Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations
Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations
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DOI：
10.1073/pnas.2015728118
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发表时间：
2021-03-23
影响因子：
11.1
通讯作者：
Perkins, Thomas T.
中科院分区：
文献类型：
--
作者：
Edwards, Devin T.;Leblanc, Marc-Andre;Perkins, Thomas T.
单分子力谱是研究蛋白质折叠的有力工具。在过去的十年中，一个关键的问题已经出现：如何改变内在的生物分子动力学的变化，通过灵活的连接器连接到微米级的力探针改变？在这里，我们研究了折叠/展开的α D-3使用原子力显微镜（AFM）为基础的力谱。α D-3提供了一个不寻常的机会，因为先前的单分子荧光共振能量转移（smFRET）研究表明，在Kramers理论的背景下，α D-3的构型扩散常数随pH值而变化。由此产生的pH依赖性为基于AFM的力光谱跟踪蛋白质折叠动力学内在变化的能力提供了测试。然而，在实验上，阿尔法D-3是具有挑战性的。在低强度下展开（
Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to mu m-scale force probes via flexible linkers? Here, we studied the folding/unfolding of alpha D-3 using atomic force microscopy (AFM)-based force spectroscopy. alpha D-3 offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed alpha D-3's configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy's ability to track intrinsic changes in protein folding dynamics. Experimentally, however, alpha D-3 is challenging. It unfolds at low force (