Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy
Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy
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DOI：
10.1016/j.jsb.2007.01.018
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发表时间：
2007-08-01
影响因子：
3
通讯作者：
Aebi, Ueli
中科院分区：
文献类型：
--
作者：
Lim, Roderick Y. H.;Koeser, Joachim;Aebi, Ueli
苯丙氨酸-甘氨酸（FG）-重复核孔蛋白（Nups）是核孔复合物（NPC）中选择性门控机制的主要组成部分。因此，一个主要的要求是了解它们如何在防止被动扩散分子的进入和促进受体结合的货物易位到NPC之间摇摆不定。为了阐明这种行为，我们研究了hNup 153的半胱氨酸修饰的富含FG的C末端结构域的纳米机械性质（即，cNUP 153）及其与importin-beta的相互作用。这是使用单分子力光谱（SMFS）与原子力显微镜（AFM）进行的。在不存在β-输入蛋白的情况下，cNup 153是高度柔性的，并且可以可逆地拉伸和松弛而不改变其固有的熵弹性，这表明缺乏FG内相互作用，即，自然展开。Importin-beta-modified AFM tips揭示了与cNup 153的复杂结合拓扑结构，并为FG-受体相互作用中的结合混杂性提供了证据。这些差异表明FG结构域之间的协同性来自FG-受体相互作用而不是FG-FG相互作用。在一个技术说明，这项工作突出了一个改进的SMFS技术，其中涉及预钝化的底层衬底表面与聚乙二醇，以减少不良的AFM针尖表面效应。随后，通过在力-体积（FV）模式下实施SMFS测量，从目标分子的低表面覆盖度获得高产量的可接受数据。(C)2007年爱思唯尔公司All rights reserved.
Phenylalanine-glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-beta. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-beta, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-beta-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG-receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG-receptor interactions instead of FG-FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force-volume (FV) mode. (C) 2007 Elsevier Inc. All rights reserved.