Kinetic Interval Measurement: A Tool to Characterize Thermal Reversion Dynamics of Light-switchable Fluorescent Proteins

Abstract

Light-switchable proteins like Light-Oxygen-Voltage (LOV) domains can be used to control protein interactions and have been applied in vivo to manipulate cell behavior. The switching between dark and light state of LOV domains depends on temperature or their chemical microenvironment and can be tuned by point mutations. Here, we present a method called Kinetic Interval Measurement (KIM) to quantify the thermal reversion dynamics of light-switchable proteins by using a custom microplate reader. We show that this versatile method can be used to determine the reversion half-life of the excited state of LOV proteins in a reproducible, fast and simple manner consuming only small amounts of protein. The sensitivity of the method allows to report on changes in temperature and imidazole concentration as well as the photoswitching dynamics of LOV proteins in living cells.