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

Song-Yi Lee; Joleen S Cheah; Boxuan Zhao; Charles Xu; Heegwang Roh; Christina K Kim; Kelvin F Cho; Namrata D Udeshi; Steven A Carr; Alice Y Ting

doi:10.1038/s41592-023-01880-5

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

Nat Methods. 2023 Jun;20(6):908-917. doi: 10.1038/s41592-023-01880-5. Epub 2023 May 15.

Authors

Song-Yi Lee^#¹, Joleen S Cheah^#², Boxuan Zhao¹, Charles Xu³, Heegwang Roh⁴, Christina K Kim^{1

5}, Kelvin F Cho^{1

6}, Namrata D Udeshi³, Steven A Carr³, Alice Y Ting^{7

8

9

10}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, USA.
² Department of Biology, Stanford University, Stanford, CA, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁵ Center for Neuroscience and Department of Neurology, University of California, Davis, CA, USA.
⁶ Amgen Research, South San Francisco, CA, USA.
⁷ Department of Genetics, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
⁸ Department of Biology, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
⁹ Department of Chemistry, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁰ Chan Zuckerberg Biohub-San Francisco, San Francisco, CA, USA. ayting@stanford.edu.

^# Contributed equally.

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.

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

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