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RET-enhanced measurement of relaxation time constants of LOV2-based optogenetic actuators (Method article)




List of AuthorsLi-Li Li, Michael J Courtney

Publication year2020

JournalProtocol exchange

eISSN2043-0116

DOIhttp://dx.doi.org/10.21203/rs.3.pex-1131/v2


Abstract

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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Last updated on 2021-24-06 at 09:43