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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.

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

  • 实验方案

  • 发表日期：

    2026年

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  • Christina Huber,
  • Merle Strack,
  • Rolf Diller,
  • Nicole Frankenberg-Dinkel

摘要

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.