Structural Origins of FRET-Observed Nascent Chain Compaction on the Ribosome.
Structural Origins of FRET-Observed Nascent Chain Compaction on the Ribosome.
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FRET 观察到的核糖体新生链压缩的结构起源。
DOI：
10.1021/acs.jpcb.8b07726
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
O'Brien,EdwardP
中科院分区：
文献类型：
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作者：
Nissley,DanielA;O'Brien,EdwardP
由Fo Prster共振能量转移过程产生的荧光信号用于监测E.大肠杆菌蛋白HemK在其合成过程中的核糖体。在翻译开始后开始10秒观察到荧光增加，表明结构域的尺寸变得更紧凑。由于荧光在每个时间点只报告一个值，它包含的信息很少的结构合奏，使其产生。在这里，我们补充了粗粒度的模拟，描述蛋白质的构象和转换的空间分辨率为3.8 μ m的实验信息。我们使用这些模拟来测试可能解释域压缩的原因的三个假设：（1）不良溶剂质量条件驱动未折叠状态压缩，（2）域在移出出口隧道时占据的空间的尺寸的变化导致压缩，或（3）域折叠导致压缩。我们发现，域折叠和尺寸崩溃都与实验数据相一致，而不良溶剂崩溃是不一致的。我们确定了替代染料标记的位置HemK，荧光后，可以区分结构域折叠和空间塌陷机制。在C-末端截短形式的蛋白质中已经观察到结构域的部分折叠。因此，很可能实验观察到的紧凑状态是一个部分折叠的中间体组成，根据我们的模拟，前三个螺旋的HemK N-末端结构域采用天然的，三级配置。通过这些模拟，我们还确定了HemK可能的共翻译折叠途径。
A fluorescence signal arising from a Förster resonance energy transfer process was used to monitor conformational changes of a domain within theE. coliprotein HemK during its synthesis by the ribosome. An increase in fluorescence was observed to begin 10 s after translation was initiated, indicating the domain became more compact in size. Since fluorescence only reports a single value at each time point it contains very little information about the structural ensemble that gives rise to it. Here, we supplement this experimental information with coarse-grained simulations that describe protein conformations and transitions at a spatial resolution of 3.8 Å. We use these simulations to test three hypotheses that might explain the cause of domain compaction: (1) that poor solvent quality conditions drive the unfolded state to compact, (2) that a change in the dimension of the space the domain occupies upon moving outside the exit tunnel causes compaction, or (3) that domain folding causes compaction. We find that domain folding and dimensional collapse are both consistent with the experimental data, while poor-solvent collapse is inconsistent. We identify alternative dye labeling positions on HemK that upon fluorescence can differentiate between the domain folding and dimensional collapse mechanisms. Partial folding of domains has been observed in C-terminally truncated forms of proteins. Therefore, it is likely that the experimentally observed compact state is a partially folded intermediate consisting, according to our simulations, of the first three helices of the HemK N-terminal domain adopting a native, tertiary configuration. With these simulations we also identify the possible cotranslational folding pathways of HemK.