Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin
Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin
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DOI：
10.1529/biophysj.107.104422
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发表时间：
2007-10-01
影响因子：
3.4
通讯作者：
Fernandez, Julio M.
中科院分区：
文献类型：
--
作者：
Garcia-Manyes, Sergi;Brujic, Jasna;Fernandez, Julio M.
单蛋白力实验依赖于基于在多蛋白链中拴系多个单蛋白结构域的分子指纹。然而，这些领域之间的相关性仍然是一个问题，在解释力谱数据，特别是在蛋白质折叠。在这里，我们首先表明，力钳光谱是一种敏感的技术，提供了一个分子指纹的基础上展开步长的四个单单体蛋白质。然后，我们测量的力依赖性的展开速率动力学的泛素和I27单体，并找到一个很好的协议，获得的数据为各自的多聚蛋白在广泛的力量，支持马尔可夫假说。此外，与一个大的统计系综在一个单一的力量，我们发现，泛素单体也表现出广泛的分布的展开时间作为一个签名的混乱折叠蛋白景观。此外，我们很容易捕获的折叠轨迹的单体，表现出相同的阶段，在折叠观察到的多蛋白质，从而消除了熵掩蔽的可能性，由其他未折叠的模块在链或结构域-结构域的相互作用。平均而言，达到I27折叠长度的时间随着淬灭力的增加以与多聚蛋白相似的速率增加。在单单体水平的力钳光谱再现使用多蛋白测量的展开和重折叠的动力学，这证明了在泛素和I27的情况下，没有将蛋白质彼此拴系的机械效应。
Single-protein force experiments have relied on a molecular fingerprint based on tethering multiple single-protein domains in a polyprotein chain. However, correlations between these domains remain an issue in interpreting force spectroscopy data, particularly during protein folding. Here we first show that force-clamp spectroscopy is a sensitive technique that provides a molecular fingerprint based on the unfolding step size of four single-monomer proteins. We then measure the force-dependent unfolding rate kinetics of ubiquitin and I27 monomers and find a good agreement with the data obtained for the respective polyproteins over a wide range of forces, in support of the Markovian hypothesis. Moreover, with a large statistical ensemble at a single force, we show that ubiquitin monomers also exhibit a broad distribution of unfolding times as a signature of disorder in the folded protein landscape. Furthermore, we readily capture the folding trajectories of monomers that exhibit the same stages in folding observed for polyproteins, thus eliminating the possibility of entropic masking by other unfolded modules in the chain or domain-domain interactions. On average, the time to reach the I27 folded length increases with increasing quenching force at a rate similar to that of the polyproteins. Force-clamp spectroscopy at the single-monomer level reproduces the kinetics of unfolding and refolding measured using polyproteins, which proves that there is no mechanical effect of tethering proteins to one another in the case of ubiquitin and I27.