In vivo-Untersuchungen von Konformations-Änderungen der Calcium-abhängigen Protein-Kinase NtCDPK2 aus Nicotiana tabacum

Ziel dieser Arbeit war die Detektion von Konformations-Änderungen der Calcium-abhängigen Protein-Kinase NtCDPK2 aus Tabak (Nicotiana tabacum), die in Abhängigkeit eines Stress-Stimulus induziert werden. Da bislang weder Röntgen- noch NMR-Strukturen für ein Gesamt-Protein einer CDPK bekannt sind,...

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Bibliographic Details
Main Author: Freymark, Gerald
Contributors: Bölker, Michael (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
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Calcium-dependent protein kinases (CDPKs) form large gene families in plants and various protists and it was proposed that members may be involved in different signalling pathways. The objective of this thesis was the detection of conformational changes of NtCDPK2 from tobacco (Nicotiana tabacum) which were induced by external stress stimuli. Because no structural data from crystallization or NMR studies are available for full length CDPKs, three independent in vivo methods were applied to investigate the conformational changes of NtCDPK2 in planta. This information should also help to elucidate the biochemical reaction mechanism of the enzyme during its in vivo activation in planta. Using the split ubiquitin (SplitUB) system intermolecular interactions of CDPK protein subdomains were first analysed in trans. The splitUB system was then further developed for the study of in vivo protein domain interactions in planta based on the Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana. An in vivo interaction between the calmodulin-like domain and the autoinhibitory junction domain of NtCDPK2 could be demonstrated. Furthermore, by investigating NtCDPK2 variants carrying point mutations in specific domains a correlation between the conformational information obtained by the SplitUB system in cis and the biological activity of the enzyme was observed. These conformational changes were shown to be dependent on kinase function either by a mutation in the active centre of the kinase or by the alteration of the structure of the autoinhibitory junction domain. In addition, integrity of the Ca2+-binding EF-hands was required for NtCDPK2 conformation. Based on a second system, bimolecular fluorescence complementation, an interaction of the N-terminal domains (VKJ) with the calmodulin-like domain of NtCDPK2 could also be shown in N. benthamiana. Third, intramolecular interactions indicating changes in the overall conformations of the protein were analysed in planta using fluorescence resonance energy transfer (FRET). Depending on the structural and thus functional properties of the autoinhibitory domain of NtCDPK2, different protein conformations could also be observed. These data support and complement a previously proposed model for CDPK in vivo activation. However, none of these systems allowed the detection of a fast kinetic of significant conformational changes for NtCDPK2 directly triggered by external stress stimuli in vivo. In an independent approach the SplitUB system was applied to analyse light-dependent conformational changes of the UV-A/blue light receptor AtCRY2 from Arabidopsis thaliana. This cryptochrome is proposed to become activated in a blue light-dependent manner. In this thesis, in vivo evidence was obtained for the first time showing a light-dependent conformational change of cryptochrome based on the application of the SplitUB system in planta.