Microsecond Folding and Domain Motions of a Spider Silk Protein Structural Switch
Microsecond Folding and Domain Motions of a Spider Silk Protein Structural Switch
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DOI：
10.1021/ja508760a
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发表时间：
2014-12-20
影响因子：
15
通讯作者：
Neuweiler, Hannes
中科院分区：
文献类型：
--
作者：
Ries, Julia;Schwarze, Simone;Neuweiler, Hannes
网蜘蛛迅速地将蛋白质单体（即所谓的蛛丝）组装成异常坚韧的丝纤维。该过程涉及ph触发的蜘蛛蛋白n端结构域（NTD）的自结合，其中包含连接蜘蛛蛋白和超分子的结构开关。单分子光谱可以探测到传统方法无法探测到的构象非均质性，但NTD的运动超出了分辨率限制。在这里，我们设计了基于光诱导电子转移到欧洲南蛛（euprothenops australis）蜘蛛的分离NTD的荧光猝灭现象的1 nm构象变化探针。单分子荧光波动的相关分析揭示了二级和三级结构的位置依赖于纳秒到微秒的运动。动力学振幅是最明显的螺旋是结合界面的一部分，而结构研究表明，大位移之间的单体和二聚体构象。位于五螺旋束中心的单个色氨酸以类似于100 μ s的方式以ph依赖的方式切换构象。平衡变性和温度跳变弛豫实验表明，仅在60 μ s内就可以实现协同折叠和超快折叠。我们推导出一种具有天然状态坚固性的自由能表面，其势垒高度与折叠和天然运动明显相似。观察到的区域内的平衡动力学表明，在蜘蛛合成丝过程中，蜘蛛通过其NTDs快速结合存在构象选择机制。
Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to supermolecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1 nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nanosecond-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in similar to 100 mu s and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 mu s. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.