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Method Article
Michael J Courtney
Turku Bioscience Centre, University of Turku and Åbo Academy University
Corresponding Author
License:
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Optogenetic actuators exist in either active or inactive states. Absorption of light drives transition of the chromophore to the activated state, whereas thermal processes typically cause gradual relaxation to the initial or dark state. Relaxation rates determine how often activation light needs to be applied to maintain the activated state, but this rate is strongly affected by temperature and sequences surrounding the photosensor domain. Application of existing cellular optogenetic actuators and optimization of new ones therefore requires knowledge of the relaxation rates under the experimental conditions in which they are used. When proteins targeted by the actuator do not generate immediately visible responses, alternative methods are required to determine relaxation times. We describe a simple yet sensitive procedure to measure the relaxation rate constant for an optogenetic actuator. By using resonance energy transfer with a fused fluorescent protein tag to detect the change in chromophore state, low amounts of whole cell lysate are sufficient to perform the measurement.
Keywords
optogenetic actuator, resonance energy transfer (RET), LOV2, relaxation time constant
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Michael J Courtney
Turku Bioscience Centre, University of Turku and Åbo Academy University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 2
Posted 16 Oct, 2020
No community comments so far
No comments provided
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Optogenetic actuators exist in either active or inactive states. Absorption of light drives transition of the chromophore to the activated state, whereas thermal processes typically cause gradual relaxation to the initial or dark state. Relaxation rates determine how often activation light needs to be applied to maintain the activated state, but this rate is strongly affected by temperature and sequences surrounding the photosensor domain. Application of existing cellular optogenetic actuators and optimization of new ones therefore requires knowledge of the relaxation rates under the experimental conditions in which they are used. When proteins targeted by the actuator do not generate immediately visible responses, alternative methods are required to determine relaxation times. We describe a simple yet sensitive procedure to measure the relaxation rate constant for an optogenetic actuator. By using resonance energy transfer with a fused fluorescent protein tag to detect the change in chromophore state, low amounts of whole cell lysate are sufficient to perform the measurement.
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