Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity
- University of Illinois at Chicago, United States
- Georgia Institute of Technology and Emory University School of Medicine, United States
- Massachusetts Institute of Technology, United States
- University of North Carolina, United States
- Koch Institute for Integrative Cancer Research at MIT, United States
Abstract
Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
Data availability
The raw mass spectrometry data and associated tables have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD018162. All data generated or analyzed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Jason E Conage-Pough
Funding
National Institutes of Health (R21CA212907)
- Jordan Fauser
National Institutes of Health (CA238720)
- Forest M White
Chicago Biomedical Consortium
- Andrei V Karginov
Army Research Office (W911NF-17-1-0395)
- Denis Tsygankov
National Institutes of Health (R21CA159179)
- Andrei V Karginov
National Institutes of Health (R01GM118582)
- Andrei V Karginov
National Institutes of Health (R21CA223915)
- Jalees Rehman
- Andrei V Karginov
National Institutes of Health (HL007829-22)
- Mark Shaaya
- Jordan Fauser
- Martin Brennan
National Institutes of Health (P01 HL060678)
- Viswanathan Natarajan
- Jalees Rehman
- Andrei V Karginov
National Institutes of Health (CA210180)
- Forest M White
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Shaaya et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity
eLife 9:e60647.
https://doi.org/10.7554/eLife.60647
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