C-terminal strand of the SH3 domain is unfolded, while preserving the remaining domain structure. Quantitative analysis of the data reveals that a mobile state of the SH3 domain confined and tethered within the cavity of GroEL, possibly through interactions with the disordered, methionine-rich C-terminal tail(s), can be detected, and that the native state of the folding intermediate mimetics is stabilized by both confinement within and binding to apo GroEL. These data provide a basis for understanding the passive activity of GroEL as a foldase/unfoldase: the unfolded state, in the absence of hydrophobic GroEL-binding consensus sequences, is destabilized within the cavity because of its larger radius of gyration compared to that of the folding intermediate, while the folding intermediate is stabilized relative to the native state because of exposure of a hydrophobic patch that favors GroEL binding.