小柯机器人

美国斯坦福大学Alice Y. Ting团队近期取得重要工作进展，他们研究利用光调节LOV-Turbo中的工程变构可实现对活细胞中邻近标记的精确时空控制。相关研究成果2023年5月15日在线发表于《自然—方法学》杂志上。

据介绍，将光响应结构域结合到工程蛋白中使得能够控制蛋白质与光的定位、相互作用和功能。

研究人员将光遗传学控制整合到邻近标记中，这是活细胞中细胞器和相互作用体的高分辨率蛋白质组学绘图的基石技术。通过结构引导筛选和定向演化，研究人员将光敏LOV结构域安装到邻近标记酶TurboID中，以低功率蓝光快速可逆地控制其标记活性。LOV Turbo可在多种环境中工作，并显著降低神经元等富含生物素环境中的背景。研究人员使用LOV Turbo进行脉冲追踪标记，发现了在细胞应激下内质网、细胞核和线粒体区室之间运输的蛋白质。研究人员还表明，LOV Turbo可以通过荧光素酶的生物发光共振能量转移来激活，而不是外部光，从而实现相互作用依赖的邻近标记。

总体而言，LOV Turbo提高了邻近标记的空间和时间精度，扩大了可以通过邻近标记解决的实验问题的范围。

附：英文原文

Title: Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells

Author: 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.

Issue&Volume: 2023-05-15

Abstract: 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.

DOI: 10.1038/s41592-023-01880-5

Source: https://www.nature.com/articles/s41592-023-01880-5

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex