Garcia-Manyes Sergi, Brujić Jasna, Badilla Carmen L, Fernández Julio M
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Biophys J. 2007 Oct 1;93(7):2436-46. doi: 10.1529/biophysj.107.104422. Epub 2007 Jun 1.
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.
单蛋白力实验依赖于一种分子指纹，该指纹基于在多蛋白链中连接多个单蛋白结构域。然而，在解释力谱数据时，尤其是在蛋白质折叠过程中，这些结构域之间的相关性仍然是一个问题。在这里，我们首先表明力钳光谱是一种灵敏的技术，它基于四种单聚体蛋白的解折叠步长提供分子指纹。然后，我们测量了泛素和I27单体的力依赖解折叠速率动力学，发现在很宽的力范围内，与相应多蛋白获得的数据有很好的一致性，这支持了马尔可夫假设。此外，在单一力下有大量统计样本时，我们表明泛素单体也表现出解折叠时间的广泛分布，作为折叠蛋白构象中无序的特征。此外，我们很容易捕捉到单体的折叠轨迹，这些单体在折叠过程中表现出与多蛋白相同的阶段，从而消除了链中其他未折叠模块或结构域间相互作用导致熵掩盖的可能性。平均而言，达到I27折叠长度的时间随着淬灭力的增加而增加，其速率与多蛋白相似。单聚体水平的力钳光谱再现了使用多蛋白测量的解折叠和再折叠动力学，这证明在泛素和I27的情况下，将蛋白相互连接没有机械效应。
