A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.
A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.
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DOI：
10.1021/jacs.6b05429
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发表时间：
2016-08-24
影响因子：
15
通讯作者：
Fernández JM
中科院分区：
文献类型：
--
作者：
Popa I;Rivas-Pardo JA;Eckels EC;Echelman DJ;Badilla CL;Valle-Orero J;Fernández JM
在生理条件下，蛋白质氧化和错误折叠发生的概率非常低，并且时间尺度很长。单分子技术提供了区分正确折叠和受损蛋白质的能力，否则这些蛋白质在整体测量中会被掩盖。然而，在生理条件下，这些罕见的事件发生的时间常数为几个小时，无法进入目前的单分子方法。在这里，我们提出了一种基于磁镊的技术，允许，第一次，在为期一周的实验中折叠的单个蛋白质的研究。该技术结合了HaloTag锚定、磁铁的亚微米定位和焦点漂移的主动校正。使用这种技术和蛋白质L作为分子模板，我们通过将磁体和测量顺磁珠之间的距离与展开/折叠步骤相关联来产生磁定律。我们证明，使用这种磁铁定律，我们可以准确地测量蛋白质的动力学在很宽的范围内的力量，最小的分散珠珠。我们还表明，力校准保持不变，在为期一周的实验应用于相同的单一蛋白质。本文中展示的方法开辟了新的令人兴奋的方法来检查“人类”时间尺度上的蛋白质，并建立了磁镊作为一种有价值的技术，研究低概率事件发生在蛋白质折叠的力量。
Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single molecule approaches. Here we present a magnetic tweezers based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micron positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template we generate a magnet-law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this article opens new exciting ways to examine proteins on the “human” time scale and establishes magnetic tweezers as a valuable technique to study low probability events that occur during protein folding under force.
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影响因子：
64.5
作者：
Alegre-Cebollada J;Kosuri P;Giganti D;Eckels E;Rivas-Pardo JA;Hamdani N;Warren CM;Solaro RJ;Linke WA;Fernández JM
通讯作者：
Fernández JM
影响因子：
15
作者：
Chen, Hu;Yuan, Guohua;Winardhi, Ricksen S.;Yao, Mingxi;Popa, Ionel;Fernandez, Julio M.;Yan, Jie
通讯作者：
Yan, Jie
影响因子：
56.9
作者：
Fernandez, JM;Li, HB
通讯作者：
Li, HB
DOI：
10.1073/pnas.1212167109
发表时间：
2012-09-04
影响因子：
11.1
作者：
Berkovich, Ronen;Hermans, Rodolfo I.;Fernandez, Julio M.
通讯作者：
Fernandez, Julio M.
影响因子：
3.4
作者：
Lipfert, Jan;Hao, Xiaomin;Dekker, Nynke H.
通讯作者：
Dekker, Nynke H.