Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.
Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.
复制标题
DOI：
10.1038/s41592-023-01880-5
复制
发表时间：
2023-06
期刊：
影响因子：
48
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Lee, Song-Yi;Cheah, Joleen S.;Zhao, Boxuan;Xu, Charles;Roh, Heegwang;Kim, Christina K.;Cho, Kelvin F.;Udeshi, Namrata D.;Carr, Steven A.;Ting, Alice Y.
将光响应结构域并入工程化蛋白质中使得能够控制蛋白质的定位、与光的相互作用和功能。我们将光遗传学控制整合到邻近标记中，邻近标记是活细胞中细胞器和相互作用组的高分辨率蛋白质组学作图的基石技术。通过结构引导筛选和定向进化，我们将光敏LOV结构域安装到邻近标记酶TurboID中，以快速可逆地控制其低功率蓝光的标记活性。“LOV-Turbo”在多种环境下工作，并大大减少了生物素丰富的环境（如神经元）中的背景。我们使用LOV-Turbo进行脉冲追踪标记以发现在细胞应激下在内质网、核和线粒体隔室之间运输的蛋白质。我们还表明，代替外部光，LOV-Turbo可以通过来自荧光素酶的生物发光共振能量转移来激活，从而实现相互作用依赖的邻近标记。总的来说，LOV-Turbo增加了邻近标记的空间和时间精度，扩大了邻近标记可以解决的实验问题的范围。
The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. ‘LOV-Turbo’ works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.
登录
查看更多内容
影响因子：
48
作者：
Kennedy, Matthew J.;Hughes, Robert M.;Peteya, Leslie A.;Schwartz, Joel W.;Ehlers, Michael D.;Tucker, Chandra L.
通讯作者：
Tucker, Chandra L.
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
14.9
作者：
Gene Ontology Consortium
通讯作者：
Gene Ontology Consortium
影响因子：
7.7
作者：
Hung V;Lam SS;Udeshi ND;Svinkina T;Guzman G;Mootha VK;Carr SA;Ting AY
通讯作者：
Ting AY
影响因子：
14.8
作者：
Gagnon, Keith T.;Li, Liande;Janowski, Bethany A.;Corey, David R.
通讯作者：
Corey, David R.