Einfluss von ATP auf die biologische Aktivität von Cryptochrom 2 aus Arabidopsis thaliana
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Philipps-Universität Marburg
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Abstract
Cryptochromes (cry) are blue light receptors that together with photolyases form the so-called
cryptochrome/photolyase family with members occuring in all three domains of life. All family
members show high structural similarities within the N-terminal photosensory (PHR,
photolyase homologous region) domain that non-covalently bind the FAD cofactor, but show
differences regarding their biological functions. Photolyases are DNA repair enzymes that
remove UV-B lesions in damaged DNA. Cryptochromes lack DNA repair activity but gained
important roles in regulating manifold biological responses. The model plant Arabidopsis
thaliana encodes two classical cryptochromes, cry1 and cry2, with main functions in regulating
photomorphogenesis and photoperiodic flowering. Prerequisite for active crys is electron
transfer towards the excited flavin cofactor allowing the formation of the protein’s signalling
state. In the case of plant crys the signalling state contains the semireduced radical form of the
flavin, FADH°. The reduction of the resting state in the dark to the flavin’s lit state is called
“photoreduction”.
For cry1, Bouly et al. (Bouly et al., 2003) demonstrated for the first time that plant
cryptochromes bind ATP. Subsequently, cry1’s metabolite binding site was determined in
crystals of the PHR domain soaked with the non-hydrolyzable ATP analog AMP-PNP
(Brautigam et al., 2004): The nucleotide is associated within the FAD cavity by a central
tyrosine. However, still not much is known about the biochemical effects of ATP-binding to crys
and consequences on their biological activity.
The central topic of this work is the role of ATP binding to cry2. The residues in cry2 necessary
for ATP-binding have not been ultimately identified by mutant studies. This is adressed in this
PhD thesis by analyzing cry2 mutants with a replacement within the predicited ATP-binding
site. Moreover, this work adresses, which effects ATP-binding has on cry2 in vitro. In addition,
it was investigated how ATP-binding may affect the biological function of cry2 in planta.
The recent results confirm that the among plant cryptochromes conserved Tyr residue 399 is
essential in cry2 for ATP binding. By using in vitro assays with non-binding mutants, it could
also been shown that ATP binding induces structural changes, especially in the C-terminal
helix that connects the PHR domain with the C-terminal extension of the protein. Moreover,
photoreduction in the cry2 wild-type protein is enhanced and FADH° is stabilized, when ATP
is present.
Complementation studies with transgenic Arabidopsis lines expressing the non-binding cry2
mutants in a cry2-deficient background showed that these proteins still retained biological
activity. However, this remaining biological activity is reduced compaired to lines that express
the wild-type protein. Consequently, ATP binding is not essential for cry2 but boosts the activity
of cry2 by stabilizing the protein’s signalling state likely through conformational changes.
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Dates
Created: 2018Issued: 2018-10-15Updated: 2018-10-15
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
ATPLight perceptionphotoreceptorPhotorezeptorATPLichtperzeption
DFG-subjects
Cryptochrom 2, Arabidopsis thaliana, Photorezeptor, ATP,
DDC-Numbers
570
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Eckel, Maike: Einfluss von ATP auf die biologische Aktivität von Cryptochrom 2 aus Arabidopsis thaliana. : Philipps-Universität Marburg 2018-10-15. DOI: https://doi.org/10.17192/z2018.0102.
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This item has been published with the following license: In Copyright