Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields
Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields
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高磁场下时间分辨电子顺磁共振触发 AsLOV2 的功能动力学
DOI：
10.1002/ange.202212832
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发表时间：
2023
影响因子：
--
通讯作者：
Sherwin, Mark S.
中科院分区：
文献类型：
--
作者：
Maity, Shiny;Price, Brad D.;Wilson, C. Blake;Mukherjee, Arnab;Starck, Matthieu;Parker, David;Wilson, Maxwell Z.;Lovett, Janet E.;Han, Songi;Sherwin, Mark S.
我们提出了 240 GHz 的时间分辨 Gd−Gd 电子顺磁共振 (TiGGER)，用于跟踪溶液状态下蛋白质机械循环期间的残基间距离。 TiGGER 利用 Gd-sTPATCN 自旋标签，与硝基氧自旋标签相比，其有利的品质包括自旋 7/2 EPR 活性中心、短接头、窄固有线宽以及在高场 (8.6 T) 下几乎没有各向异性。使用 TiGGER，我们确定光激活后，AsLOV2 的 C 端和 N 端在不到 1 秒的时间内分离，并以大约 60 秒的时间常数松弛回到平衡。 TiGGER 揭示，AsLOV2 的 Q513A 变体中光激活的长程机械运动减慢，并且与最近文献中描述的光激发发色团的类似减慢弛豫相关。 TiGGER 有潜力对蛋白质触发功能动力学研究的现有方法进行有价值的补充。
We present time‐resolved Gd−Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter‐residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd‐sTPATCN spin labels, whose favorable qualities include a spin‐7/2 EPR‐active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C‐terminus and N‐terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light‐activated long‐range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.