A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics
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- 作者：Ionel Popa ; Jaime Andrés Rivas-Pardo ; Edward C. Eckels ; Daniel J. Echelman ; Carmen L. Badilla ; Jessica Valle-Orero ; Julio M. Fernández
- 刊名：Journal of the American Chemical Society
- 出版年：2016
- 出版时间：August 24, 2016
- 年：2016
- 卷：138
- 期：33
- 页码：10546-10553
- 全文大小：578K
- 年卷期：0
- ISSN：1520-5126
文摘
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-micrometer 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.