Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein.
Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein.
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DOI：
10.1038/ncomms7861
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发表时间：
2015-04-17
影响因子：
16.6
通讯作者：
Marqusee S
中科院分区：
文献类型：
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作者：
Guinn EJ;Jagannathan B;Marqusee S
蛋白质折叠的一个基本问题是蛋白质是通过一个还是多个轨迹折叠。虽然大多数实验表明一个单一的途径，模拟表明蛋白质可以通过许多平行的途径折叠。在这里，我们使用化学变性剂，机械力和定点突变的组合，以证明在一个简单的，两个状态的折叠蛋白质的存在下，多个展开途径。我们发现，这些多个途径具有结构上不同的过渡态，并且蛋白质序列和环境中看似微小的变化可以强烈地调节途径之间的通量。这些结果表明，在体内，拥挤的细胞环境可以强烈影响蛋白质折叠和去折叠的机制。我们的研究解决了实验和理论研究之间明显的二分法，并强调了使用多管齐下的方法来揭示蛋白质自由能景观的复杂性的优势。 虽然大多数蛋白质折叠实验可以用单一途径来解释，但理论证据表明存在多个途径。在这里，作者使用力，化学变性和诱变的组合来调节平行途径之间的通量来解决这个问题。
A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape. Although most protein folding experiments can be explained by a single pathway, theoretical evidence suggests the presence of multiple pathways. Here, the authors resolve this using a combination of force, chemical denaturation and mutagenesis to modulate the flux between parallel pathways.