Analyzing In Vitro Autokinase Activity of Microbial Phytochromes Under Well-Defined Light Conditions

Microbial phytochromes primarily function as sensor histidine kinases in two-component regulatory systems, with light-triggered conformational changes driving varying autokinase outputs. Analyzing these responses under different light conditions is key to understanding phytochrome functions in host organisms.Here, we introduce a novel method for providing quantitative correlation between kinase activity and calculated Pr/Pfr fractions as determined by spectroscopic measurements. The approach includes data analysis for calculating Pr/Pfr content using UV/Vis absorption spectroscopy. It also introduces a Kinase Illumination Module (KIM) that creates a controlled light environment during autokinase assays, replicating conditions from UV/Vis spectroscopy to ensure consistent photostationary states. The method provides a versatile platform for investigating the dynamics of phytochrome signaling pathways, which is applicable to a wide range of experimental conditions.