The Effect of Intrachain Electrostatic Repulsion on Conformational Disorder and Dynamics of the Sic1 Protein
The Effect of Intrachain Electrostatic Repulsion on Conformational Disorder and Dynamics of the Sic1 Protein
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DOI：
10.1021/jp500776v
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发表时间：
2014-04-17
影响因子：
3.3
通讯作者：
Gradinaru, Claudiu C.
中科院分区：
文献类型：
--
作者：
Liu, Baoxu;Chia, Darius;Gradinaru, Claudiu C.
酵母细胞周期蛋白依赖性激酶抑制剂Sic 1是一种无序蛋白，在多位点磷酸化后，与SCF泛素连接酶的Cdc 4亚基形成动态复合物。为了了解多位点磷酸化依赖的Sic 1：Cdc 4相互作用，这最终导致一个尖锐的细胞周期转变，无序的Sic 1 N-末端靶向区域的构象特性进行了研究，使用单分子荧光光谱。通过在非变性盐和离子变性剂中进行实验，确定了对电荷筛选具有不同灵敏度的多个构象群体。端到端的距离和流体动力学半径都随着盐浓度的增加而单调减小，并且观察到在高变性剂条件下链尺寸的翻转。将数据拟合到双链结合-筛选模型，得到诸如不带电链的排除体积和对变性剂的结合常数的参数。拟合数据需要类似于1.2的总体比例因子，这意味着Sic 1不能用随机高斯链近似。荧光相关光谱揭示了Sic 1结构的波动发生在快速（10-100 ns）和缓慢（类似于10 ms）的时间尺度上，与快速相缺乏低盐溶液。这项研究的结果提供了直接的证据，长程链内静电排斥是一个重要因素的构象景观的Sic 1，并支持静电的作用，在确定整体形状和流体动力学性质的内在无序的蛋白质。
The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants. Both the end-to-end distance and the hydrodynamic radius decrease monotonically with increasing the salt concentration, and a rollover of the chain dimensions in high denaturant conditions is observed. The data were fit to the polyelectrolyte binding-screening model, yielding parameters such as the excluded volume of the uncharged chain and the binding constant to denaturant. An overall scaling factor of similar to 1.2 was needed for fitting the data, which implies that Sic1 cannot be approximated by a random Gaussian chain. Fluorescence correlation spectroscopy reveals Sic1 structure fluctuations occurring on both fast (10-100 ns) and slow (similar to 10 ms) time scales, with the fast phase absent in low salt solutions. The results of this study provide direct evidence that long-range intrachain electrostatic repulsions are a significant factor for the conformational landscape of Sic1, and support the role of electrostatics in determining the overall shape and hydrodynamic properties of intrinsically disordered proteins.