Engineered bacteriophytochrome heterodimers for research and applications

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Proteins often function as dimers, with bacteriophytochromes (BphPs) being homodimeric photoreceptors.
• Bacteriophytochromes possess a photosensory module (PSM) for light detection and a histidine kinase (HK) output module for signal transduction.

Purpose of the Study:

• To engineer monomeric bacteriophytochrome PSMs capable of forming stable heterodimers.
• To investigate the role of dimerization in the activity of output modules, specifically histidine kinase (HK) and phosphatase domains.
• To demonstrate the application of light-inducible heterodimerization for controlling cellular processes.

Main Methods:

• Modification of salt bridges at the dimerization interface of Deinococcus radiodurans phytochrome (DrBphP) to create monomeric PSMs.
• Characterization of heterodimer formation upon mixing engineered monomeric PSMs.
• Assessment of HK and phosphatase activity in response to red light in heterodimeric constructs.
• Implementation of heterodimeric variants in a red light-regulated gene expression system.

Main Results:

• Successfully generated monomeric bacteriophytochrome PSMs that form stable heterodimers when mixed.
• Confirmed that these heterodimeric PSMs can modulate output HK activity in a light-dependent manner.
• Demonstrated that dimerization is essential for the kinase activity of the FixL HK module but not necessarily for the phosphatase activity of DrBphP.
• Showcased the combined control of gene expression using light-activated heterodimerization.

Conclusions:

• Engineered bacteriophytochrome heterodimers offer a novel mechanism for light-controlled protein interactions.
• This system provides a powerful tool for optogenetics and receptor protein research, enabling precise control over cellular functions.
• The findings highlight the critical role of dimerization in the signaling pathways of bacteriophytochromes and related systems.