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

Abstract

<p>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.<br /></p>