Using Aureochrome to Control Protein-Protein Interactions with Light Public Deposited

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  • March 22, 2019
  • Zou, Xin
    • Affiliation: School of Medicine, Department of Pharmacology
  • Protein-protein interactions occurring with precise timing and subcellular localization are critical for regulating various cellular behaviors, yet it is difficult to study these behaviors because there are no practical means to generate protein-protein interactions at precise times and places in live cells. Photoactivatable proteins provide a way to manipulate protein-protein interactions with light in vivo. Recently, a blue light receptor Aureochrome was discovered in stramenopile algae Vaucheria frigida. It has a basic region domain, leucine zipper domain and a LOV (Light, Oxygen, Voltage) domain. Blue light treatment strongly enhances Aureochrome binding to target DNA, implying that Aureochrome is a blue light-regulated transcription factor. To control protein-protein interactions by taking advantage of Aureochrome, we characterized the light-regulated dimerization of Aureochrome. With co-immunoprecipitation assays, we showed that a leucine zipper coupled with a LOV domain (F144-K348) is sufficient for light-dependent dimerization. Critical mutations or deletion of the leucine zipper destroy the dimerization, indicating that the leucine zipper domain is critical for dimerization. Mutation of the LOV domain also disrupts the response to light. Deletion of 25 amino acids at the C-terminus leads to light-independent dimerization, implying that an autoinhibition mechanism is involved. By introducing a salt bridge mutation in the leucine zipper domain, we are able to re-engineer Aureochrome to generate homodimerization rather than heterodimerization, which could potentially be valuable in many applications. Further to verification of the dimerization of AUREO1 in living cells, we expect AUREO1 to be useful for precisely controlling protein-protein interactions temporally and spatially with light.
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  • ... in partial fulfillment of the requirements for the degree of Master of Science in the Department of Pharmacology.
  • Hahn, Klaus

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