Nanomechanics of HaloTag tethers.
Nanomechanics of HaloTag tethers.
复制标题
HALOTAG TETHERS的纳米力学。
DOI：
10.1021/ja4056382
复制
发表时间：
2013-08-28
影响因子：
15
通讯作者：
Fernandez JM
中科院分区：
文献类型：
--
作者：
Popa I;Berkovich R;Alegre-Cebollada J;Badilla CL;Rivas-Pardo JA;Taniguchi Y;Kawakami M;Fernandez JM
卤代烷烃脱氢酶（HaloTag）酶的活性位点可以共价连接到氯代烷烃配体，提供机械上强的系链，抵抗大的拉力。在这里，我们证明了共价拴系的蛋白L和I27多聚蛋白之间的AFM悬臂和玻璃表面使用HaloTag锚定在一端，和硫醇化学在另一端。通过观察全长多蛋白解折叠，结合高达~2000 pN的高分离力，明确证实了共价束缚。我们使用这些共价锚定的多聚蛋白来研究HaloTag蛋白的显着机械性能。我们发现，当施加的力的方向从C-末端改变到N-末端时，触发HaloTag蛋白展开的力表现出四倍的增加，从131 pN增加到491 pN。力钳实验表明，HaloTag蛋白的解折叠对拉力的敏感性是蛋白L的两倍，并且以较慢的速度重新折叠。我们展示了这些特性如何允许在高作用力下长期观察蛋白质折叠-展开周期，而不受HaloTag系链的干扰。
The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an AFM cantilever and a glass surface using HaloTag anchoring at one end, and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ~2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a four-fold increase, from 131 pN to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice more sensitive to pulling force compared to protein L, and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.