Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations
Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations
复制标题
通过单分子力谱和计算机模拟对 NanoLuc 进行机械展开和重折叠
DOI：
10.1021/acs.biomac.2c00997
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发表时间：
2022
影响因子：
6.2
通讯作者：
Marszalek, Piotr E.
中科院分区：
文献类型：
--
作者：
Apostolidou, Dimitra;Zhang, Pan;Yang, Weitao;Marszalek, Piotr E.
纳米发光蛋白（NanoLuc, Nluc）是一种高度生物发光的蛋白，自诞生以来，在生物检测中得到了广泛应用。我们最近设计了一种NanoLuc多蛋白，它具有高生物发光性，但在机械展开后表现出强烈的错误折叠倾向。在此，我们利用原子力显微镜（AFM）进行了单分子力谱（SMFS）研究，并利用分子动力学（SMD）模拟了由Nluc和I91 titin结构域组成的两个新的杂交蛋白结构，即I91-I91-Nluc-I91-I91-I91 （I912-Nluc-I914）和I91-Nluc-I91-Nluc-I91-Nluc-I91 -Nluc-I91-Nluc-I91，以详细表征Nluc的展开行为，并进一步研究了我们之前在i912 - nluc3 - i912结构域中观察到的Nluc的错误折叠特性。我们的SMFS结果证实，Nluc的展开过程在所有结构中都相似；然而，Nluc的重折叠在这些结构中是不同的，当Nluc是单体或被I91结构域分开时，它的错误折叠是最小的。我们对单体Nluc、Nluc二偶体和Nluc三元体的模拟确定了其机械稳定性的起源，并捕获了有趣的展开中间体，这也是我们在实验中观察到的。
A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912construct. Our SMFS results confirm that Nluc’s unfolding proceeds similarly in all constructs; however, Nluc’s refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.