Unfolding mechanics of holo- and apocalmodulin studied by the atomic force microscope
Unfolding mechanics of holo- and apocalmodulin studied by the atomic force microscope
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DOI：
10.1110/ps.3600102
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发表时间：
2002-06-01
期刊：
影响因子：
8
通讯作者：
Ikai, A
中科院分区：
文献类型：
--
作者：
Hertadi, R;Ikai, A
钙调素(CaM)的结构稳定性已经通过化学和热方法进行了研究。钙负载形式的CaM被发现非常稳定，因为它可以暴露在&90℃的温度或9M尿素溶液中，而其三级结构没有明显变化，因此无法通过实验使用传统分析方法进行展开研究。在这项研究中，我们开发了一种使用原子力显微镜(AFM)通过拉伸蛋白质的N-末端和C-末端残基来测量机械打开CaM的力的系统，我们成功地获得了apo和Holo形式的CaM的力-拉伸(F-E)曲线。在我们的实验中，当apoCaM和holoCaM完全伸展时，得到了可区分的F-E曲线。在apoCaM拉伸时观察到的非常低的力表明apo形式的柔韧性相对较高。相反，在HoloCaM的全拉伸过程中，出现了相对较高的去折叠力和特征力峰。后一种形式的CaM的F-E曲线很可能反映了比apoCaM更刚性和更有组织的全息CaM构象。这些实验证实，原子力显微镜能够根据机械特性清楚地区分两种不同形式的凸轮。
The structural stability of calmodulin (CaM) has been investigated previously by chemical and thermal methods. The calcium-loaded form of CaM has been found to be exceptionally stable, because it can be exposed to temperatures >90degreesC or to a 9 M urea solution without a marked change in its tertiary structure, and is therefore not experimentally accessible for unfolding studies using conventional analytical methods. In this study, we have developed a system for measuring the force for mechanically unfolding CaM using an atomic force microscope (AFM) by stretching the protein from its N- and C-terminal residues, we have been successful in obtaining force versus extension (F-E) curves for both apo and holo forms of CaM. In our experiment, distinguishable F-E curves have been obtained upon stretching of apoCaM and holoCaM to their full extensions. A very low force observed upon stretching of apoCaM indicated a relatively high flexibility of the apo form. On the contrary, a relatively high unfolding force and the appearance of a characteristic force peak were noted during full stretching of holoCaM. The F-E curve of the latter form of CaM most likely reflects a more rigid and probably more organized conformation of holoCaM than that of apoCaM. These experiments confirmed that the AFM is able to clearly distinguish two functionally distinct forms of CaM in terms of their mechanical properties.