This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
RET-enhanced measurement of relaxation time constants of LOV2-based optogenetic actuators
Michael J Courtney
Turku Bioscience Centre, University of Turku and Åbo Academy University
Corresponding Author
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.
Keywords
optogenetic actuator, resonance energy transfer (RET), LOV2, relaxation time constant
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 2
Posted 16 Oct, 2020
No community comments so far
No comments provided
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
RET-enhanced measurement of relaxation time constants of LOV2-based optogenetic actuators
Michael J Courtney
Turku Bioscience Centre, University of Turku and Åbo Academy University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
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.