Aktivierung und Deaktivierung von Cryptochrom 1 aus Arabidopsis thaliana
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Philipps-Universität Marburg
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Abstract
Cryptochromes and photolyases are flavoproteins which belong to the Cryptochrome/Photolyase-Superfamily (CPF). Even though all CPFs show a high sequence and structure similarity they can be distinguished by their physiological functionality. Generally, cryptochromes function as blue-light photoreceptor, while photolyases repair CPD- or (6-4)-DNA damages in single and/or double stranded DNA. However, the borders between the classes of proteins inside of the CPF can be blurred over the different organisms in which they are occurring. Except of animal type II cryptochromes, CPFs contain FAD as redox active cofactor. The FAD is known to absorb blue-light and in combination with an external reducing agent it is reduced to FADH° in cryptochromes or FADH- in photolyases. In photolyases the blue-light activated FADH-* transfers its electron to the DNA leasion, which is essential for DNA repair. In cryptochromes formation of the FADH° is proposed to lead to a conformational change, but the molecular mechanism is less clear.
This work focused on the very early steps of the molecular activation mechanisms of cryptochrome 1 (cry1) from Arabidopsis thaliana. It could be shown that cry1 forms a tetrameric structure under blue-light condition in vitro. By investigating the dependencies of the cry1 oligomer formation it turned out that beside blue-light and an external reducing agent also molecular oxygen is required. This novel finding led to the conclusion that not FADH° is the active FAD state but the reoxidation of this cofactor is crucial for the cry1 activation. Through enzymatic assays for superoxide (O2·-) and hydrogenperoxide (H2O2) activities it could be also shown that cry1 produces reactive oxygen species (ROS) in the presence of blue-light. Interestingly, this work also proofed that cry1 oligomere can be received by external ROS species without blue-light. About the physiological relevance of this finding can only be speculated, but a novel mechanism of redox-regulation for cry1 is feasible.
In cooperation with Dr. Stephan Bohn, Dr. Thomas Heimerl, Dr. Jürgen Plitzko, Dr. Gert Bange and Dr. Jan Schuller we were able to obtain a cryoEM structure of the blue-light activated tetrameric complex of cry1.
Blue-light inhibitor of cryptochromes (BICs) are proteins which were discovered recently by Wang et al. in 2016. In Arabidopsis thaliana BIC1 and BIC2 are responsible for inhibiting cry1 and cry2. In this study BIC1 was extensively studied to reveal the mechanism how it inhibits cry1. It could be shown that BIC1 has peroxidase activity and contains a covalently bound heme cofactor in vitro. Besides its peroxidase activity BIC1 promotes the monomerization of cry1 under blue-light conditions and in the dark. Taken both mechanisms together they utilize BIC1 as a potent inhibitor of cry1.
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Created: 2021Issued: 2022-02-03Updated: 2022-02-03
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
cryogenic electron microscopyCryoEMCryptochromesCry1photoreceptorreactive oxygen speciesROSblue light
DFG-subjects
ROSKryoelektronenmikroskopieReaktive SauerstoffspeziesPhotorezeptorBlue-light inhibitors of cryptochromesCry1BIC,CryptochromeBlaulicht
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570
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Niemann, Nils: Aktivierung und Deaktivierung von Cryptochrom 1 aus Arabidopsis thaliana. : Philipps-Universität Marburg 2022-02-03. DOI: https://doi.org/10.17192/z2021.0242.
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This item has been published with the following license: In Copyright