State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, 21002, China.
Sci Rep. 2019 Jul 19;9(1):10518. doi: 10.1038/s41598-019-47004-y.
Protein (un)folding is a complex and essential process. With the rapid development of single-molecule techniques, we can detect multiple and transient proteins (un)folding pathways/intermediates. However, the observation of multiple multistep (>2) unfolding scenarios for a single protein domain remains limited. Here, we chose metalloprotein with relatively stable and multiple metal-ligand coordination bonds as a system for such a purpose. Using AFM-based single-molecule force spectroscopy (SMFS), we successfully demonstrated the complex and multistep protein unfolding scenarios of the β-domain of a human protein metallothionein-3 (MT). MT is a protein of ~60 amino acids (aa) in length with 20 cysteines for various metal binding, and the β-domain (βMT) is of ~30 aa with an MS metal cluster. We detected four different types of three-step protein unfolding scenarios from the Cd-βMT, which can be possibly explained by the rupture of Cd-S bonds in the complex CdS metal cluster. In addition, complex unfolding scenarios with four rupture peaks were observed. The Cd-S bonds ruptured in both single bond and multiple bonds modes. Our results provide not only evidence for multistep protein unfolding phenomena but also reveal unique properties of metalloprotein system using single-molecule AFM.
蛋白质的折叠和解折叠是一个复杂而重要的过程。随着单分子技术的快速发展，我们可以检测到多个瞬时的蛋白质折叠和解折叠途径/中间体。然而，对于单个蛋白质结构域，观察到多个多步骤（>2）的折叠情况仍然有限。在这里，我们选择相对稳定且具有多个金属配体配位键的金属蛋白酶作为该目的的系统。使用基于原子力显微镜的单分子力谱（SMFS），我们成功地证明了人类金属硫蛋白-3（MT）β-结构域的复杂和多步骤蛋白质折叠情况。MT 是一种约 60 个氨基酸（aa）长度的蛋白质，具有 20 个用于各种金属结合的半胱氨酸，β-结构域（βMT）约 30 aa 长，有一个 MS 金属簇。我们从 Cd-βMT 中检测到四种不同类型的三步蛋白质折叠情况，这可能可以用复杂的 CdS 金属簇中 Cd-S 键的断裂来解释。此外，还观察到具有四个断裂峰的复杂展开情况。Cd-S 键以单键和多键模式断裂。我们的结果不仅为多步蛋白质折叠现象提供了证据，还利用单分子原子力显微镜揭示了金属蛋白酶系统的独特性质。
