Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores
Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores
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DOI：
10.1021/ac3035025
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发表时间：
2013-02-19
影响因子：
7.4
通讯作者：
Edel, Joshua B.
中科院分区：
文献类型：
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作者：
Japrung, Deanpen;Dogan, Jakob;Edel, Joshua B.
部分或完全无序的蛋白质是信号转导途径的工具，然而，这些蛋白质的许多机制方面还没有很好地理解。例如，沿着结合途径的中间状态的数量和性质仍然是一个激烈争论的话题。为了揭示无序蛋白质结构域及其复合物的构象异质性，我们通过将无序蛋白质通过嵌入薄介电膜内的纳米孔进行单分子实验。该平台允许在不需要荧光标记或其他修饰基团的情况下生成单分子统计。这些研究是在两种不同的内在无序蛋白质结构域上进行的，一种是来自甲状腺激素和类维生素A受体（ACTR）的激活剂的结合结构域，另一种是CREB结合蛋白（NCBD）的核辅激活剂结合结构域，沿着它们的双分子复合物。我们的研究结果表明，ACTR和NCBD填充不同的构象易位后通过纳米孔。另一方面，两个无序结构域的折叠复合物作为一种构象移位。令人惊讶的是，我们发现NCBD在高盐浓度下发生电荷反转。这通过易位统计以及通过测量C-电位来验证。静电相互作用以前已经被认为在本质上无序的蛋白质的缔合中起着关键作用，并且观察到的行为进一步增加了它们的结合反应的复杂性。
Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the C-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.